Methods and compositions for T cell differentiation

Abstract

The technology described herein is directed to a stroma-free method of T cell differentiation using soluble Notch ligands. Soluble Notch oligomeric complexes and compositions thereof, as well as methods for making them, are also described herein. Further described herein are immune cells differentiated using stroma-free methods and compositions comprising such immune cells. In some embodiments, the immune cells may be genetically modified. In some embodiments, the immune cells or compositions comprising the immune cells may be administered to a patient as cell replacement therapy to treat a condition.

Description

Detailed Description of the Invention

[0001] [Technical field] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 413,377, filed October 5, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Government support This invention was made with government support under grant numbers HL007574, DK120535, and CA220340 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] The technology described herein relates to immune cell differentiation methods. [Background technology]

[0004] There is a shortage of functional immune cells for in vivo cell replacement therapy, therapies for numerous diseases, disorders, and conditions, as well as for disease modeling, drug screening, and in vitro studies of hematologic disorders. T cells are a critical component of the human adaptive immune system and have great therapeutic potential. However, current T cell-mediated therapies rely on autologous T cells, which limits their broader application. Human induced pluripotent stem cells (iPSCs) represent an ideal source for off-the-shelf, scalable production of cell therapy. However, generating mature, functional T cells from iPSCs has proven challenging. In addition, iPSC differentiation routinely requires coculture with mouse stromal cells, which limits the translational potential of iPSC-derived T cells. Therefore, a high-yield, clinically applicable T cell differentiation method is needed. [Summary of the Invention]

[0005] Embodiments of the technology described herein include methods for differentiating T cells using novel formulations of soluble Notch ligands. In one aspect, the methods described herein are stroma-free T cell differentiation methods, i.e., methods that do not involve co-culturing with stromal cells or any other type of support cells using soluble Notch ligands. Co-culturing with stromal cells, such as murine stromal cells, limits the translational potential of iPSC-derived T cells, for example, by complicating production protocols and adding the risk of transplant rejection due to the presence of stromal cells. Furthermore, T cells differentiated using stromal cells in the past have exhibited an innate-like phenotype (e.g., as measured by TCRgd expression, a marker for gamma delta T cells). Preferably, the T cells exhibit an adaptive phenotype, characterized, for example, by expression of TCRα and β.

[0006] One embodiment provided herein describes a soluble Notch ligand oligomeric complex comprising at least two Notch ligand monomers and a scaffold.

[0007] In one embodiment of any aspect described herein, the Notch ligand monomer is selected from the group consisting of Delta-like-1 (DLL1), Delta-like-3 (DLL3), Delta-like-4 (DLL4), Jagged1 (JAG1), and Jagged2 (JAG2). In one embodiment of any aspect described herein, the Notch ligand monomer is a DLL4 ligand.

[0008] In one embodiment of any aspect described herein, the complex comprises a single type of Notch ligand monomer, e.g., the complex comprises only multiple DLL4 monomers.

[0009] In one embodiment of any aspect described herein, the complex comprises at least two types of Notch ligand monomers, eg, the complex comprises both DLL1 and DLL4 monomers.

[0010] In one embodiment of any aspect described herein, the scaffold is a naturally occurring scaffold or a synthetic scaffold.

[0011] In one embodiment of any aspect described herein, the complex is a dimer, trimer, tetramer, pentamer, hexamer, octamer, icosahedron, or any other higher order 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 a trimer.

[0012] In one embodiment of any aspect described herein, the DLL4 ligand monomer comprises a sequence selected from SEQ ID NOs: 6-9.

[0013] Another embodiment provided herein describes a composition comprising any of the soluble notch ligand oligomeric complexes described herein.

[0014] Another aspect provided herein describes a method of making a soluble Notch ligand oligomeric complex, the method comprising contacting a plurality of Notch ligand monomers with a population of scaffolds for a time sufficient to promote the formation of the complex.

[0015] In one embodiment of any aspect described herein, the plurality of Notch ligand monomers further comprises a GS linker.

[0016] In one embodiment of any aspect described herein, the multiple Notch ligand monomers are fused to the SpyTag via a GS linker.

[0017] In one embodiment of any aspect described herein, the population of scaffolds further comprises a GGSGGS linker (SEQ ID NO: 49).

[0018] In one embodiment of any aspect described herein, the population of scaffolds is fused to SpyCatcher via a GGSGGS linker (SEQ ID NO: 49).

[0019] Another embodiment provided herein describes a soluble Notch ligand oligomeric complex produced from any of the methods described herein.

[0020] Another embodiment provided herein describes a composition of soluble Notch ligand oligomers produced from any of the methods described herein.

[0021] Another aspect provided herein describes a method of activating Notch signaling in a population of cells, the method comprising contacting the population of cells with any of the soluble Notch ligand oligomeric complexes or compositions thereof described herein.

[0022] In one embodiment of any aspect describe herein, the population of cells is not adhered to a substrate.

[0023] In one embodiment of any aspect describe herein, the population of cells is adhered to a substrate.

[0024] In one embodiment of any aspect described herein, 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.

[0025]

[0014] In one embodiment of any aspect described herein, the cell is a mammalian cell.

[0015] In one embodiment of any aspect described herein, the cell is a non-mammalian cell.

[0026] Another embodiment provided herein describes a method comprising: a) differentiating a population of pluripotent stem cells in aggregation medium for a time sufficient to promote differentiation into a population of CD34+ hemogenic endothelium; b) inhibiting histone methyltransferase in the resulting population of CD34+ hemogenic endothelium; and c) differentiating the resulting population of CD34+ hemogenic endothelium in CD3+ T cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of CD3+ T cells.

[0027] Another embodiment provided herein describes a method comprising: a) differentiating a population of pluripotent stem cells in aggregation medium for a time sufficient to promote differentiation into a population of CD34+ hemogenic endothelium; b) inhibiting an epigenetic regulator in the resulting population of CD34+ hemogenic endothelium; and c) differentiating the resulting population of CD34+ hemogenic endothelium in CD3+ T cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of CD3+ T cells.

[0028] Another aspect provided herein describes a method comprising: a) differentiating a population of pluripotent stem cells in aggregation medium for a time sufficient to promote differentiation into a population of CD34+ hemogenic endothelium; b) inhibiting G9a and / or GLP in the resulting population of CD34+ hemogenic endothelium; and c) differentiating the resulting population of CD34+ hemogenic endothelium in CD3+ T cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of CD3+ T cells.

[0029] Another embodiment provided herein describes a method comprising: a) differentiating a population of pluripotent stem cells in aggregation medium for a time sufficient to promote differentiation into a population of CD34+ hemogenic endothelium; and b) differentiating the resulting population of CD34+ hemogenic endothelium in CD3+ T cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of CD3+ T cells.

[0030] In one embodiment of any aspect described herein, differentiating the hemogenic endothelium in the presence of a soluble Notch ligand does not include co-culturing with stromal cells that express a Notch ligand.

[0031] In one embodiment of any aspect described herein, differentiating the hemogenic endothelium in the presence of a soluble Notch ligand does not include co-culturing with OP9-DL1 or OP9-DL4 cells.

[0032] In one embodiment of any aspect described herein, the time sufficient to promote differentiation into a population of CD3+ T cells is at least 4 weeks.

[0033]

[0013] In one embodiment of any aspect described herein, the CD3+ T cell differentiation medium is serum-free.

[0034]

[0013] In one embodiment of any aspect described herein, the CD3+ T cell differentiation medium comprises FLT3 and IL7.

[0035]

[0014] In one embodiment of any aspect described herein, the CD3+ T cell differentiation medium comprises 15 ng / ml FLT3 and 25 ng / ml IL7.

[0036]

[0014] In one embodiment of any aspect described herein, the CD3+ T cell differentiation medium further comprises 5 ng / mL thrombopoietin (TPO) and / or 30 ng / ml SCF for at least the first 2 weeks of differentiation in the CD3+ T cell differentiation medium.

[0037] In one embodiment of any aspect described herein, the CD3+ T cell differentiation medium comprising TPO promotes differentiation into a population of CD5+CD7+ ProT cells.

[0038]

[0013] In one embodiment of any aspect described herein, the population of CD3+ T cells comprises a population of CD4+CD8+ T cells.

[0039]

[0013] In one embodiment of any aspect described herein, the method further includes differentiating the population of CD4+CD8+ T cells in single positive T cell differentiation medium for a time sufficient to promote differentiation into a population of CD4+ cells and a population of CD8+ cells.

[0040] In one embodiment of any aspect described herein, the time sufficient to promote differentiation of a population of CD4+CD8+ T cells into a population of CD4+ T cells and a population of CD8+ cells is at least one week.

[0041] In one embodiment of any aspect described herein, the time sufficient to promote differentiation of the population of CD34+ hemogenic endothelium into a population of CD4+ T cells and a population of CD8+ cells is at least 5 weeks.

[0042] In one embodiment of any aspect described herein, the single positive T cell differentiation medium comprises 10 ng / mL of IL-15 and a T cell activator.

[0043]

[0013] In one embodiment of any aspect described herein, the T cell activator comprises 10 ul / ml of CD3 / CD28 T cell activator.

[0044] In one embodiment of any aspect described herein, the T cell activator comprises one CD3 / CD28 T cell activator Dynabead per cell.

[0045] In one embodiment of any aspect described herein, the method further comprises a step of CD4+ cell enrichment and / or CD8+ cell enrichment at least one week later.

[0046] In one embodiment of any aspect described herein, the population of pluripotent stem cells comprises induced pluripotent stem cells (iPS cells) or embryonic stem cells (ESCs).

[0047] In one embodiment of any aspect described herein, the induced pluripotent stem cells are generated by introducing only the reprogramming factors OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28 into mature cells.

[0048] In one embodiment of any aspect described herein, the induced pluripotent stem cells are generated by introducing reprogramming factors into mature cells two or more times.

[0049] In one embodiment of any aspect described herein, the population of pluripotent stem cells is differentiated into a population of CD34+ hemogenic endothelium using embryoid bodies or 2D adherent cultures.

[0050] In one embodiment of any aspect described herein, the time sufficient to promote differentiation into a population of CD34+ hemogenic endothelium is at least 8 days.

[0051]

[0013] In one embodiment of any aspect describe herein, the aggregation medium comprises BMP4, SB-431542, CHIR99021, bFGF, VEGF, IL-6, IL-11, IGF-1, SCF, and EPO.

[0052]

[0014] In one embodiment of any aspect described herein, the aggregation medium includes 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.

[0053] In one embodiment of any aspect described herein, the method further comprises selecting or isolating the resulting population of CD34+ hemogenic endothelium using expression of surface markers on the population of CD34+ hemogenic endothelium.

[0054] In one embodiment of any aspect described herein, 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.

[0055] In one embodiment of any aspect described herein, the method further comprises genetically modifying the obtained population of CD34+ hemogenic endothelium or the obtained population of CD3+ T cells.

[0056] In one embodiment of any aspect described herein, the genetic modification is to edit endogenous HLA, to remove endogenous TCR, and / or to express a chimeric antigen receptor (CAR).

[0057] In one embodiment of any aspect described herein, the histone methyltransferase catalyzes the addition of a methyl group to histone 3 lysine residue 9 (H3K9) and / or histone 3 lysine residue 27 (H3K27).

[0058]

[0013] In one embodiment of any aspect described herein, the histone methyltransferases H3K9 and / or H3K27 are inhibited by a small molecule inhibitor or a nucleic acid inhibitor.

[0059] In one embodiment of any aspect described herein, the histone methyltransferase H3K9 and / or H3K27 small molecule inhibitor is a heteroorganic or organometallic compound.

[0060]

[0014] In one embodiment of any aspect described herein, 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), EI1, GSK343, GSK126, and UNC1999.

[0061] In one embodiment of any aspect described herein, the nucleic acid inhibitor is a nucleic acid that targets the expression of a histone methyltransferase.

[0062] In one embodiment of any aspect described herein, the nucleic acid inhibitor is an RNA interference inhibitor or drug.

[0063] In one embodiment of any aspect described herein, the nucleic acid inhibitor is an EZH1-specific nucleic acid selected from the group consisting of an aptamer that binds to EZH1, an EZH1-specific RNA interfering agent, and a vector encoding an EZH1-specific RNA interfering agent, and the RNA interfering agent comprises one or more nucleotide sequences selected from SEQ ID NOs: 11-19.

[0064] In one embodiment of any aspect described herein, the epigenetic regulator is a DNA methyltransferase (DNMT), a methyl-CpG binding domain (MBD) protein, a DNA demethylase, a histone methyltransferase (HMT), a methylhistone binding protein, a histone demethylase, a histone acetyltransferase (HAT), an acetyl-binding protein, or a histone deacetylase (HDAC).

[0065]

[0013] In one embodiment of any aspect described herein, the inhibitor of an epigenetic regulator is selected from the group consisting of UNC0224, MC1568, and CAY10591.

[0066] In one embodiment of any aspect described herein, the inhibitor of an epigenetic regulator is provided at a concentration of at least 500 nM.

[0067] In one embodiment of any aspect described herein, the time sufficient to promote differentiation of the population of CD34+ cells to a population of CD5+CD7+ proT cells is about 14 days.

[0068]

[0014] In one embodiment of any aspect described herein, the 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.

[0015] In one embodiment of any aspect described herein, the G9a and / or GLP inhibitor is UNC0224.

[0069] In one embodiment of any aspect described herein, the G9a and / or GLP inhibitor is provided at a concentration of 300 nM to 5 uM.

[0070] In one embodiment of any aspect described herein, the time sufficient to promote differentiation of the population of CD34+ cells to a population of CD5+CD7+ proT cells is about 14 days.

[0071] Another aspect provided herein describes a method comprising: a) differentiating a population of pluripotent stem cells in aggregation medium for a time sufficient to promote differentiation into a population of CD34+ hemogenic endothelium; and b) differentiating the resulting population of CD34+ hemogenic endothelium in CD3+ T cell differentiation medium comprising 15 ng / ml FLT3 and 25 ng / ml IL7 in the presence of a soluble Notch ligand for at least four weeks to promote differentiation into a population of CD3+ T cells, wherein the CD3+ T cell differentiation medium further comprises 5 ng / mL TPO and 30 ng / ml SCF for at least the first two weeks.

[0072] Another aspect provided herein describes a method comprising: a) differentiating a population of pluripotent stem cells in aggregation medium for a time sufficient to promote differentiation into a population of CD34+ hemogenic endothelium; and b) differentiating the resulting population of CD34+ hemogenic endothelium in CD3+ T cell differentiation medium comprising 15 ng / ml FLT3 and 25 ng / ml IL7 in the presence of a soluble Notch ligand for at least four weeks to promote differentiation into a population of CD3+ T cells, wherein the CD3+ T cell differentiation medium further comprises 5 ng / mL TPO, 30 ng / ml SCF, and G9a / GLP inhibitor for at least the first two weeks.

[0073] In one embodiment of any aspect described herein, the population of CD3+ T cells exhibits a gene expression profile most similar to that of alpha beta T cells. In one embodiment of any aspect described herein, the population of CD3+ T cells exhibits a gene expression profile at least 10%, 20%, 30%, 40% or more similar to that of alpha beta T cells.

[0074]

[0014] In one embodiment of any aspect described herein, the population of CD3+ T cells exhibits a gene expression profile with a Pearson correlation coefficient compared to peripheral blood alpha beta T cells of at least 0.85.

[0015] In one embodiment of any aspect described herein, the population of CD3+ T cells exhibits a productive Simpson clonality value of about 0.025.

[0075]

[0013] In one embodiment of any aspect described herein, the population of CD3+ T cells exhibits T cell receptor (TCR) complementarity determining regions (CDRs) that are at least 3 nucleotides longer than immune cells differentiated without methyltransferase inhibition or using stromal cells.

[0076] In one embodiment of any aspect described herein, the soluble Notch ligand is used at a concentration of 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM or more.

[0077] Another aspect provided herein describes an immune cell produced by any of the methods described herein.

[0078] In one embodiment of any aspect described herein, the immune cells exhibit a gene expression profile that is at least 10%, 20%, 30%, 40% or more similar to an alpha beta T cell.

[0079]

[0013] In one embodiment of any aspect described herein, the immune cells exhibit a gene expression profile with a Pearson correlation coefficient compared to peripheral blood alpha beta T cells of at least 0.85.

[0080]

[0013] In one embodiment of any aspect described herein, the immune cells exhibit a productivity Simpson clonality value of about 0.025.

[0081]

[0013] In one embodiment of any aspect described herein, the immune cells exhibit T cell receptor (TCR) complementarity determining regions (CDRs) that are at least 3 nucleotides longer than immune cells differentiated using stromal cells without methyltransferase inhibition.

[0082] A composition comprising any of the immune cells or populations thereof produced by the methods described herein.

[0083] In one embodiment of any aspect described herein, the composition further comprises a pharmaceutically acceptable carrier.

[0084] Another aspect provided herein describes a pharmaceutical composition comprising an immune cell or population thereof produced by any of the methods described herein and a pharmaceutically acceptable carrier.

[0085] In one embodiment of any aspect described herein, the pharmaceutical composition is for use in cell replacement therapy in a subject.

[0086] Another aspect provided herein describes a method of cell replacement therapy, the method comprising administering to a recipient subject in need thereof an immune cell or population thereof produced by any method described herein, or any composition or pharmaceutical composition described herein.

[0087] In one embodiment of any aspect described herein, the recipient subject is undergoing chemotherapy and / or radiation.

[0088] In one embodiment of any aspect described herein, the recipient subject has a deficiency in immune function and / or lymphocyte reconstitution.

[0089] In one embodiment of any aspect described herein, prior to transplantation, the immune cells or populations thereof are treated ex vivo with prostaglandin E2 and / or the antioxidant N-acetyl-L-cysteine ​​(NAC) to promote subsequent engraftment in the recipient subject.

[0090] In one embodiment of any aspect described herein, the immune cells or population thereof are autologous to the recipient subject. In one embodiment of any aspect described herein, the immune cells or population thereof are HLA-type matched to the recipient subject.

[0091] Another embodiment provided herein describes a cell having activated Notch expression produced by any of the methods described herein.

[0092] In one embodiment of any aspect described herein, the cells have increased Notch expression relative to a suitable control.

[0093] In one embodiment of any aspect described herein, Notch expression in the cells is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more compared to a suitable control.

[0094] Another aspect provided herein describes a composition comprising any of the cells or populations thereof described herein.

[0095] Another aspect provided herein describes a pharmaceutical composition comprising any of the cells or populations thereof described herein and a pharmaceutically acceptable carrier.

[0096] Another aspect provided herein is a method, comprising: a) detecting CD34 + Inhibiting histone methyltransferases in hemogenic endothelial populations and b) CD34 + The hemogenic endothelial population was identified as CD56 + and differentiating in natural killer (NK) cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of NK cells. [Brief explanation of the drawings]

[0097] [Figure 1A]A schematic diagram illustrating Notch activation for generating T cells using engineered Notch ligand proteins with various scaffold proteins is presented. Figure 1A) Engineering of the Notch ligand delta-like ligand 4 (DLL4) protein complex: DLL4 is genetically fused to the SpyTag (ST) peptide, while the oligomeric protein complex is genetically fused to the SpyCatcher (SC) protein. SpyTag and SpyCatcher form a covalent conjugate upon mixing; therefore, oligomeric DLL4 protein complexes can be generated by simply mixing purified DLL4-ST with oligomeric-SC. The resulting protein complex is oligomeric DLL4. Figure 1B) A cartoon illustrating cell-cell interactions promoted by soluble oligomeric-DLL4, which brings neighboring cells together and induces Notch synapse formation, activating downstream signaling. This results in the efficient generation of T cells from human induced pluripotent stem cells (iPSCs). Red arrows indicate the direction of signaling. Figure 1C) PDB models showing all oligomeric protein complexes (scaffolds) used in this study: C3: trimer, C5: pentamer, C6: hexamer, C8: octamer, Icos: icosahedron that can be either a 60-mer or a 120-mer, DLL4: monovalent. [Figure 1B]A schematic diagram illustrating Notch activation for generating T cells using engineered Notch ligand proteins with various scaffold proteins is presented. Figure 1A) Engineering of the Notch ligand delta-like ligand 4 (DLL4) protein complex: DLL4 is genetically fused to the SpyTag (ST) peptide, while the oligomeric protein complex is genetically fused to the SpyCatcher (SC) protein. SpyTag and SpyCatcher form a covalent conjugate upon mixing; therefore, oligomeric DLL4 protein complexes can be generated by simply mixing purified DLL4-ST with oligomeric-SC. The resulting protein complex is oligomeric DLL4. Figure 1B) A cartoon illustrating cell-cell interactions promoted by soluble oligomeric-DLL4, which brings neighboring cells together and induces Notch synapse formation, activating downstream signaling. This results in the efficient generation of T cells from human induced pluripotent stem cells (iPSCs). Red arrows indicate the direction of signaling. Figure 1C) PDB models showing all oligomeric protein complexes (scaffolds) used in this study: C3: trimer, C5: pentamer, C6: hexamer, C8: octamer, Icos: icosahedron that can be either a 60-mer or a 120-mer, DLL4: monovalent. [Figure 1C]A schematic diagram illustrating Notch activation for generating T cells using engineered Notch ligand proteins with various scaffold proteins is presented. Figure 1A) Engineering of the Notch ligand delta-like ligand 4 (DLL4) protein complex: DLL4 is genetically fused to the SpyTag (ST) peptide, while the oligomeric protein complex is genetically fused to the SpyCatcher (SC) protein. SpyTag and SpyCatcher form a covalent conjugate upon mixing; therefore, oligomeric DLL4 protein complexes can be generated by simply mixing purified DLL4-ST with oligomeric-SC. The resulting protein complex is oligomeric DLL4. Figure 1B) A cartoon illustrating cell-cell interactions promoted by soluble oligomeric-DLL4, which brings neighboring cells together and induces Notch synapse formation, activating downstream signaling. This results in the efficient generation of T cells from human induced pluripotent stem cells (iPSCs). Red arrows indicate the direction of signaling. Figure 1C) PDB models showing all oligomeric protein complexes (scaffolds) used in this study: C3: trimer, C5: pentamer, C6: hexamer, C8: octamer, Icos: icosahedron that can be either a 60-mer or a 120-mer, DLL4: monovalent. [Figure 2] A bar graph showing soluble DLL4 ligand-mediated activation of Notch signaling in a reporter cell line (U2OS-Notch1-Gal4) is presented. Notch activity was measured via firefly luciferase activity upon treatment of U2OS-Notch1-Gal4 cells with various concentrations of various soluble oligomeric DLL4 protein complexes. Note that 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 modulation of the signal. The interstitial control was coculture of U2OS-Notch1-Gal4 with U2OS-DLL4 cells. [Figure 3A]Data are presented below demonstrating in vitro T cell differentiation experiments. Figure 3A) Schematic diagram showing the in vitro T cell differentiation experiment. Umbilical cord blood (CB)-CD34+ HSPCs were differentiated into proT cell precursor (proT) cells and seeded into 96-well plates for suspension culture. The proT cells were then treated with different types of soluble DLL4 ligands and induced to differentiate into CD4 / CD8 double-positive (DP) T cells. Figure 3B) Screening of soluble DLL4 protein complexes shows that C3-DLL4 activates Notch signaling to efficiently generate DP T cells from proT cells in a concentration-dependent manner. All other constructs failed to generate DP T cells. Figure 3C) Flow cytometry results showing the generation of DP T cells using the engineered soluble C3-DLL4 ligand, whereas free DLL4 ligand alone failed to support proT differentiation. [Figure 3B] Data are presented below demonstrating in vitro T cell differentiation experiments. Figure 3A) Schematic diagram showing the in vitro T cell differentiation experiment. Umbilical cord blood (CB)-CD34+ HSPCs were differentiated into proT cell precursor (proT) cells and seeded into 96-well plates for suspension culture. The proT cells were then treated with different types of soluble DLL4 ligands and induced to differentiate into CD4 / CD8 double-positive (DP) T cells. Figure 3B) Screening of soluble DLL4 protein complexes shows that C3-DLL4 activates Notch signaling to efficiently generate DP T cells from proT cells in a concentration-dependent manner. All other constructs failed to generate DP T cells. Figure 3C) Flow cytometry results showing the generation of DP T cells using the engineered soluble C3-DLL4 ligand, whereas free DLL4 ligand alone failed to support proT differentiation. [Figure 3C]Data are presented below demonstrating in vitro T cell differentiation experiments. Figure 3A) Schematic diagram showing the in vitro T cell differentiation experiment. Umbilical cord blood (CB)-CD34+ HSPCs were differentiated into proT cell precursor (proT) cells and seeded into 96-well plates for suspension culture. The proT cells were then treated with different types of soluble DLL4 ligands and induced to differentiate into CD4 / CD8 double-positive (DP) T cells. Figure 3B) Screening of soluble DLL4 protein complexes shows that C3-DLL4 activates Notch signaling to efficiently generate DP T cells from proT cells in a concentration-dependent manner. All other constructs failed to generate DP T cells. Figure 3C) Flow cytometry results showing the generation of DP T cells using the engineered soluble C3-DLL4 ligand, whereas free DLL4 ligand alone failed to support proT differentiation. [Figure 4A] We demonstrate that cell-cell interactions induced by the C3-DLL4 complex lead to the formation of cell clusters. Figure 4A) Various concentrations of soluble C3-DLL4 complex were added to K562 cells expressing engineered Notch receptors (K562-N1-Gal4 reporter line), and cell interactions were monitored. After 24 h of incubation, cell clusters were observed in a concentration-dependent manner, with the highest cluster formation occurring at a C3-DLL4 concentration of 10 nM. Figure 4B) Cells grown without soluble DLL4 complex or (Figure 4C) those grown on 2D-DLL4-coated plates did not exhibit cluster formation. D-F) The mechanism underlying concentration-dependent cluster formation. Figure 4D) K562-N1-Gal4 cells were incubated with various concentrations of C3-DLL4 for 1 h, then stained with an antibody detecting C3-DLL4, followed by flow cytometry analysis. The intensity of C3-DLL4 indicates the degree of binding to the cell surface. (Figure 4E) At high concentrations of C3-DLL4 (i.e., 1 uM), cells may be saturated with the protein and therefore there is no cell-cell interaction, whereas (Figure 4F) at low concentrations of C3-DLL4 (i.e., 10 nM), cells are not fully saturated with C3-DLL4 and therefore there is enough room for cell-cell interaction. [Figure 4B]We demonstrate that cell-cell interactions induced by the C3-DLL4 complex lead to the formation of cell clusters. Figure 4A) Various concentrations of soluble C3-DLL4 complex were added to K562 cells expressing engineered Notch receptors (K562-N1-Gal4 reporter line), and cell interactions were monitored. After 24 h of incubation, cell clusters were observed in a concentration-dependent manner, with the highest cluster formation occurring at a C3-DLL4 concentration of 10 nM. Figure 4B) Cells grown without soluble DLL4 complex or (Figure 4C) those grown on 2D-DLL4-coated plates did not exhibit cluster formation. D-F) The mechanism underlying concentration-dependent cluster formation. Figure 4D) K562-N1-Gal4 cells were incubated with various concentrations of C3-DLL4 for 1 h, then stained with an antibody detecting C3-DLL4, followed by flow cytometry analysis. The intensity of C3-DLL4 indicates the degree of binding to the cell surface. (Figure 4E) At high concentrations of C3-DLL4 (i.e., 1 uM), cells may be saturated with the protein and therefore there is no cell-cell interaction, whereas (Figure 4F) at low concentrations of C3-DLL4 (i.e., 10 nM), cells are not fully saturated with C3-DLL4 and therefore there is enough room for cell-cell interaction. [Figure 4C]We demonstrate that cell-cell interactions induced by the C3-DLL4 complex lead to the formation of cell clusters. Figure 4A) Various concentrations of soluble C3-DLL4 complex were added to K562 cells expressing engineered Notch receptors (K562-N1-Gal4 reporter line), and cell interactions were monitored. After 24 h of incubation, cell clusters were observed in a concentration-dependent manner, with the highest cluster formation occurring at a C3-DLL4 concentration of 10 nM. Figure 4B) Cells grown without soluble DLL4 complex or (Figure 4C) those grown on 2D-DLL4-coated plates did not exhibit cluster formation. D-F) The mechanism underlying concentration-dependent cluster formation. Figure 4D) K562-N1-Gal4 cells were incubated with various concentrations of C3-DLL4 for 1 h, then stained with an antibody detecting C3-DLL4, followed by flow cytometry analysis. The intensity of C3-DLL4 indicates the degree of binding to the cell surface. (Figure 4E) At high concentrations of C3-DLL4 (i.e., 1 uM), cells may be saturated with the protein and therefore there is no cell-cell interaction, whereas (Figure 4F) at low concentrations of C3-DLL4 (i.e., 10 nM), cells are not fully saturated with C3-DLL4 and therefore there is enough room for cell-cell interaction. [Figure 4D]We demonstrate that cell-cell interactions induced by the C3-DLL4 complex lead to the formation of cell clusters. Figure 4A) Various concentrations of soluble C3-DLL4 complex were added to K562 cells expressing engineered Notch receptors (K562-N1-Gal4 reporter line), and cell interactions were monitored. After 24 h of incubation, cell clusters were observed in a concentration-dependent manner, with the highest cluster formation occurring at a C3-DLL4 concentration of 10 nM. Figure 4B) Cells grown without soluble DLL4 complex or (Figure 4C) those grown on 2D-DLL4-coated plates did not exhibit cluster formation. D-F) The mechanism underlying concentration-dependent cluster formation. Figure 4D) K562-N1-Gal4 cells were incubated with various concentrations of C3-DLL4 for 1 h, then stained with an antibody detecting C3-DLL4, followed by flow cytometry analysis. The intensity of C3-DLL4 indicates the degree of binding to the cell surface. (Figure 4E) At high concentrations of C3-DLL4 (i.e., 1 uM), cells may be saturated with the protein and therefore there is no cell-cell interaction, whereas (Figure 4F) at low concentrations of C3-DLL4 (i.e., 10 nM), cells are not fully saturated with C3-DLL4 and therefore there is enough room for cell-cell interaction. [Figure 4E]We demonstrate that cell-cell interactions induced by the C3-DLL4 complex lead to the formation of cell clusters. Figure 4A) Various concentrations of soluble C3-DLL4 complex were added to K562 cells expressing engineered Notch receptors (K562-N1-Gal4 reporter line), and cell interactions were monitored. After 24 h of incubation, cell clusters were observed in a concentration-dependent manner, with the highest cluster formation occurring at a C3-DLL4 concentration of 10 nM. Figure 4B) Cells grown without soluble DLL4 complex or (Figure 4C) those grown on 2D-DLL4-coated plates did not exhibit cluster formation. D-F) The mechanism underlying concentration-dependent cluster formation. Figure 4D) K562-N1-Gal4 cells were incubated with various concentrations of C3-DLL4 for 1 h, then stained with an antibody detecting C3-DLL4, followed by flow cytometry analysis. The intensity of C3-DLL4 indicates the degree of binding to the cell surface. (Figure 4E) At high concentrations of C3-DLL4 (i.e., 1 uM), cells may be saturated with the protein and therefore there is no cell-cell interaction, whereas (Figure 4F) at low concentrations of C3-DLL4 (i.e., 10 nM), cells are not fully saturated with C3-DLL4 and therefore there is enough room for cell-cell interaction. [Figure 4F]We demonstrate that cell-cell interactions induced by the C3-DLL4 complex lead to the formation of cell clusters. Figure 4A) Various concentrations of soluble C3-DLL4 complex were added to K562 cells expressing engineered Notch receptors (K562-N1-Gal4 reporter line), and cell interactions were monitored. After 24 h of incubation, cell clusters were observed in a concentration-dependent manner, with the highest cluster formation occurring at a C3-DLL4 concentration of 10 nM. Figure 4B) Cells grown without soluble DLL4 complex or (Figure 4C) those grown on 2D-DLL4-coated plates did not exhibit cluster formation. D-F) The mechanism underlying concentration-dependent cluster formation. Figure 4D) K562-N1-Gal4 cells were incubated with various concentrations of C3-DLL4 for 1 h, then stained with an antibody detecting C3-DLL4, followed by flow cytometry analysis. The intensity of C3-DLL4 indicates the degree of binding to the cell surface. (Figure 4E) At high concentrations of C3-DLL4 (i.e., 1 uM), cells may be saturated with the protein and therefore there is no cell-cell interaction, whereas (Figure 4F) at low concentrations of C3-DLL4 (i.e., 10 nM), cells are not fully saturated with C3-DLL4 and therefore there is enough room for cell-cell interaction. [Figure 5]Confocal microscopy studies reveal Notch synapse formation induced by C3-DLL4-mediated cell-cell interactions. Notch1 expression in K562-N1-Gal4 cells was induced by doxycycline (Dox), followed by treatment with 10 nM C3-DLL4 for either 1 or 24 hours. Cells were then fixed, stained for C3-DLL4 (anti-His tag) and Notch1-ECD (anti-Flag tag), and imaged by confocal microscopy. (Figure 5A) Dox-treated cells show Notch1 expression (middle panel), but C3-DLL4 does not. (Figure 5B) Cells treated with C3-DLL4 for 1 hour showed a homogenous distribution of C3-DLL4 on the cell surface, colocalizing with Notch1-ECD. Note that no cell-cell interactions were observed at this stage. FIG. 5C) Cells treated with C3-DLL4 for 24 hours showed cell-cell interactions, and C3-DLL4 and Notch1 co-localized at cell-cell junctions, indicating Notch synapse formation. [Figure 6A] Figures 6A-6D show activation of endogenous Notch by C3-DLL4. (Figures 6A-6D) MDA-MB-231 cells. Figure 6A) Upregulation of Notch target genes (Hes1, Hey1, Hey2, HeyL, and NRARP) in MDA-MB-231 cells as measured by RT-qPCR. Figure 6B) Western blot shows time-dependent Notch1 intracellular domain (N1ICD) cleavage in MDA-MB-231 cells upon C3-DLL4-mediated activation. Figure 6C) Confocal microscopy showing Notch synapse formation mediated by C3-DLL4 in MDA-MB-231 cells. (Figures 6D-6F) SVG-A cells. Figure 6D) Upregulation of Notch target genes (Hes1, Hey1, Hey2, HeyL) in SVG-A cells as measured by RT-qPCR. Western blot experiments showing C3-DLL4-mediated (FIG. 6E) N2ICD and (FIG. 6F) N1ICD cleavage. [Figure 6B]Figures 6A-6D show activation of endogenous Notch by C3-DLL4. (Figures 6A-6D) MDA-MB-231 cells. Figure 6A) Upregulation of Notch target genes (Hes1, Hey1, Hey2, HeyL, and NRARP) in MDA-MB-231 cells as measured by RT-qPCR. Figure 6B) Western blot shows time-dependent Notch1 intracellular domain (N1ICD) cleavage in MDA-MB-231 cells upon C3-DLL4-mediated activation. Figure 6C) Confocal microscopy showing Notch synapse formation mediated by C3-DLL4 in MDA-MB-231 cells. (Figures 6D-6F) SVG-A cells. Figure 6D) Upregulation of Notch target genes (Hes1, Hey1, Hey2, HeyL) in SVG-A cells as measured by RT-qPCR. Western blot experiments showing C3-DLL4-mediated (FIG. 6E) N2ICD and (FIG. 6F) N1ICD cleavage. [Figure 6C] Figures 6A-6D show activation of endogenous Notch by C3-DLL4. (Figures 6A-6D) MDA-MB-231 cells. Figure 6A) Upregulation of Notch target genes (Hes1, Hey1, Hey2, HeyL, and NRARP) in MDA-MB-231 cells as measured by RT-qPCR. Figure 6B) Western blot shows time-dependent Notch1 intracellular domain (N1ICD) cleavage in MDA-MB-231 cells upon C3-DLL4-mediated activation. Figure 6C) Confocal microscopy showing Notch synapse formation mediated by C3-DLL4 in MDA-MB-231 cells. (Figures 6D-6F) SVG-A cells. Figure 6D) Upregulation of Notch target genes (Hes1, Hey1, Hey2, HeyL) in SVG-A cells as measured by RT-qPCR. Western blot experiments showing C3-DLL4-mediated (FIG. 6E) N2ICD and (FIG. 6F) N1ICD cleavage. [Figure 6D]Figures 6A-6D show activation of endogenous Notch by C3-DLL4. (Figures 6A-6D) MDA-MB-231 cells. Figure 6A) Upregulation of Notch target genes (Hes1, Hey1, Hey2, HeyL, and NRARP) in MDA-MB-231 cells as measured by RT-qPCR. Figure 6B) Western blot shows time-dependent Notch1 intracellular domain (N1ICD) cleavage in MDA-MB-231 cells upon C3-DLL4-mediated activation. Figure 6C) Confocal microscopy showing Notch synapse formation mediated by C3-DLL4 in MDA-MB-231 cells. (Figures 6D-6F) SVG-A cells. Figure 6D) Upregulation of Notch target genes (Hes1, Hey1, Hey2, HeyL) in SVG-A cells as measured by RT-qPCR. Western blot experiments showing C3-DLL4-mediated (FIG. 6E) N2ICD and (FIG. 6F) N1ICD cleavage. [Figure 6E] Figures 6A-6D show activation of endogenous Notch by C3-DLL4. (Figures 6A-6D) MDA-MB-231 cells. Figure 6A) Upregulation of Notch target genes (Hes1, Hey1, Hey2, HeyL, and NRARP) in MDA-MB-231 cells as measured by RT-qPCR. Figure 6B) Western blot shows time-dependent Notch1 intracellular domain (N1ICD) cleavage in MDA-MB-231 cells upon C3-DLL4-mediated activation. Figure 6C) Confocal microscopy showing Notch synapse formation mediated by C3-DLL4 in MDA-MB-231 cells. (Figures 6D-6F) SVG-A cells. Figure 6D) Upregulation of Notch target genes (Hes1, Hey1, Hey2, HeyL) in SVG-A cells as measured by RT-qPCR. Western blot experiments showing C3-DLL4-mediated (FIG. 6E) N2ICD and (FIG. 6F) N1ICD cleavage. [Figure 6F]Figures 6A-6D show activation of endogenous Notch by C3-DLL4. (Figures 6A-6D) MDA-MB-231 cells. Figure 6A) Upregulation of Notch target genes (Hes1, Hey1, Hey2, HeyL, and NRARP) in MDA-MB-231 cells as measured by RT-qPCR. Figure 6B) Western blot shows time-dependent Notch1 intracellular domain (N1ICD) cleavage in MDA-MB-231 cells upon C3-DLL4-mediated activation. Figure 6C) Confocal microscopy showing Notch synapse formation mediated by C3-DLL4 in MDA-MB-231 cells. (Figures 6D-6F) SVG-A cells. Figure 6D) Upregulation of Notch target genes (Hes1, Hey1, Hey2, HeyL) in SVG-A cells as measured by RT-qPCR. Western blot experiments showing C3-DLL4-mediated (FIG. 6E) N2ICD and (FIG. 6F) N1ICD cleavage. [Figure 7A] We demonstrate that protein structure-function relationships can determine soluble Notch activation. Figure 7A) Various shapes of oligomers (C3, planar; C5, concave; C6 and C8, cylindrical helical bundles; I53, spherical). Figure 7B) Planar C3 exhibits high Notch activation, whereas concave and cylindrical shapes do not. [Figure 7B] We demonstrate that protein structure-function relationships can determine soluble Notch activation. Figure 7A) Various shapes of oligomers (C3, planar; C5, concave; C6 and C8, cylindrical helical bundles; I53, spherical). Figure 7B) Planar C3 exhibits high Notch activation, whereas concave and cylindrical shapes do not. DETAILED DESCRIPTION OF THE INVENTION

[0098] Adoptive T cell therapy holds great promise for the treatment of immunodeficiency, viral infection, autoimmunity, and cancer. T cells are typically collected from a patient's peripheral blood and then manipulated and expanded ex vivo before being reinjected into the patient for disease treatment. However, broader application has been hindered by cumbersome and labor-intensive protocols for engineering autologous, patient-specific cells. Human induced pluripotent stem cells (iPSCs) represent an attractive source for scalable manufacturing of cell therapy and, when combined with strategies for immune matching or cloaking, may represent an "off-the-shelf" product. Previous studies have derived T cells from human iPSCs and engineered them to express chimeric antigen receptors (CARs), demonstrating proof-of-principle applications for cancer immunotherapy. However, iPSC-derived CAR T cells (iPSC-CAR T cells) can exhibit innate-like gamma-delta T cell characteristics and do not function as robustly as primary alpha-beta T cells. Described herein is a novel technology involving the use of soluble Notch ligands that enabled the efficient generation of mature alpha-beta T cells for off-the-shelf iPSC-CAR T cells for adoptive cancer therapy.

[0099] Notch signaling is essential for long-term, definitive hematopoietic stem and progenitor cell (HSPC) and T lymphocyte (T cell) development.

[0100] Soluble Notch Ligands In nature, hematopoietic stem cells (HSCs) in the bone marrow give rise to multipotent progenitors (MPPs), which then differentiate into common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs). CLPs migrate from the bone marrow to the thymus, where thymic epithelial cells expressing delta-like ligand 4 (DLL4) induce canonical Notch1 signaling in early thymic progenitors (ETPs). This Notch1 signaling is essential for T cell lineage commitment and is required further during the early stages of thymocyte differentiation, up to the double negative 3 (DN3) stage. Active Notch signaling during these early stages of T cell development inhibits other lineage potentials, such as B cell and myeloid cell (including dendritic cell (DC)) potential. During β selection, Notch signaling is turned off as a result of pre-T cell receptor signaling. Thus, subsequent stages of T cell development exhibit very low levels of Notch signaling. Notch also regulates regulatory T (T Reg ) cells (specifically, thymic T RegIt has been suggested that Notch signaling affects the development of various cell types (e.g., vertebrates, mammalian cells). Notch signaling is mediated by multiple Notch receptors. The Notch signaling pathway is highly conserved in both vertebrate and invertebrate species and regulates the fate decisions of many different cells. It is important for developmental patterning, such as neurogenesis, angiogenesis, and myogenesis, and regulates T cell development and stem cell maintenance. Notch signaling is also involved in cellular processes throughout adulthood. Notch-mediated signaling occurs between adjacent cells, and both the receptor and its ligand are transmembrane proteins. For example, see Schmitt TM, Zuniga-Pflucker JC (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 Dll1- and Dll4-Mediated Notch Activation Levels Shows Differential Lymphomyeloid Lineage Commitment Outcomes. J Immunol. 185(2):867-76; Ohishi K et al. (These are incorporated herein by reference.) Delta-1 enhances marrow and thymus repopulating ability of human CD34( + )CD38( - ) cord blood cells. J Clin Invest. 2002 Oct;110(8):1165-74, and Dallas MH et al. Density of the Notch ligand Delta1 determines generation of B and T cell precursors from hematopoietic stem cells J Exp Med. 2005 May 2;201(9):1361-1366, which are incorporated herein by reference.

[0101] Notch ligands are single-pass transmembrane proteins with a DSL (Delta, Serrate, LAG-2) domain and a variable number of EGF-like repeats. Two classes of canonical Notch ligands exist: the Delta / Delta-like and the Serrate / Jagged class. The latter have an additional domain of cysteine-rich repeats near the transmembrane domain. Five mammalian canonical Notch ligands exist: Jagged-1, Jagged-2, DLL1, DLL3, and DLL4. These can bind to four Notch receptors, Notch1-4. DLL1, also known as Notch delta ligand or Delta-like 1, is a protein that interacts with the NOTCH2 receptor. See, for example, Shimizu K, et al., 2001, J. Biol. Chem. 276(28):25753-8; Blaumueller CM, et al., 1997, Cell 90(2):281-91; Shimizu K, et al., 2000, Mol. Cell. Biol. 20(18):6913-22. In humans, DLL1 is a protein encoded by the DLL1 gene. DLL1 is the human homolog of the Notch delta ligand.

[0102] Provided herein is a soluble Notch ligand oligomeric complex comprising at least a Notch ligand monomer and a scaffold.

[0103] It is well established in the art that Notch activation requires mechanical pulling of the DLL4 ligand by the Notch receptor, and therefore, for signal transduction to occur, the DLL4 ligand must be tethered either to the cell surface or in a plate-bound form (see, e.g., Siebel, C. & Lendahl, U. Notch Signaling in Development, Tissue Homeostasis, and Disease. Physiol. Rev. 97, 1235-1294 (2017), the contents of which are incorporated herein in their entirety). Therefore, it was not predicted that a soluble form of a Notch ligand would be sufficient to activate signal transduction. Specifically, it has not previously been shown or predicted in the art that a soluble Notch ligand complex can both bind to a receptor and exert mechanical force when not bound to a cell (e.g., a stromal cell) or a solid substrate.

[0104] In one embodiment, the oligomeric complex comprises a Notch ligand selected from the group consisting of Delta-like-1 (DLL1), Delta-like-3 (DLL3), Delta-like-4 (DLL4), Jagged1 (JAG1), and Jagged2 (JAG2). In one embodiment, the Notch ligand is DLL4.

[0105] In some embodiments, 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 maintains the same function (e.g., binding and / or activation of a Notch receptor) as SEQ ID NO: 6-9.

[0106] SEQ ID NO: 6, DLL4 Delta-like canonical Notch ligand 4 [Homo sapiens (human)], Gene ID: 54567, NCBI Reference Sequence: NG_046974.1, 9734 bp

[0107] SEQ ID NO: 7, Homo sapiens Delta-like canonical Notch ligand 4 (DLL4), mRNA, NCBI Reference Sequence: NM_019074.4, 3426 bp

[0108] SEQ ID NO: 8, Homo sapiens Delta-like canonical Notch ligand 4 (DLL4), CDS mRNA, NCBI Reference Sequence: NM_019074.4, 2058 bp

[0109] In some embodiments, 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 maintains the same function (e.g., binding and / or activation of a Notch receptor) as SEQ ID NO:4.

[0110] SEQ ID NO: 9, Delta-like protein 4 precursor [Homo sapiens], NCBI Reference Sequence: NP_061947.1, 685 amino acids MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCYTDLSTDTFVCNCPYGFVGSRCEFPVGLPPSFPWVAVSLGVGLAVLLVLLGMVAVAVRQLRLRRPDDGSREAMNNLSDFQKDNLIPAAQLKNTNQKKELEVDCGLDKSNCGKQQNHTLDYNLAPGPLGRGTMPGKFPHSDKSLGEKAPLRLHSEKPECRISAICSPRDSMYQSVCLISEERNECVIATEV

[0111] In some embodiments, the Notch ligand comprises the extracellular domain of human DLL4 corresponding to amino acids 25-405 of DLL4, or 1-526 of DLL4, or amino acids 1-524 of DLL4, or amino acids 27-524 of DLL4 (see, e.g., SEQ ID NO:9 for the full-length sequence of DLL4). In some embodiments, the Notch ligand comprises the extracellular domain of human DLL4 corresponding to amino acids 25-405 of DLL4 (see, e.g., SEQ ID NO:9 for the full-length sequence of DLL4). In some embodiments, the extracellular domain of human DLL4 comprises 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:10, or SEQ ID NO:5, and maintains the same function (e.g., binding and / or activation of a Notch receptor) as SEQ ID NO:10.

[0112] SEQ ID NO: 10, human DLL4 extracellular domain, 526 amino acids MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRP EALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGT CSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNF TGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCYTDLSTDTFVCNCPYGFVGSRCEFPVGLPPS

[0113] In some embodiments, 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 a sequence of SEQ ID NO:1-3 that maintains the same function (e.g., binding and / or activation of a Notch receptor) as SEQ ID NO:1-3.

[0114] SEQ ID NO: 1, DLL1 Delta-like canonical Notch ligand 1 [Homo sapiens (human)], Gene ID: 28514, NCBI Reference Sequence: NG_027940.1, 8873 bp

[0115] SEQ ID NO: 2 Homo sapiens Delta-like canonical Notch ligand 1 (DLL1), mRNA, NCBI Reference Sequence: NM_005618.4, 3779 bp

[0116] SEQ ID NO: 3 Homo sapiens Delta-like canonical Notch ligand 1 (DLL1), CDS mRNA, NCBI Reference Sequence: NM_005618.4, 2172 bp

[0117] In some embodiments, the amino acid sequence of the Notch ligand is DLL1 and comprises 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 SEQ ID NO: 4, or the sequence of SEQ ID NO: 4 that maintains the same function (e.g., binding and / or activation of a Notch receptor).

[0118] SEQ ID NO: 4 Delta-like protein 1 precursor [Homo sapiens], NCBI Reference Sequence: NP_005609.3, 723 aa MGSRCALALAVLSALLCQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVGYSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHERGHRYVCECARGYGGPNCQFLLPELPPGPAVVDLTEKLEGQGGPFPWVAVCAGVILVLMLLLGCAAVVVCVRLRLQKHRPPADPCRGETETMNNLANCQREKDISVSIIGATQIKNTNKKADFHGDHSADKNGFKARYPAVDYNLVQDLKGDDTAVRDAHSKRDTKCQPQGSSGEEKGTPTTLRGGEASERKRPDSGCSTSKDTKYQSVYVISEEKDECVIATEV

[0119] In some embodiments, the Notch ligand is Delta1ext-IgG and comprises the extracellular domain of human DLL1 corresponding 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 the full-length sequence of DLL1). In some embodiments, 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 maintains the same function (e.g., binding and / or activation of a Notch receptor) as SEQ ID NO:5.

[0120] SEQ ID NO:5, human DLL1 extracellular domain, 536 amino acids MGSRCALALAVLSALLCQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDD LATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTC QQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVGYSGFNC EKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHERGHRYVCECARGYGGPNCQFLLPELPPGPAVVDLTEKL

[0121] There are several ways to provide a Notch ligand, for example, by providing a purified recombinant form of the Notch ligand or a Notch receptor-binding fragment, which receptor-binding fragment is sufficient to trigger a cell signaling event in vivo upon contact and binding to an extracellular Notch receptor on these cells. In one embodiment, the Notch ligand is not attached to a solid substrate, for example, using a covalent or non-covalent bond or linkage. In one embodiment, the Notch ligand is not attached to a cell culture dish.

[0122] In one embodiment, the oligomeric complex is a homo-oligomeric complex. In other words, the oligomeric complex contains a single type of Notch monomer. For example, the oligomeric complex contains only DLL4 monomers.

[0123] In one embodiment, the oligomeric complex is a hetero-oligomeric complex. In other words, the oligomeric complex comprises at least two types of Notch monomers. For example, the oligomeric complex comprises both DLL1 and DLL4 monomers.

[0124] In one embodiment, the oligomeric complex exists as a dimer, trimer, tetramer, pentamer, hexamer, octamer, icosahedron, or any other higher order oligomeric state.

[0125] In one embodiment, the oligomeric complex is a 60-mer or a 120-mer.

[0126] In one embodiment, the oligomeric complex comprises a scaffold that binds to the ligand of the complex.

[0127] The scaffold used herein may be a synthetic scaffold. For example, non-limiting examples of synthetic scaffolds that can be used include dimers (e.g., novel dimer C2_HD-1092), trimers (e.g., novel trimer C3_HD-1069), tetramers (e.g., novel tetramer C4_nat_HF-7900), and pentamers (e.g., novel pentamers C5_HF-2101).

[0128] Alternatively, the scaffold used herein may be a naturally occurring scaffold. For example, non-limiting examples of naturally occurring scaffolds that can be used include any naturally occurring protein: dimer (e.g., IgG Fc fragment), trimer (e.g., 2-dehydro-3-deoxy-phosphogluconate aldolase), and pentamer (e.g., lumazine synthase).

[0129] In one embodiment, the scaffold is a nucleic acid scaffold, a protein scaffold, or a lipid scaffold.

[0130] Those skilled in the art will be able to identify scaffolds for the purpose of practicing the claimed inventions described herein. Such scaffolds are further described, for example, in International Patent Applications Nos. 2002 / 032925 and 2004 / 113521, U.S. Patent Applications Nos. 14 / 615,296, 16 / 740,248, and 17 / 564,467, and U.S. Patent Nos. 8,293,482, 9,176,129, 9,217,011, 9,447,170, 10,844,370, and 11,161,893, the contents of each of which are incorporated herein by reference in their entirety.

[0131] The ligand monomer can be immobilized to the scaffold using methods known in the art. For example, in some embodiments, the Notch ligand further comprises a domain that immobilizes the Notch ligand to the scaffold. As a non-limiting example, the Notch ligand monomer comprises a first member of an affinity pair, and the scaffold comprises a second member of the affinity pair. In one embodiment, the first and second members of the affinity couple 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 (e.g., FLAG and anti-FLAG monoclonal antibodies, sequences known in the art) in combination with a corresponding antibody or binding portion or fragment thereof, digoxigenin and anti-digoxigenin, mouse immunoglobulin and goat anti-mouse immunoglobulin, non-immunological binding pairs, hormones and hormone binding proteins, thyroxine and cortisol hormone binding proteins, receptors and receptor agonists, receptors and receptor antagonists, acetylcholine receptors and acetylcholine or analogs thereof, lectins and carbohydrates, enzymes and enzyme cofactors, enzymes and enzyme inhibitors, complementary oligonucleotide pairs capable of forming nucleic acid duplexes, and a negatively charged first molecule and a positively charged second molecule.

[0132] In one embodiment, the binding of the affinity pair is not reversible.

[0133] In one embodiment, the binding of the affinity pair is reversible.

[0134] In one embodiment, the Notch ligand monomer is immobilized on the scaffold via a synthetic heterodimer, eg, LHD101A / B, or a coil-coil heterodimer.

[0135] In one embodiment, the affinity pair used to immobilize the monomers on the scaffold is SpyTag and SpyCatcher. In one embodiment, the plurality of Notch ligand monomers further comprises a GS linker. In one embodiment, SpyTag is fused to the plurality of Notch ligand monomers via a GS linker. In one embodiment, the population of scaffolds further comprises a GGSGGS linker (SEQ ID NO: 49). In one embodiment, the population of scaffolds is fused to SpyCatcher via a GGSGGS linker (SEQ ID NO: 49).

[0136] One aspect provided herein is a composition of any of the oligomeric conjugates described herein.

[0137] Another aspect provided herein is a method of making a Notch ligand oligomeric complex as described herein, the method comprising contacting a plurality of Notch ligand monomers with a population of scaffolds for a time sufficient to promote the formation of the complex.

[0138] In one embodiment, the time sufficient to promote complex formation 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 more.

[0139] Methods for analyzing and screening oligomeric complexes formed via the methods described herein are well known in the art and can be performed by one of ordinary skill in the art.

[0140] Yet another aspect is the composition of any oligomeric complex produced via the methods described herein.

[0141] Notch activation via soluble Notch ligands The soluble Notch ligand complexes and compositions thereof described herein can be used in place of bound Notch ligand (e.g., substrate-bound ligand) in any application in which bound Notch ligand is used. For example, exemplary applications for activation of Notch signaling, T cell differentiation, and natural killer (NK) cell differentiation are described herein. Furthermore, it is fully contemplated herein that the soluble Notch ligand complexes and compositions thereof described herein can be used in applications not described herein that are not known to utilize bound Notch ligand.

[0142] Thus, provided herein is a method for activating Notch signaling in a cell, comprising contacting the cell with a soluble Notch oligomeric complex described herein for a period of time sufficient to induce Notch activation.Those skilled in the art can determine whether Notch has been activated using standard methods in the art, such as those described herein in the Examples.

[0143] In one embodiment, the time sufficient to activate Notch signaling is at least 1 hour.

[0144] In one embodiment, the time sufficient to activate Notch signaling is 24 hours or less.

[0145] In one embodiment, the time sufficient to activate Notch signaling 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 more.

[0146] In one embodiment, the cell is a human cell.

[0147] In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. For example, the cell can be derived from human internal organs, skin, bone, blood, and connective tissue. The cell can be a fibroblast, keratinocyte, muscle cell (e.g., myocyte), cumulus cell, neuronal cell, mammary cell, hepatocyte, and pancreatic islet cell. Furthermore, the cell can be an epithelial, endothelial, neural, adipose, cardiac, skeletal muscle, skin, immune cell (e.g., B lymphocyte (B cell), T lymphocyte (T cell), liver, spleen, lung, peripheral circulating blood cell, gastrointestinal, renal, bone marrow, or pancreatic cell.

[0148] In one embodiment, the cell is a non-human cell. Non-limiting examples of non-human cells include any cell derived from a mouse, cow, monkey, pig, horse, or sheep.

[0149] In one embodiment, the contacting cells are adhered to a substrate, eg, a solid substrate or cells.

[0150] In one embodiment, the contacted cells are in suspension.

[0151] In one embodiment, the soluble Notch ligand is provided at a concentration of 1 nM to 100 nM, or at a concentration of 5 nM to 15 nM.

[0152] In one embodiment, 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 more.

[0153] In one embodiment, 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 more.

[0154] In one embodiment, the soluble Notch ligand is provided at a concentration of at least 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM or more.

[0155] In a preferred embodiment, the soluble Notch ligand is provided at a concentration of 10 nM.

[0156] In one embodiment, the level of Notch activation is at least 10% higher compared to an appropriate control. As used herein, a "suitable control" is an otherwise identical population that has not been contacted with soluble Notch oligomeric complexes, has been contacted for a shorter period of time, or has been contacted with a reduced amount of Notch oligomeric complexes. One skilled in the art can determine the level of Notch activation achieved after contact using standard methods, such as those described herein in the Examples.

[0157] In one embodiment, the level of Notch activation is at least 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%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 1 %,38%,39%,40%,41%,42%,43%,44%,45%,46%,47%,48%,49%,50%,51%,52%,53%,54%,55%,56%,57%,58%,59%,60%,61%,62%,63%,64%,65%,66%,67%,68%,69%,70%,71%,72%,73%,74%,75%,76%,77%,78%,79 ...0%,71%,72%,73%,74%,75%,76%,77%,78%,79%,70%,71%,72%,73%,74%,75%,76%,77%,78%,79%,70%,71%,72%,73%,74%,75%,76%,77%,78%,79 3%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or at least 5-fold, 10-fold, compared to an appropriate control; 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 50x, 100x, 150x, 200x, 250x, 300x, 350x, 400x, 450x, 500x, 550x, 600x, 650x, 700x, 750x, 800x, 850x, 900x, 950x, 1000x or more.

[0158] Differentiation of cells into NK cells via the use of Notch ligands is known in the art and is further described, for example, in International Patent Application No. 2022 / 187682(A2), the contents of which are incorporated herein by reference in their entirety. Provided herein are methods for differentiating cells into NK cells using the soluble Notch ligands described herein.

[0159] Thus, there is provided a method comprising: a) detecting CD34 + Inhibiting histone methyltransferases in hemogenic endothelial populations and b) CD34 + The hemogenic endothelial population was identified as CD56 + and differentiating in natural killer (NK) cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of NK cells.

[0160] Also provided is a method comprising: a) culturing a population of pluripotent stem cells by immunofluorescence analysis using CD34 + b) allowing the resulting CD34 + Inhibiting histone methyltransferases in hemogenic endothelial populations and c) the resulting CD34 + The hemogenic endothelial population was identified as CD56 + and differentiating in natural killer (NK) cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of NK cells.

[0161] Also, there is provided a method comprising: a) detecting CD34 + by inhibiting epigenetic regulators of hemogenic endothelial populations and b) CD34 + The hemogenic endothelial population was identified as CD56 + and differentiating in NK cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of NK cells.

[0162] Also provided is a method comprising: a) culturing a population of pluripotent stem cells by immunofluorescence analysis using CD34 + b) allowing the resulting CD34 + Inhibiting epigenetic regulators in the hemogenic endothelial population and c) the resulting CD34 + The hemogenic endothelial population was identified as CD56 + and differentiating in NK cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of NK cells.

[0163] Also, there is provided a method comprising: a) detecting CD34 + a) inhibiting G9a and / or GLP in the hemogenic endothelial population; and b) CD34 + The hemogenic endothelial population was identified as CD56 + and differentiating in NK cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of NK cells.

[0164] Also provided is a method comprising: a) culturing a population of pluripotent stem cells by immunofluorescence analysis using CD34 + b) allowing the resulting CD34 + c) inhibiting G9a and / or GLP in the hemogenic endothelial population; and + The hemogenic endothelial population was identified as CD56 + and differentiating in NK cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of NK cells.

[0165] Also, the method comprises: + The hemogenic endothelial population was identified as CD56 + Methods are provided herein that include differentiating in NK cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of NK cells.

[0166] Also provided is a method comprising: a) culturing a population of pluripotent stem cells by immunofluorescence analysis using CD34 + b) allowing the resulting CD34 + The hemogenic endothelial population was identified as CD56 + and differentiating in NK cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of NK cells.

[0167] Differentiation method In one aspect, a method is described herein that includes: (a) differentiating a population of pluripotent stem cells in aggregation medium for a time sufficient to promote differentiation into a population of CD34+ hemogenic endothelium; and (b) differentiating the resulting population of CD34+ hemogenic endothelium in CD3+ T cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of CD3+ T cells.

[0168] In some embodiments, the method further comprises inhibiting histone methyltransferase in the resulting population of CD34+ hemogenic endothelium. Such inhibition can increase the efficiency of differentiation into T cells. Accordingly, in one aspect, a method is described herein comprising: (a) differentiating a population of pluripotent stem cells in aggregation medium for a time sufficient to promote differentiation into a population of CD34+ hemogenic endothelium; (b) inhibiting histone methyltransferase in the resulting population of CD34+ hemogenic endothelium; and (c) differentiating the resulting population of CD34+ hemogenic endothelium in CD3+ T cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of CD3+ T cells.

[0169] In one embodiment, CD34 + The hemogenic endothelial population is cultured for at least 4 weeks in a CD3 medium containing 100 ng / ml SCF, 100 ng / ml FLT3, and 50 ng / ml IL7 in the presence of, for example, a 10 nM concentration of soluble Notch ligand. + Cultivate in T cell differentiation medium and differentiate into CD3+ Promotes differentiation into T cell populations.

[0170] In one embodiment, CD34 + The hemogenic endothelial population is cultured for at least 4 weeks in a CD3 endothelial cell line containing 100 ng / ml FLT3 and 50 ng / ml IL7 in the presence of a soluble Notch ligand at a concentration of, for example, 10 nM. + Cultivate in T cell differentiation medium and differentiate into CD3 + Promotes differentiation into T cell populations.

[0171] In one embodiment, CD34 + The hemogenic endothelial population is cultured for at least 4 weeks in a CD3 medium containing 30 ng / ml SCF, 15 ng / ml FLT3, and 25 ng / ml IL7 in the presence of, for example, a 10 nM concentration of soluble Notch ligand. + Cultivate in T cell differentiation medium and differentiate into CD3 + Promotes differentiation into T cell populations.

[0172] In one embodiment, CD34 + The hemogenic endothelial population is cultured for at least 4 weeks in a CD3 endothelial cell line containing 15 ng / ml FLT3 and 25 ng / ml IL7 in the presence of a soluble Notch ligand at a concentration of, for example, 10 nM. + Cultivate in T cell differentiation medium and differentiate into CD3 + Promotes differentiation into T cell populations.

[0173] In one aspect, a method comprises: (a) culturing a population of pluripotent stem cells by immunofluorescence analysis using a CD34 + (b) allowing the resulting CD34 + A population of hemogenic endothelium is cultured for at least 4 weeks in the presence of, for example, 10 nM of soluble Notch ligand, in the presence of CD3+, 15 ng / ml FLT3, and 25 ng / ml IL7. + Differentiate in T cell differentiation medium to differentiate CD3 + and promoting differentiation into a population of CD3 T cells. +Methods are described herein wherein the T cell differentiation medium further comprises 5 ng / mL TPO and 30 ng / ml SCF for at least the first two weeks.

[0174] In another aspect, a method comprises: (a) culturing a population of pluripotent stem cells by immunofluorescence analysis using a method comprising: + (b) allowing the resulting CD34 + A population of hemogenic endothelium is cultured for at least 4 weeks in the presence of, for example, 10 nM of soluble Notch ligand, in the presence of CD3+, 15 ng / ml FLT3, and 25 ng / ml IL7. + Differentiate in T cell differentiation medium to differentiate CD3 + and promoting differentiation into a population of CD3 T cells. + The methods described herein further comprise, for at least the first two weeks, T cell differentiation medium comprising 5 ng / mL TPO, 30 ng / ml SCF, and G9a inhibitor.

[0175] In another aspect, a method comprises: (a) culturing a population of pluripotent stem cells by immunofluorescence analysis using a method comprising: + (b) allowing the resulting CD34 + A population of hemogenic endothelium is cultured for at least 4 weeks in a CD3 medium containing 100 ng / ml SCF, 100 ng / ml FLT3, and 50 ng / ml IL-7 in the presence of, for example, a 10 nM concentration of soluble Notch ligand. + Differentiate in T cell differentiation medium to differentiate CD3 + and promoting differentiation into a population of CD3 T cells. + The methods described herein further comprise TPO (50 ng / mL) for at least the first two weeks.

[0176] In another aspect, a method comprises: (a) culturing a population of pluripotent stem cells by immunofluorescence analysis using a method comprising: + (b) allowing the resulting CD34 +A population of hemogenic endothelium is cultured for at least 4 weeks in a CD3 medium containing 100 ng / ml SCF, 100 ng / ml FLT3, and 50 ng / ml IL-7 in the presence of, for example, a 10 nM concentration of soluble Notch ligand. + Differentiate in T cell differentiation medium to differentiate CD3 + and promoting differentiation into a population of CD3 T cells. + The methods described herein further comprise TPO (50 ng / mL) and a G9a / GLP inhibitor for at least the first two weeks.

[0177] In another aspect, a method comprises: (a) culturing a population of pluripotent stem cells by immunofluorescence analysis using a method comprising: + (b) allowing the resulting CD34 + A population of hemogenic endothelium is cultured for at least 4 weeks in the presence of, for example, 10 nM of soluble Notch ligand, in the presence of CD3+, 100 ng / ml FLT3, and 50 ng / ml IL7. + Differentiate in T cell differentiation medium to differentiate CD3 + and promoting differentiation into a population of CD3 T cells. + The methods described herein further comprise T cell differentiation medium at least for the first two weeks, at least comprising 50 ng / mL TPO and 100 ng / ml SCF.

[0178] In another aspect, a method comprises: (a) culturing a population of pluripotent stem cells by immunofluorescence analysis using a method comprising: + (b) allowing the resulting CD34 + A population of hemogenic endothelium is cultured for at least 4 weeks in the presence of, for example, 10 nM of soluble Notch ligand, in the presence of CD3+, 100 ng / ml FLT3, and 50 ng / ml IL7. + Differentiate in T cell differentiation medium to differentiate CD3 + and promoting differentiation into a population of CD3 T cells. +The methods described herein further comprise, for at least the first two weeks, 50 ng / mL TPO, 100 ng / ml SCF, and G9a inhibitor.

[0179] pluripotent stem cells In some embodiments, the stromal-free T cell differentiation methods described herein comprise differentiating a population of pluripotent stem cells. Pluripotent stem cells (PSCs) have the potential to give rise to all somatic tissues. In one embodiment of any method, cell, or composition described herein, the population of pluripotent stem cells is induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). iPSCs and ESCs can be produced by any method known in the art. In some embodiments, the population of pluripotent stem cells comprises embryonic stem cells (ESCs). Embryonic stem cells (ESCs) are stem cells derived from the undifferentiated inner mass cells of a human embryo.

[0180] Directed differentiation of PSCs aims to recapitulate embryonic development and generate patient-matched tissues by specifying the three germ layers. A common theme in directed differentiation across all germ layers is the tendency of PSCs to give rise to embryonic- and fetal-like cell types, which pose challenges to integration and function in adult recipients. This feature is particularly prominent in the hematopoietic system, which emerges in temporally and spatially separated waves during ontogeny. The earliest "primitive" precursors emerge in the yolk sac at 8.5 dpc and give rise to a limited repertoire of macrophages, megakaryocytes, and nucleated erythrocytes. These early embryonic-like precursors are largely bone marrow-based and are unable to functionally repopulate the bone marrow of adult recipients. In contrast, "definitive" cells with hematopoietic stem cell (HSC) potential later emerge in the arterial endothelium within the aorta-gonad-mesonephros (AGM) and other anatomical sites. Directed differentiation of PSCs gives rise to hematopoietic precursors similar to those found in the early embryonic yolk sac. They lack functional reconstitution capacity, are biased toward the myeloid lineage, and express embryonic globins. Therefore, understanding the key fate decision mechanisms that drive the development of either primitive or definitive lineages is crucial for identifying HSCs and other adult-like cell types (e.g., erythroid) from PSCs.

[0181] In some embodiments, the population of pluripotent stem cells (PSCs) comprises induced pluripotent stem cells (iPS cells). In some embodiments, the induced pluripotent stem cells are generated by introducing only the reprogramming factors OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28 into mature cells. In some embodiments, the induced pluripotent stem cells are generated by introducing the reprogramming factors into mature cells two or more times.

[0182] In some embodiments, the pluripotent stem cells (PSCs) described herein are induced pluripotent stem cells (iPSCs). An advantage of using iPSCs is that the cells can be derived from the same subject into which the final immune cells will be reintroduced. That is, somatic cells can be obtained from the subject, reprogrammed into induced pluripotent stem cells, and then transfected and differentiated into modified immune cells (e.g., autologous cells) that are administered to the subject. Because the precursors are essentially derived from an autologous source, the risk of engraftment rejection or allergic response is reduced compared to the use of cells from another subject or group of subjects. In some embodiments, the cells for generating iPSCs are derived from a non-autologous source. Additionally, the use of iPSCs does not require cells obtained from an embryonic source. Thus, in one embodiment, the PSCs used in the disclosed methods are not embryonic stem cells.

[0183] Although differentiation is generally irreversible under physiological conditions, several methods have recently been developed for reprogramming somatic cells into induced pluripotent stem cells. Exemplary methods are known to those skilled in the art and are briefly described herein below.

[0184] As used herein, the term "reprogramming" refers to a process that changes or reverses the differentiation state of a differentiated cell (e.g., a somatic cell). In other words, reprogramming refers to a process that reverts the differentiation of a cell to a more undifferentiated or more primitive type of cell. It should be noted that placing many primary cells in culture may result in some loss of fully differentiated characteristics. Therefore, simply culturing such cells included in the term differentiated cells does not convert them into non-differentiated (e.g., undifferentiated) or pluripotent cells. The transition of differentiated cells to pluripotency requires a reprogramming stimulus that exceeds the stimulus that results in partial loss of differentiation characteristics in culture. Reprogrammed cells also generally possess the characteristic of the ability to be passaged for extended periods without loss of proliferative capacity, as opposed to their parent primary cells, which are capable of only a limited number of divisions in culture.

[0185] Cells to be reprogrammed can be either partially or terminally differentiated prior to reprogramming. In some embodiments, reprogramming involves the complete restoration of a differentiated cell (e.g., a somatic cell) to a pluripotent or multipotent state. In some embodiments, reprogramming involves the complete or partial restoration of a differentiated cell (e.g., a somatic cell) to an undifferentiated state (e.g., an embryonic-like cell). Reprogramming can result in the expression of specific genes by the cell, which expression further contributes to reprogramming. In certain embodiments described herein, reprogramming a differentiated cell (e.g., a somatic cell) causes the differentiated cell to adopt an undifferentiated state (e.g., an undifferentiated cell). The resulting cell is referred to as a "reprogrammed cell" or "induced pluripotent stem cell (iPSC or iPS cell)."

[0186] Reprogramming can involve altering, e.g., reversing, at least some of the inherited patterns of nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, and the like that occur during cell differentiation. Reprogramming is distinct from simply maintaining the existing undifferentiated state of an already pluripotent cell or maintaining the existing, fully undifferentiated state of an already multipotent cell (e.g., a common bone marrow stem cell). Reprogramming is also distinct from promoting self-renewal or proliferation of already pluripotent or multipotent cells, although the compositions and methods described herein can also, in some embodiments, be used for such purposes.

[0187] The particular approach or method used to generate pluripotent stem cells from somatic cells (broadly referred to as "reprogramming") is not necessarily critical to the described methods. Thus, any method of reprogramming somatic cells to a pluripotent phenotype would be suitable for use in the methods described herein.

[0188] Reprogramming methodologies for generating pluripotent cells using defined combinations of transcription factors have been described to induce pluripotent stem cells from somatic cells. Yamanaka and Takahashi converted mouse somatic cells into ES cell-like cells with expanded developmental potential by direct transduction of Oct4, Sox2, Klf4, and optionally c-Myc. See U.S. Patent Nos. 8,058,065 and 9,045,738 to Yamanaka and Takahashi. iPSCs resemble ES cells because they restore much of the pluripotency-associated transcriptional circuitry and epigenetic landscape. Furthermore, mouse iPSCs fulfill all standard assays for pluripotency, specifically, in vitro differentiation into cell types of the three germ layers, teratoma formation, chimera contribution, germline transmission, and tetraploid complementation.

[0189] Subsequent studies have shown that human iPS cells can be obtained using similar transduction methods, and that the transcription factor trio, OCT4, SOX2, and NANOG, have been established as a core set of transcription factors governing pluripotency. The generation of iPS cells can be achieved by the introduction of nucleic acid sequences encoding stem cell-associated genes into adult somatic cells using viral vectors.

[0190] OCT4, SOX2, KLF4, and c-MYC are the original four transcription factors identified for reprogramming mouse fibroblasts into iPSCs. These same four factors were also sufficient to generate human iPSCs. OCT3 / 4 and SOX2 function as core transcription factors in the pluripotency network by regulating the expression of pluripotency-related genes. Kruppel-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. While OCT4 and SOX2 are required, KLF4 and c-MYC can be replaced by NANOG, a homeobox protein important for maintaining pluripotency in both ES cells and early embryos, and LIN28, an RNA-binding protein. A combination of OCT4, SOX2, NANOG, and LIN28 reprogramming factors has also been reported to be sufficient to generate human iPSCs.

[0191] In one embodiment of any method, cell, or composition described herein, iPSCs are generated by introducing exogenous copies of only three reprogramming factors, e.g., OCT4, SOX2, and KLF4, into mature or somatic cells. In one embodiment of any method, cell, or composition described herein, c-MYC, or nanog and / or LIN28, are further introduced into iPSCs carrying exogenous genes encoding copies of OCT4, SOX2, and KLF4 to differentiate into mature or somatic cells. In one embodiment of any method, cell, or composition described herein, iPSCs are generated by introducing exogenous copies of reprogramming factors OCT4, SOX2, and KLF4, and optionally carrying c-MYC, or nanog, and / or LIN28, for differentiation into mature or somatic cells.

[0192] In one embodiment of any method, cell, or composition described herein, iPSCs are generated by contacting mature cells with at least one vector, the at least one vector carrying exogenous genes encoding copies of reprogramming factors OCT4, SOX2, and KLF4, and optionally c-MYC or nanog and / or LIN28 for differentiation into mature or somatic cells, and the reprogramming factors are expressed in vivo in the contacted mature or somatic cells. The contacting can be in vitro or ex vivo. All reprogramming factors required for differentiation can be expressed by a single vector (e.g., a vector carrying exogenous genes encoding copies of OCT4, SOX2, KLF4, and c-MYC). Alternatively, the reprogramming factors can be expressed by two or more vectors, each used to contact the iPSCs. For example, iPSCs can be contacted with a first vector carrying exogenous gene-encoding copies of OCT4, SOX2, and a second vector carrying exogenous gene-encoding copies KLF4 and c-MYC.

[0193] In one embodiment of any disclosed method, the iPS cells comprise at least an exogenous copy of a nucleic acid sequence encoding a reprogramming factor selected from the group consisting of the genes Oct4 (Pou5f1), Sox2, cMyc, Klf4, Nanog, Lin28, and Glis1. In some embodiments, a combination of reprogramming factors is used, such as 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 Lin28.

[0194] In one embodiment of any method, cell, or composition described herein, iPSCs are generated by introducing the disclosed reprogramming factors or any combination of reprogramming factors into mature or somatic cells more than once. In one embodiment, the combination of reprogramming factors is different when the combination is introduced into iPSCs more than once, e.g., a combination of Oct4(Pou5f1), Sox2, cMyc, Klf4, and Nanog is first introduced into iPSCs, and a combination of Oct4(Pou5f1), Sox2, and cMyc is subsequently introduced into iPSCs. In one embodiment of any method, cell, or composition described herein, iPSCs are generated into mature / somatic cells by contacting mature cells with the disclosed vector factors more than once.

[0195] In some embodiments, the population of pluripotent stem cells (e.g., iPSCs) is not differentiated in the presence of a Notch ligand, e.g., a soluble Notch ligand. In some embodiments, the aggregation medium used to promote the differentiation of a population of pluripotent stem cells (e.g., iPSCs) into a population of CD34+ hemogenic endothelium does not contain a Notch ligand, e.g., a soluble Notch ligand. In some embodiments, the cell culture vessel used during the differentiation of a population of pluripotent stem cells (e.g., iPSCs) into a population of CD34+ hemogenic endothelium does not contain a Notch ligand, e.g., a soluble Notch ligand.

[0196] iPS cells can be generated or derived from terminally differentiated somatic cells, and adult stem cells, or somatic stem cells. That is, non-pluripotent progenitor cells can be made pluripotent or multipotent by reprogramming. In such cases, it may not be necessary to include the number of reprogramming factors required to reprogram the terminally differentiated cells. Furthermore, reprogramming can be induced by non-viral introduction of reprogramming factors, such as introduction of the protein itself, or introduction of a nucleic acid encoding the reprogramming factor, or introduction of messenger RNA that produces the reprogramming factor upon translation (see, e.g., Warren et al., Cell Stem Cell, 2010 Nov 5;7(5):618-30, which is incorporated herein by reference in its entirety). Reprogramming can be achieved by introducing a combination of nucleic acids encoding stem cell-associated genes, including, for example, Oct-4 (also known as Oct-3 / 4 or Pouf51), Sox1, Sox2, Sox3, Sox15, Sox18, NANOG, Klf1, Klf2, Klf4, Klf5, NR5A2, c-Myc, l-Myc, n-Myc, Rem2, Tert, and LIN28. In one embodiment, reprogramming using the methods and compositions described herein can further include introducing one or more of Oct-3 / 4, a member of the Sox family, a member of the Klf family, and a member of the Myc family into somatic cells. In one embodiment, the methods and compositions described herein further include introducing one or more of each of Oct-4, Sox2, Nanog, c-MYC, and Klf4 for reprogramming. As noted above, the precise method used for reprogramming is not necessarily critical to the methods and compositions described herein. However, when cells differentiated from the reprogrammed cells are to be used, for example, in human therapy, in one embodiment, the reprogramming is not affected by methods that alter the genome, and thus, in such an embodiment, reprogramming is achieved without the use of, for example, viral or plasmid vectors.

[0197] The efficiency of reprogramming (i.e., the number of reprogrammed cells) from a population of starting cells can be enhanced by the addition of various small molecules, as shown in 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 their entirety). Thus, agents or combinations of agents that enhance the efficiency or speed of induced pluripotent stem cell generation can be used to generate patient-specific or disease-specific iPSCs. Some non-limiting examples of agents that enhance reprogramming efficiency include soluble Wnt, Wnt-conditioned medium, BIX-01294 (G9a histone methyltransferase), PD0325901 (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.

[0198] Other non-limiting examples of reprogramming enhancers include suberoylanilide hydroxamic acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, depudecin (e.g., (-)-depudecin), HC toxin, Nullscript (4-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), phenylbutyrate (e.g., sodium phenylbutyrate) and valproic acid ((VPA) 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 or FK228), benzamides (e.g., CI-994 (e.g., N-acetyldinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA (m-carboxynamic acid bishydroxamic acid), JNJ16241199, tubacine, A-161906, proxamide, oxamflatin, 3-Cl-UCHA (e.g., 6-(3-chlorophenylureido)caproic hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31, and CHAP50. Other reprogramming enhancers include, for example, dominant negative forms of HDACs (e.g., catalytically inactive forms), siRNA inhibitors of HDACs, and antibodies that specifically bind to HDACs. Such inhibitors are available from, for example, BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Aton Pharma, Titan Pharmaceuticals, Schering AG, Pharmion, MethylGene, and Sigma Aldrich.

[0199] To confirm the derivation of pluripotent stem cells for use with the methods described herein, isolated clones can be tested for the expression of stem cell markers. Such expression in cells derived from somatic cells identifies the cells as induced pluripotent stem cells. Stem cell markers may be selected from the non-limiting group including SSEA3, SSEA4, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Nat1. In one embodiment, cells expressing Oct4 or Nanog are identified as pluripotent. Methods for detecting the expression of such markers include, for example, RT-PCR and immunological methods that detect the presence of encoded polypeptides, such as Western blot or flow cytometry analysis. In some embodiments, detection does not involve only RT-PCR but also includes detection of protein markers. Intracellular markers can best be identified via RT-PCR, whereas cell surface markers are readily identified by, for example, immunocytochemistry.

[0200] The pluripotent stem cell properties of isolated cells can be confirmed by testing the iPSCs to differentiate into cells of each of the three germ layers. As an example, teratoma formation in nude mice can be used to evaluate the pluripotent properties of isolated clones. The cells are introduced into nude mice, and histology and / or immunohistochemistry is performed on tumors arising from the cells. For example, the growth of tumors containing cells from all three germ layers further indicates that the cells are pluripotent stem cells.

[0201] Many U.S. patents and published patent applications teach and describe methods for generating iPSCs and related subject matter, for example, U.S. Patent Nos. 8,058,065, 9,347,044, 9,347,042, 9,347,045, 9,340,775, 9,341,625, 9,340,772, 9,250,230, 9,132,152, 9,045,738, 9,005,975, 9,005,976, 8,927,277, 8,993,329, 8,900,871, 8,852,941, 8,802,438, 8,691, 8,711, 8,721, 8,731, 8,741, 8,751, 8,761, 8,771, 8,781, 8,852,941, 8,802,438, 8,861, 8,911, 8,921, 8,931, 8,941, 8,951, 8,961, 8,971, 8,981, 8,991, 9,991, 10,102, 10,111, 10,121, 10,131, 10,141, 10,151, 10,161, 10,171, 10,181, 10,191, 10,192, 10,193, 10,194, 10,195, 10,196, 10,1 574, 8735150, 8765470, 8058065, 8048675, and U.S. Patent Publication Nos. 2009 / 0227032, 2010 / 0210014, 2011 / 0250692, 2011 / 0201110, 2011 / 0200568, 2011 / 0223669, 2011 / 0306516, 2010 / 0021437, 2011 / 0256626, Same No. 2011 / 0044961, No. 2012 / 0276070, No. 2012 / 0214243, No. 2012 / 0263689, No. 2012 / 0128655, No. 2012 / 0100568, No. 20 13 / 0295064, 2013 / 0029866, 2013 / 0059386, 2013 / 0183759, 2013 / 0189786, 2013 / 0295579, 2013 / 0 Nos. 130387, 2013 / 0157365, 2014 / 0234973, 2014 / 0227736, 2014 / 0093486, 2014 / 0301988, 2014 / 0170746, 2014 / 0178989, 2014 / 0349401, 2014 / 0065227, and 2015 / 0140662, all of which are incorporated by reference in their entireties.

[0202] In some embodiments, iPSCs can be derived from somatic cells. Somatic cells, as the term is used herein, refer to any cell that forms the body of an organism, except for germ cells. All cell types in the mammalian body are differentiated somatic cells, except for sperm and eggs, the cells that give rise to them (gametocytes), and undifferentiated stem cells. For example, internal organs, skin, bone, blood, and connective tissue are all composed of differentiated somatic cells. In one embodiment of any method, cell, or composition described herein, the mature cells that give rise to iPS cells include any somatic cell, such as B lymphocytes (B cells), T lymphocytes (T cells), and fibroblasts and keratinocytes.

[0203] Additional somatic cell types for use with the compositions and methods described herein include fibroblasts (e.g., primary fibroblasts), muscle cells (e.g., myocytes), cumulus cells, neural cells, mammary cells, hepatocytes, and pancreatic islet cells. In some embodiments, the somatic cells are primary cell lines or are the progeny of primary or secondary cell lines. In some embodiments, the somatic cells are obtained from a human sample, such as a hair follicle, a blood sample, a biopsy (e.g., a skin biopsy or fat biopsy), a swab sample (e.g., an oral swab sample), and are therefore human somatic cells.

[0204] Some non-limiting examples of differentiated somatic cells include, but are not limited to, epithelial, endothelial, neural, adipose, cardiac, skeletal muscle, skin, immune cells, liver, spleen, lung, peripheral circulating blood cells, gastrointestinal, renal, bone marrow, and pancreatic cells. In some embodiments, somatic cells can be primary cells isolated from any somatic tissue, including, but not limited to, brain, liver, gastrointestinal tract, stomach, intestine, adipose, muscle, uterus, skin, spleen, endocrine organs, bone, etc. Furthermore, somatic cells can be derived from any mammalian species, with non-limiting examples including murine, bovine, simian, porcine, equine, ovine, or human cells. In some embodiments, somatic cells are human somatic cells.

[0205] When reprogrammed cells are used to generate progenitor cells for use in the therapeutic treatment of a disease, it is desirable, but not necessary, to use somatic cells isolated from the patient to be treated. For example, somatic cells involved in the disease and somatic cells relevant to the therapeutic treatment of the disease may be used. In some embodiments, methods for selecting reprogrammed cells from a heterogeneous population containing reprogrammed cells and the somatic cells from which they were derived or generated can be carried out by any known means. For example, reprogrammed cells can be isolated using a selectable marker, such as a drug resistance gene, as an indicator, such as a selectable marker gene.

[0206] The reprogrammed somatic cells disclosed herein can express any number of pluripotent cell markers, including: alkaline phosphatase (AP), ABCG2, stage-specific embryonic antigen-1 (SSEA-1), SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Tra-2-49 / 6E, ERas / ECAT5, E-cadherin, beta-III-tubulin, alpha-smooth muscle actin (α-S MA), fibroblast growth factor 4 (Fgf4), Cripto, Dax1, zinc finger protein 296 (Zfp296), N-acetyltransferase-1 (Nat1), embryonic stem cell-associated transcript 1 (ECAT1), ESG1 / DPPA5 / ECAT2, ECAT3, ECAT6, ECAT7, ECAT8, ECAT9, ECAT10, ECAT15-1, ECAT15-2, Fth117, Sal14, blastocyst metastasis Transcription factors (Utf1), Rex1, p53, G3PDH, telomerase including TERT, silent X chromosome genes, Dnmt3a, Dnmt3b, TRIM28, F-box-containing protein 15 (Fbx15), Nanog / ECAT4, Oct3 / 4, Sox2, Klf4, c-Myc, Esrrb, TDGF1, GABRB3, Zfp42, FoxD3, GDF3, CYP25A1, developmental pluripotency associated 2 (DPPA2), T cell lymphoma Such markers include tumor breakpoint 1 (Tcl1), DPPA3 / Stella, DPPA4, and other common markers of pluripotency. Other markers may include Dnmt3L, Sox15, Stat3, Grb2, β-catenin, and Bmil. Such cells may also be characterized by downregulation of markers characteristic of the somatic cells from which the induced pluripotent stem cells are derived. In one embodiment, iPSCs are derived from mature, differentiated somatic cells.

[0207] In some embodiments, 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. In some embodiments, the population of pluripotent stem cells does not comprise stem cells purified or isolated from a cord blood or bone marrow sample. In some embodiments, the population of pluripotent stem cells is not derived from stem cells isolated from a patient sample (e.g., cord blood or bone marrow). In preferred embodiments, the population of pluripotent stem cells comprises 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.

[0208] hematopoietic endothelium In some embodiments, the methods described herein comprise differentiating a population of pluripotent stem cells (e.g., iPSCs) into a population of hematopoietic cells. In some embodiments, the population of hematopoietic cells comprises hemogenic endothelial cells and / or hematopoietic stem cells (HSCs). Hematopoietic cells (e.g., hemogenic endothelial cells, HSCs) can be generated using any method known in the art.

[0209] One exemplary approach to generating HSCs from hPSCs is to identify HSCs from their ontogenetic precursors. It is widely accepted that HSCs originate from hemogenic endothelium (HE) in the aorta-gonad-mesonephros (AGM) and arterial endothelium in other anatomical sites. Recent studies on directed differentiation of HE from hPSCs have provided valuable insights into some of the signaling pathways that control the emergence of primitive or definitive populations. However, the endothelial-to-hematopoietic transition (e.g., HE to HSC) remains incompletely understood in human hematopoietic development.

[0210] As used herein, the term "hemogenic endothelium" refers to a unique subset of endothelial cells scattered within blood vessels that can differentiate into hematopoietic cells. In developing mice, HSCs arise from a small population of endothelial cells with hematopoietic potential (hemogenic endothelium) located within the aorta-gonad-mesonephros region, beginning at embryonic day 10.5. In a process known as endothelial-to-hematopoietic transition (EHT), endothelial cells at the base of the aorta converge and sprout into the extravascular space, subsequently re-entering the circulation via the inferior vein. In some embodiments, a population of cells comprising hemogenic endothelial properties is differentiated in vitro from a population of pluripotent stem cells (e.g., iPSCs). Such "cells comprising hemogenic endothelial properties" may also be referred to herein as hemogenic endothelium.

[0211] Attempts to derive HSCs from pluripotent stem cells (PSCs) are complicated by the fact that embryonic hematopoiesis consists of two programs, primitive and definitive, but only definitive hematopoiesis generates HSCs and therefore lymphoid lineages. Definitive hematopoiesis, as measured by T lymphocyte potential, emerges after the establishment of the primitive hematopoietic program and develops from a progenitor population that exhibits characteristics of the hemogenic endothelium.

[0212] In some embodiments, the T cell differentiation methods described herein comprise differentiating a population of pluripotent stem cells in aggregation medium for a time sufficient to promote differentiation into a population of CD34+ hemogenic endothelium. In some embodiments, the resulting CD34+ hemogenic endothelium is capable of undergoing definitive hematopoiesis and / or exhibits lymphoid potential. In some embodiments, the hemogenic endothelium differentiates or is differentiated into hematopoietic stem cells (HSCs).

[0213] In some embodiments, a population of pluripotent stem cells (e.g., iPSCs) 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 that are generated and cultured in vitro in the presence of serum. EBs can generate a mixture of primitive and definitive hematopoietic progenitor cell types. While primitive precursors are equivalent to those naturally occurring in vivo during early stages of embryonic development, at later stages of maturation, the embryonic population gives rise to definitive precursor cells that behave similarly to cells typical of adult hematopoiesis.

[0214] In some embodiments, the time sufficient 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 time sufficient 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.

[0215] In some embodiments, the aggregation medium comprises BMP4, SB-431542, CHIR99021, bFGF, VEGF, IL-6, IL-11, IGF-1, SCF, and EPO, or any combination thereof. In some embodiments, the aggregation medium 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 provided in WO 2021 / 150919(A1)).

[0216] In some embodiments, the components of the aggregation medium are varied during differentiation of pluripotent stem cells into hemogenic endothelium. As a non-limiting example, 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. See, e.g., Sturgeon et al., Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells, Nat Biotechnol. 2014 Jun;32(6):554-561, which is incorporated herein by reference.

[0217] In some embodiments, the aggregation medium comprises a BMP (e.g., 10 ug / mL of BMP) during differentiation days 0, 1, and / or 2. In some embodiments, the aggregation medium does not comprise a BMP during differentiation days 3, 4, 5, 6, 7, or 8.

[0218] In some embodiments, the aggregation medium contains 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 Wnt agonist. Inhibition of activin-nodal signaling and activation of Wnt signaling promotes the development of lymphoid definitive progenitors (KDRs). + CD235a - (See, e.g., Sturgeon 2014, supra, which is incorporated herein by reference.) In some embodiments, the aggregation medium does not include SB-431542 and / or CHIR99021 during days 0, 1, 3, 4, 5, 6, 7, and / or 8 of differentiation.

[0219] In some embodiments, the aggregation medium comprises bFGF (e.g., 5 ng / ml bFGF) during differentiation days 1, 2, 3, 4, 5, 6, 7, and / or 8. In some embodiments, the aggregation medium does not comprise bFGF during differentiation day 0.

[0220] In some embodiments, the aggregation medium 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 medium does not comprise VEGF during days 0, 1, or 2 of differentiation.

[0221] In some embodiments, the aggregation medium comprises hematopoietic cytokines during differentiation days 6, 7, and / or 8. In some embodiments, the aggregation medium does not comprise hematopoietic cytokines during differentiation days 0, 1, 2, 3, 4, or 5. In some embodiments, 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).

[0222] In some embodiments, the differentiation method further comprises selecting or isolating the resulting population of CD34+ hemogenic endothelium using expression of a surface marker 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). In some embodiments, the surface marker for hemogenic endothelium is CD34 (e.g., high CD34 surface expression).

[0223] In some embodiments, additional positive or negative markers for hemogenic endothelium may include, but are not limited to, CD45, CD38, KDR, CD235, and CD43. In some embodiments, the population of CD34+ hemogenic endothelium is CD45 negative / low. In some embodiments, the population of CD34+ hemogenic endothelium is CD38 negative / low. In some embodiments, the population of CD34+ hemogenic endothelium is KDR+. In some embodiments, the population of CD34+ hemogenic endothelium is CD235 negative / low. In some embodiments, the population of CD34+ hemogenic endothelium is CD43 negative / low.

[0224] In some embodiments, hemogenic endothelium and / or HSCs are generated using any method known in the art. As a non-limiting example, methods for differentiating PSCs into hemogenic endothelium can include the introduction of transcription factors such as ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, and / or SPI1. See, e.g., International Application No. 2018 / 048828; U.S. 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 entirety.

[0225] In some embodiments, hemogenic endothelium is not derived from PSCs, but rather is derived directly from endothelial cells. For example, endothelial cells (e.g., from lung, brain, and other tissues) can be directly reprogrammed into hemogenic endothelium by transduction with transcription factors (e.g., Fosb, Gfi1, Runx1, and Spi1) and co-culture with immortalized endothelial cell lines, and the endothelial cells can be further exposed to cell-extrinsic factors (e.g., serum, SB-431542, and / or endothelial mitogens). See, e.g., Lis et al., Nature. 2017 May 25, 545(7655):439-445; Blaser and Zon, Blood. 2018 Sep 27;132(13):1372-1378, which are incorporated herein by reference.

[0226] Inhibition of epigenetic regulators In some aspects, methods of T cell differentiation are described herein that include inhibiting at least one epigenetic regulator. As used herein, the term "epigenetic regulator" refers to a factor, e.g., a polypeptide, e.g., an enzyme, that affects DNA methylation and / or histone modifications (e.g., histone acetylation, histone methylation), thereby affecting the transcription level of a gene without altering (e.g., substituting or deleting) the nucleotide sequence of the genome. Non-limiting examples of epigenetic regulators include DNA methyltransferases (DNMTs, e.g., DNMT1, DNMT3a, DNMT3b), methyl-CpG binding domain (MBD) proteins (e.g., MeCP2, MBD1, MBD2, MCD4, KAISO, ZBTB4, ZBTB38, UHRHRF2), DNA demethylases (e.g., 5'-methylcytokinin hydroxylase, TET1, TET2, TET3), histone methyltransferases (HMTs, e.g., SUV39, SET1, EZH1, EZH2, Set2, PRDM, SMYD, DOT1L, PRMT, G9a, GLP), methyl-histone binding proteins (e.g., HP1, Chd1, BPTF, L3MBTL1, ING2), and the like. , BHC80, JMJD2A), histone demethylases (e.g., KDMs, e.g., LSD, JHDM, JMJD, JARID, Uts, PHF), histone acetyltransferases (HATs, e.g., HAT1, GCN5, PCAF, MYST, p300, CBP, SRC / p160), acetyl-binding proteins (e.g., BROMO domain, DPF domain, or YEATS domain-containing proteins), histone deacetylases (HDACs, e.g., HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, Sirt1, Sirt2, Sirt3, Sirt4, Sirt5, Sirt6, Sirt7). See, for example, Cheng et al., Signal Transduction and Targeted Therapy volume 4, Article number: 62 (2019), the contents of which are incorporated herein by reference in their entirety.

[0227] In some embodiments, the method comprises inhibiting an epigenetic regulator in the resulting population of CD34+ hemogenic endothelium after differentiating the population of pluripotent stem cells in aggregation medium for a time sufficient to promote differentiation into a population of CD34+ hemogenic endothelium. In some embodiments, the method comprises inhibiting an epigenetic regulator in the population of CD34+ hemogenic endothelium before differentiating the population of CD34+ hemogenic endothelium in CD3+ T cell differentiation medium in the presence of a Notch ligand for a time sufficient to promote differentiation into a population of CD3+ T cells.

[0228] Thus, in one aspect, a method is described herein that includes: (a) differentiating a population of pluripotent stem cells in aggregation medium for a time sufficient to promote differentiation into a population of CD34+ hemogenic endothelium; (b) inhibiting an epigenetic regulator in the resulting population of CD34+ hemogenic endothelium; and (c) differentiating the resulting population of CD34+ hemogenic endothelium in CD3+ T cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of CD3+ T cells.

[0229] In some embodiments, the CD34+ hemogenic endothelium is treated with an inhibitor of an epigenetic regulator. Exemplary inhibitors of epigenetic regulators include inhibitors of at least one of the following: DNMTs, MBDs, DNA demethylases, HMTs, methylhistone-binding proteins, histone demethylases, HATs, acetyl-binding proteins, or HDACs. In some embodiments, the epigenetic regulator is an H3K9 methyltransferase. H3K9 methylation in humans is primarily dependent on members of the Suv39 family, namely, EHMT1 / GLP, EHMT2 / G9a, SUV39H1, SUV39H2, SETDB1, and SETDB2, as well as the non-Suv39 enzymes PRDM2 and ASH1L.

[0230] Non-limiting examples of DNMT inhibitors include azacitidine, decitabine, guadecitabine, and hydralazine. Non-limiting examples of HMT inhibitors include pinometostat, tazemetostat, GSK2816126, CPI-1205, TCP, ORY-2001, GSK2879552, and 4SC-202. Non-limiting examples of HDAC inhibitors include valproic acid, phenylbutyrate, vorinostat, trichostatin A, belinostat, entinostat, panobinostat, mocetinostat, CI-994, romidepsin, nicotinamide, suramin, PRI-724, GSK525762, CPI-0610, RO6870810, and MK-8628.

[0231] In some embodiments, the inhibitor of an epigenetic regulator is selected from Table 1. In some embodiments, the inhibitor of an epigenetic regulator is selected from the group consisting of: SB939 (prasinostat), 4-iodo-SAHA, scriptaid, oxaflatin (i.e., oxamflatin), s-HDAC-42, UNC0224, piroxamide, MC1568, CAY10398, CAY10591, SAHA (vorinostat) (SIH-359), SGI-1027, and rucaparib (Rubraca™). In some embodiments, the inhibitor of an epigenetic regulator is selected from the group consisting of SB939 (prasinostat), 4-iodo-SAHA, scriptaid, oxaflatin (i.e., oxamflatin), s-HDAC-42, UNC0224, pyroxamide, MC1568, CAY10398, CAY10591, and SAHA (vorinostat) (SIH-359). See, e.g., Figure 7 and Table 2 of International Patent Publication No. WO 2021 / 150919(A1).

[0232] [Table 1] JPEG2025535084000003.jpg191158JPEG2025535084000004.jpg210158JPEG2025535084000005.jpg227157

[0233] In some embodiments, 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.

[0234] In some embodiments, the inhibitor of an epigenetic regulator is UNC0224 or 5-methyl-2'-deoxycytidine (see, e.g., the structure of Formula I below). In some embodiments, the inhibitor of an epigenetic regulator is 5-methyl-2'-deoxycytidine. 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).

[0235] [ka]

[0236] In some embodiments, 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 30 ...0 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 90 nM, at least 100 nM, at least 150 nM, at least 200 In some embodiments, the inhibitor of an epigenetic regulator is provided at a concentration of 1 nM to 10 nM, 10 nM to 50 nM, 50 nM to 100 nM, 100 nM to 500 nM, 500 nM to 1 uM, 1 uM to 500 nM, 500 nM to 1 uM, 1 uM to 5 uM, or 5 uM to 10 uM.

[0237] In some embodiments, cells (e.g., CD34+ hemogenic endothelium) are cultured with exposure to an inhibitor of an epigenetic regulator until development of CD5+CD7+ proT cells. In some embodiments, cells (e.g., CD34+ hemogenic endothelium) are cultured with exposure to an inhibitor of an epigenetic regulator for about 14 days. In some embodiments, cells (e.g., CD34+ hemogenic endothelium) are cultured with exposure 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, or at least The cells are exposed to and cultured with an inhibitor of an epigenetic regulator for 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 46 days, at least 47 days, at least 48 days, at least 49 days, at least 50 days, or more.

[0238] Inhibition of G9a and / or GLP In some aspects, described herein are methods of T cell differentiation that include inhibiting G9a and / or GLP. In some aspects, described herein are methods of T cell differentiation that include inhibiting G9a. G9a may also be referred to interchangeably as euchromatin histone lysine methyltransferase 2 (EHMT2), histone H3-K9 methyltransferase 3, KMT1C, lysine N-methyltransferase 1C, BAT8, or NG36. G9a is a methyltransferase that methylates lysine residues on histone H3 (see, e.g., NCBI Gene ID: 10919, SEQ ID NOs: 45-46, or sequences at least 95% identical and maintain the same function, or functional fragments thereof). In some aspects, described herein are methods of T cell differentiation that include inhibiting G9a-like protein (GLP). GLP may also be referred to interchangeably as euchromatic histone lysine methyltransferase 1 (EHMT1), KMT1D, Eu-HMTase1, or histone-lysine N-methyltransferase, H3 lysine-9 specific 5 (see, e.g., NCBI Gene ID: 79813, SEQ ID NOs: 47-48, or sequences at least 95% identical and maintain the same function, or functional fragments thereof).

[0239] G9a and GLP exist primarily as a G9a-GLP heteromeric complex. G9a and GLP are the primary enzymes for mono- and dimethylation of histone H3 at Lys9 (H3K9me1 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, whereas its histone methyltransferase activity is not required for DNA methylation, suggesting that these two activities function independently. G9a / GLP is likely targeted to histone H3 by various DNA-binding proteins, such as E2F6, MGA, MAX, and / or DP1. In addition to histone methyltransferase activity, G9a / GLP also methylates non-histone proteins, for example, by dimethylating Lys-373 of p53 / TP53.

[0240] G9a also mediates the monomethylation of Lys-56 on histone H3 (H3K56me1) during the G1 phase, promoting the interaction between histone H3 and PCNA and regulating DNA replication. Nevertheless, G9a also methylates histone H1. G9a also methylates CDYL, WIZ, ACIN1, DNMT1, HDAC1, ERCC6, KLF12, and itself. During the G0 phase, GLP may contribute to the silencing of MYC- and E2F-responsive genes, suggesting a role in the G0 / G1 transition in the cell cycle. In addition to histone methyltransferase activity, GLP also methylates nonhistone proteins: it mediates the dimethylation of Lys-373 on p53 / TP53.

[0241] SEQ ID NO: 45, Homo sapiens euchromatin histone lysine methyltransferase 2 (EHMT2), transcript variant 1, mRNA, NCBI reference sequence: NM_001289413.1 (region 5-3706), 3702 bp

[0242] SEQ ID NO: 46, Histone-lysine N-methyltransferase EHMT2 isoform c (Homo sapiens), NCBI Reference Sequence: NP_001276342.1, 1233 aa

[0243] SEQ ID NO: 47, Homo sapiens euchromatin histone lysine methyltransferase 1 (EHMT1), transcript variant 2, mRNA, NCBI reference sequence: NM_001145527.2 (region 25-2451), 2427 bp

[0244] SEQ ID NO: 48, Histone-lysine N-methyltransferase EHMT1 isoform 2 (Homo sapiens), NCBI Reference Sequence: NP_001138999.1, 808 aa MAAADAEAVPARGEPQQDCCVKTELLGEETPMAADEGSAEKQAGEAHMAADGETNGSCENSDASSHANAAKHTQDSARVNPQDGTNTLTRIAENGVSERDSEAAKQNHVTADDFVQTSVIGSNGYILNKPALQAQPLRTTSTLASSLPGHAAKTLPGGAGKGRTPSAFPQTPAAPPATLGEGSADTEDRKLPAPGADVKVHR ARKTMPKSVVGLHAASKDPREVREARDHKEPKEEINKNISDFGRQQLLPPFPSLHQSLPQNQCYMATTKSQTACLPFVLAAAVSRKKKRRMGTYSLVPKKKTKVLKQRTVIEMFKSITHSTVGSKGEKDLGASSLHVNGESLEMDSDEDDSEELEEDDGHGAEQAAAFPTEDSRTSKESMSEADRAQKMDGESEEEQESVDT GEEEEGGDESDLSSESSIKKKFLKRKGKTDSPWIKPARKRRRRSRKKPSGALGSESYKSSAGSAEQTAPGDSTGYMEVSLDSLDLRVKGILSSQAEGLANGPDVLETDGLQEVPLCSCRMETPKSREITTLANNQCMATESVDHELGRCTNSVVKYELMRPSNKAPLLVLCEDHRGRMVKHQCCPGCGYFCTAGNFMECQPE SSISHRFHKDCASRVNNASYCPHCGEESSKAKEVTIAKADTTSTVTPVPGQEKGSALEGRADTTTGSAAGPPLSEDDKLQGAASHVPEGFDPTGPAGLGRPTPGLSQGPGKETLESALIALDSEKPKKLRFHPKQLYFSARQGELQKVLLMLVDGIDPNFKMEHQNKRSPLHAAAEAGHVDICHMLVQFCRLGSPRRSRGCLW

[0245] In some embodiments, the method comprises inhibiting G9a and / or GLP in the resulting population of CD34+ hemogenic endothelium after differentiating the population of pluripotent stem cells in aggregation medium for a time sufficient to promote differentiation into a population of CD34+ hemogenic endothelium. In some embodiments, the method comprises inhibiting G9a and / or GLP in the population of CD34+ hemogenic endothelium before differentiating the population of CD34+ hemogenic endothelium in CD3+ T cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of CD3+ T cells.

[0246] Thus, in one aspect, a method is provided, comprising: (a) culturing a population of pluripotent stem cells by transfecting the population with CD34 + (b) allowing the resulting CD34 + (c) inhibiting G9a and / or GLP in the hemogenic endothelial population; and (d) the resulting CD34 + The hemogenic endothelial population was identified as CD3 + in the presence of soluble Notch ligand for a time sufficient to promote differentiation into a population of T cells, CD3 + and differentiating in a T cell differentiation medium.

[0247] In one embodiment, the inhibitor is a G9a / GLP inhibitor. In one embodiment, the G9a / GLP inhibitor is selected from the compounds listed in Table 3, or derivatives or analogs thereof. In one embodiment, 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. In one embodiment, the G9a / GLP inhibitor is selected from the group consisting of UNC0224, UNC0638, A366, BRD4770, BIX01294, and UNC0642 (see, e.g., Figures 8, 10B, 12B, 13B, 13D-13F in International Patent Publication No. WO 2021 / 150919(A1)). In some embodiments, the G9a / GLP inhibitor is selected from the group consisting of UNC0224, UNC0638, BRD4770, BIX01294, and UNC0642 (see, e.g., Figures 8, 10B, 12B, 13B, 13D-13F in International Patent Publication No. WO 2021 / 150919(A1)).

[0248] In some embodiments, 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, BIX01294, UNC0642, UNC0631, UNC0646, UNC0321, and E72. In some embodiments, 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. In some embodiments, the G9a / GLP inhibitor is a type III G9a / GLP inhibitor such as chaetocin. In some embodiments, 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., Figures 13D-13F presented in International Patent No. 2021 / 150919(A1).) All references cited in Table 2 are specifically incorporated herein by reference in their entirety.

[0249] [Table 2] JPEG2025535084000008.jpg239158JPEG2025535084000009.jpg156158JPEG2025535084000010.jpg166158JPEG2025535084000011.jpg155158

[0250] In some embodiments, 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, In some embodiments, the G9a / GLP inhibitor is provided at a concentration of 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 uM, at least 1.25 uM, at least 1.5 uM, at least 1.75 uM, at least 2.0 uM, at least 2.5 uM, at least 3 uM, at least 4 uM, at least 5 uM, at least 6 uM, at least 7 uM, at least 8 uM, at least 9 uM, or at least 10 uM. In some embodiments, the G9a / GLP inhibitor is provided at a concentration of 1 nM to 10 nM, 10 nM to 50 nM, 50 nM to 100 nM, 100 nM to 500 nM, 500 nM to 1 uM, 1 uM to 5 uM, or 5 uM to 10 uM.

[0251] In some embodiments, 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.

[0252] In some embodiments, cells (e.g., CD34+ hemogenic endothelium) are cultured with exposure to a G9a / GLP inhibitor until development of CD5+CD7+ proT cells. In some embodiments, cells (e.g., CD34+ hemogenic endothelium) are cultured with exposure to a G9a / GLP inhibitor for about 14 days. In some embodiments, cells (e.g., CD34+ hemogenic endothelium) are cultured with exposure 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, or 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 46 days, at least 47 days, at least 48 days, at least 49 days, at least 50 days, or more.

[0253] In some embodiments, culturing cells (e.g., CD34+ hemogenic endothelium) in the presence of a G9a / GLP inhibitor increases the number of resulting 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%, or at least 50% compared to cells not cultured in the presence of a G9a / GLP inhibitor. %, 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 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold or more higher.

[0254] In some embodiments, culturing cells (e.g., CD34+ hemogenic endothelium) in the presence of a G9a / GLP inhibitor increases or decreases the number of erythroid or myeloid lineage cells (e.g., red blood cells, macrophages, granulocytes, megakaryocytes) 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%, or at least 60% compared to cells not cultured in the presence of a G9a / GLP inhibitor. %, 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 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold or more.

[0255] In some embodiments, culturing cells (e.g., CD34+ hemogenic endothelium) in the presence of a G9a / GLP inhibitor increases 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%, or at least or by 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 by at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold or more.

[0256] In some embodiments, culturing cells (e.g., CD34+ hemogenic endothelium) in the presence of a G9a / GLP inhibitor increases the percentage of the resulting target 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) out of 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%, or at least or 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 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold or more.

[0257] In some embodiments, the methods for differentiating T cells described herein (e.g., T cell differentiation with G9a / GLP inhibition and no stroma) produce 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). In some embodiments, the methods for differentiating T cells described herein (e.g., T cell differentiation with G9a / GLP inhibition and no stroma) produce a population that comprises at least 15% CD5+CD7+Pro-T cells.

[0258] For example, Greiner et al.Nature Chemical Biology1(3),143-145(2005), Liu et al.Journal of Medicinal Chemistry54(17),6139-6150(2011), Liu et al.J Med Chem.2010 Aug 12;53(15):5844-5857, Liu et al.,J Med Chem.2009 Dec 24;52(24):7950-7953, Kondengaden et al.,Eur J Med Chem.2016 Oct 21,122:382-393, Yuan et al.ACS Chem Biol.2012 Jul 20;7(7):1152-1157, Chang et al.J Mol Biol.2010 Jul 2;400(1):1-7, Christman et al. Proceedings of the National Academy of Sciences of the United States of America 92(16), 7347-7351 (1995), Cheng et al., Signal Transduction and Targeted Therapy volume 4, Article number: 62 (2019), the contents of each of which are incorporated herein by reference in their entirety.

[0259] Inhibition of histone methyltransferases In some embodiments, the differentiation method can include inhibiting histone methyltransferase. Inhibiting histone methyltransferase (e.g., EZH1 knockdown) can increase differentiation efficiency (e.g., of T cells). Thus, in some embodiments, the differentiation method includes, for example, inhibiting histone methyltransferase in the resulting population of CD34+ hemogenic endothelial cells. Methods for inhibiting histone methyltransferase are known in the art. See, for example, International Application No. 2018 / 048828, U.S. Application No. 2019 / 0225940, Doulatov et al., Cell Stem Cell. 2013 October 3, 13(4), and Vo et al., Nature 2018, 553(7689):506-510, the contents of each of which are incorporated herein by reference in their entirety.

[0260] However, the step of inhibiting histone methyltransferase (e.g., EZH1 knockdown) is not required. Thus, in some embodiments, the differentiation method does not include inhibiting histone methyltransferase, for example, in the resulting population of CD34+ hemogenic endothelium.

[0261] During these experiments, we discovered that inhibition of specific histone-modifying enzymes targeting H3K9 and H3K27 promotes the lymphoid potential of hematopoietic precursors derived from pluripotent stem cells. 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 in epigenetic signaling. Trimethylation of lysine 9 (H3K9) of histone H3 mediates chromatin recruitment of HP1, heterochromatin condensation, and gene silencing. Similarly, methylation of H3K27 and H4K20 is associated with a repressive state of chromatin, while expressed genes are methylated at H3K4, H3K36, and H3K79. In humans, H3K9 methylation is primarily dependent on members of the Suv39 family, namely, EHMT1 / GLP, EHMT2 / G9a, SUV39H1, SUV39H2, SETDB1, and SETDB2, as well as the 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). In contrast, H3K27 methylation is carried out by Polycomb Repressive Complex 2 (PRC2).

[0262] Di- / trimethylation of H3K9 is primarily catalyzed by the conserved SUV39H1 / 2 histone methyltransferase, while 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 contains EZH1 / 2 catalytic subunits, SUZ12, EED, and RBBP7 / 4 (see, e.g., Margueron R, and Reinberg D, 2011).

[0263] It is specifically contemplated herein that inhibition of histone lysine methyltransferases that target H3K9 and H3K27 relieves transcriptional repression resulting from histone H3 methylation, thereby promoting gene expression that promotes cell differentiation, particularly T cell specificity.

[0264] In one embodiment, the histone methyltransferase catalyzes the addition of a methyl group to histone H3 lysine residue 9 (H3K9) and / or histone H3 lysine residue 27 (H3K27).

[0265] In one embodiment, the histone methyltransferase inhibitor inhibits the G9a / GLP heteromeric complex.

[0266] 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.

[0267] GLP (EC 2.1.1.43) (UniProtKB: Q9H9B1) is also known as EHMT1 (euchromatic histone-lysine N-methyltransferase 1), G9a-like protein 1, and GLP1.

[0268] In one embodiment, the histone methyltransferase inhibitor inhibits EZH1 (Enhancer of Zeste1 Polycomb repressive complex 2 subunit).

[0269] In one embodiment, the H3K27 histone methyltransferase is EZH1 (EC:2.1.1.43) (UniproKB Q92800-1).

[0270] In one embodiment, the H3K27 histone methyltransferase is not EZH2 (EC:2.1.1.43) (Unipro Q15910-1).

[0271] In one embodiment, the inhibitor of histone methyltransferase inhibits the gene expression or protein catalytic activity of histone methyltransferase.

[0272] In one embodiment, histone methyltransferases H3K9 and / or H3K27 are inhibited by small molecules, or nucleic acids, or CRISPR-mediated targeted gene interference.

[0273] In some embodiments, histone methyltransferase H3K9 and / or H3K27 is inhibited by a small molecule inhibitor or a nucleic acid inhibitor. In one embodiment of any of the methods, cells, or compositions described, 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 having a molecular weight of less than about 10,000 grams per mole (i.e., including heteroorganic and organometallic compounds), organic or inorganic compounds having a molecular weight of less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 5000 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. In some embodiments, the small molecule is a heteroorganic compound or an organometallic compound.

[0274] In one embodiment, histone methyltransferase small molecule inhibitors 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)HCl, UNC1999, MM-102, SGC0946, entacapone, EPZ015666, UNC0379, EI1, MI-2 (menin-MLL inhibitor), MI-3 (menin-MLL inhibitor), PFI-2, GSK126, or EPZ004777.

[0275] In one embodiment, the histone methyltransferase small molecule inhibitor is selected from the group consisting of UNC0631, BRD4770, UNC1999, CPI-360, and BIX01294.

[0276] In one embodiment, the nucleic acid inhibitor is a nucleic acid that targets the expression of histone methyltransferase. For example, it targets the mRNA or primary transcript of the histone methyltransferase EZH1, thereby inhibiting the protein expression of the enzyme. Histone-lysine N-methyltransferase, also known as Enhancer of Polycomb Repressive Complex 2 Subunit Zeste1 (EZH1) or EC 2.1.1.43, is a component of the non-canonical Polycomb Repressive Complex 2 (PRC2), which mediates the methylation of histone H3 (see MIM602812) lys27 (H3K27) and functions in maintaining embryonic stem cell pluripotency and plasticity. The external identification of the human EZH1 gene is as follows: HGNC:3526, Entrez Gene:2145, Ensembl:ENSG00000108799, OMIM:601674, UniProtKB:Q92800, EMBL:AB002386 mRNA and corresponding mRNA translation:BAA20842.2, GENBANK:BT009782 mRNA and corresponding mRNA translation:AAP88784.1.

[0277] In one embodiment, the nucleic acid inhibitor targets human EZH1 mRNA.

[0278] In one embodiment, the nucleic acid inhibitor is an RNA interference inhibitor or a CRISPR-mediated gene interference inhibitor.The RNA interference inhibitor can be designed using the mRNA of EZH1 as a target, using the predictive RNAi software found at Whitehead Institute, MIT, the siRNA website, Invitrogen / ThermoFisher's BLOCK-iT™ RNAi Designer, and other online siRNA design tools on RNAiWeb.

[0279] Similarly, Crisper guide RNAs 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 / sgrna-design), dna20, Clontech, AddGene, e-crisp, and Innovative Genomic, using the EZH1 mRNA or genomic gene as a target.

[0280] CRISPR (clustered regularly interspaced short palindromic repeats) Cas9-mediated gene disruption has been widely used to generate loss-of-function mutations in diverse organisms, including mammals (reviewed in Cong et al., 2013, Science, 339(6121):819-23; Hsu et al., 2014, Cell, 157(6):1262-78). Cas9-based knockout screening has been applied to identify essential genes and genes involved in drug resistance in various cell lines.For general information about CRISPR-Cas systems, their components, and delivery of such components, including methods, materials, delivery vehicles, vectors, particles, AAVs, and their production and use, including in amounts and formulations, all useful in the practice of the present invention, see U.S. Patent Nos. 8,999,641; 8,993,233; 8,945,839; 8,932,814; 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359; No. 2014 / 0310830, No. 2014 / 0287938, No. 2014 / 0273234, No. 2014 / 0273232, No. 2014 / 0273231 No. 2014 / 0256046, No. 2014 / 0248702, No. 2014 / 0242700, No. 2014 / 0242699, No. 2014 / 02426 64, 2014 / 0234972, 2014 / 0227787, 2014 / 0189896, 2014 / 0186958, 2014 / 0186919, 2014 / 0186843, 2014 / 0179770, 2014 / 0179006, 2014 / 0170753, European Patent No. 784 162(B1) and 2 771 468(B1), European Patent Applications Nos. 2 771 468 (EP13818570.7), 2 764 103 (EP13824232.6), and 2 784 162 (EP14170383.5), and International Application No. 2014 / 093661, all of which are incorporated herein by reference in their entirety.

[0281] CRISPR / Cas systems contemplated for use in the context of the present invention can use any suitable CRISPR enzyme. In some embodiments, the CRISPR enzyme is a Type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes, or S. thermophilus Cas9, and can include mutated Cas9s from these organisms. The enzyme can be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in eukaryotic cells.

[0282] As described herein, CRISPR / Cas systems are used to specifically target multiple sequences within a contiguous genomic region of interest. Targeting typically involves 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 that includes at least one Cas protein and one or more guide RNAs from a guide RNA library described herein.

[0283] In these methods, the Cas protein and one or more guide RNAs, which may be on the same or different vectors of the system, are integrated into each cell, thereby targeting each guide sequence to a sequence within the contiguous genomic region within each cell in the population of cells. The Cas protein is operably linked to a regulatory element to ensure expression in the cell, more specifically, to a promoter suitable for expression in the cells of the population of cells. In certain embodiments, the promoter is an inducible promoter, such as a doxycycline-inducible promoter. When transcribed within the cells of the population of cells, the guide RNA containing the guide sequence directs sequence-specific binding of the CRISPR-Cas system to the target sequence in the contiguous genomic region. Typically, binding of the CRISPR-Cas system induces cleavage of the contiguous genomic region by the Cas protein.

[0284] RNA interference (RNAi), mediated by short interfering RNA (siRNA) or microRNA (miRNA), is a powerful method for post-transcriptional regulation of gene expression. RNAi has been widely used to study biological processes in mammalian cells and may constitute a therapeutic approach for human diseases in which selective regulation of gene expression would be desirable. Depending on the degree of complementarity between the miRNA and target mRNA sequence, loss of gene expression occurs by inducing degradation of the cognate mRNA or by translational decay. Endogenous miRNAs are transcribed as primary transcripts and subsequently processed by the RNAse III enzyme Drosha to create a stem-loop structure. Nuclear export and cleavage by Dicer generate mature short double-stranded molecules (siRNAs) separated into guide and passenger strands. The guide strand is loaded into the RNA-induced silencing complex (RISC), and an effector complex mediates cleavage of the target mRNA with the functional guide strand binding to RISC proteins, while the passenger strand is degraded. Loading of the guide strand versus the passenger strand into RISC is highly dependent on the stability of the 5' end of the siRNA, with less stable strands preferentially incorporated into RISC, although the precise regulation in mammalian cells is not fully understood. The 5' end of the guide strand contains a "seed region" that is important for target specificity. Accurate cleavage by Drosha and Dicer is crucial for generating guide RNAs with defined seed regions that mediate efficient binding to the appropriate target mRNA. While inaccurate processing can result in binding to off-target molecules, shifts in the cleavage site also alter the nucleotide composition of the duplex end, which can have profound effects on strand loading into RISC.

[0285] Inhibition of the expression of a selected target polypeptide is mediated by the use of an RNA interference agent. RNA interference (RNAi) uses small interfering RNA (siRNA) duplexes to target messenger RNA encoding the target polypeptide for selective degradation. siRNA-dependent post-transcriptional silencing of gene expression involves cleaving the target messenger RNA molecule at a site guided by the siRNA. RNAi is an evolutionarily conserved process in which the expression or introduction of RNA with a sequence identical or highly similar to that of a target gene results in sequence-specific degradation or specific post-transcriptional gene silencing (PTGS) of the messenger RNA (mRNA) transcribed from the target gene (see, e.g., Coburn, G. and Cullen, B. (2002) J. Virology 76(18):9225), thereby inhibiting the expression of the target gene. In one embodiment, the RNA is double-stranded RNA (dsRNA). This process has been described in plants, invertebrates, and mammalian cells. In nature, RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes the processive cleavage of long dsRNA into double-stranded fragments called siRNAs. The siRNAs are incorporated into a protein complex (called the "RNA-induced silencing complex" or "RISC") that recognizes and cleaves target mRNAs. RNAi can also be initiated by introducing nucleic acid molecules, such as synthetic siRNAs or RNA interference agents, to inhibit or silence the expression of target genes. As used herein, "inhibition of target gene expression" includes any reduction in the expression, activity, or level of a target gene or a protein encoded by the target gene compared to the situation in which RNA interference is not induced. This reduction can be 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, compared to the expression of a target gene not targeted by an RNA interference agent or the activity or level of a protein encoded by the target gene.

[0286] The terms "RNA interfering agent" and "RNA interference," as 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 that functions to inhibit expression of a target gene, for example, by RNAi. siRNA may be chemically synthesized, produced by in vitro transcription, or produced within a host cell. In one embodiment, the siRNA is a double-stranded RNA (dsRNA) molecule about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides, more preferably about 19, 20, 21, 22, or 23 nucleotides in length, and may contain 3' and / or 5' overhangs on each strand having lengths of about 0, 1, 2, 3, 4, or 5 nucleotides. The lengths of the overhangs are independent between the two strands; i.e., the length of the overhang on one strand is independent of the length of the overhang on the second strand. Preferably, the siRNA is capable of promoting RNA interference through degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA).

[0287] siRNA also includes small hairpin (also called stem-loop) RNAs (shRNAs). In one embodiment, these shRNAs are composed of a short (e.g., about 19 to about 25 nucleotides) antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and a similar sense strand. Alternatively, the sense strand may precede the nucleotide loop structure, and the antisense strand may follow. These shRNAs can be contained in plasmids, retroviruses, and lentiviruses and can be expressed, for example, from the pol III U6 promoter or another promoter (see, e.g., Stewart, et al. (2003) RNA April;9(4):493-501, incorporated herein by reference in its entirety). The target gene or sequence of the RNA interfering agent can be a cellular gene or genomic sequence, such as a G9a / GLP or EZH1 sequence. The siRNA can be substantially homologous to the target gene or genomic sequence, or a fragment thereof. In this context, the term "homologous" is defined as being substantially identical to, sufficiently complementary to, or similar to a target mRNA or a fragment thereof to achieve targeted RNA interference. In addition to natural RNA molecules, RNA suitable for inhibiting or interfering with the expression of a target sequence includes RNA derivatives and analogs. Preferably, the siRNA is identical to its target. Preferably, the siRNA targets only one sequence. Each RNA interference agent, such as an siRNA, can be screened for potential off-target effects, for example, by expression profiling. Such methods are known to those skilled in the art and are described, for example, in Jackson et al. Nature Biotechnology 6:635-637, 2003. In addition to expression profiling, potential target sequences can be screened for similar sequences in a sequence database to identify potential sequences that may have off-target effects. For example, 15, or perhaps as few as 11 consecutive nucleotides of sequence identity are sufficient to direct silencing of non-target transcripts.Therefore, proposed siRNAs can be first screened to avoid potential off-target silencing using sequence identity analysis by any known sequence comparison method, such as BLAST. The siRNA sequence is selected to maximize the incorporation of the antisense (guide) strand of the siRNA into RISC, thereby maximizing the ability of RISC to target G9a / GLP or EZH1 mRNA for degradation. This can be achieved by scanning for sequences with the lowest binding free energy at the 5' end of the antisense strand. Lower free energy results in enhanced unwinding of the 5' end of the antisense strand of the siRNA duplex, thereby ensuring that the antisense strand is incorporated into RISC and directs sequence-specific cleavage of human G9a / GLP or EZH1 mRNA. siRNA molecules need not be limited to those molecules containing only RNA, but also include, for example, chemically modified nucleotides and non-nucleotides, including molecules in which the ribose sugar molecule is replaced with another sugar molecule or a molecule that performs a similar function. Furthermore, non-natural linkages between nucleotide residues, such as phosphorothioate linkages, can be used. The RNA strand can be derivatized with a reactive functional group of a reporter group, such as a fluorophore. Particularly useful derivatives are modified at the end of the RNA strand, typically the 3' end of the sense strand. For example, the 2'-hydroxyl at the 3' end can be easily and selectively derivatized with various groups. Other useful RNA derivatives incorporate 2'O-alkylated residues or nucleotides with modified carbohydrate moieties, such as 2'-O-methylribosyl derivatives and 2'-O-fluororibosyl derivatives. The RNA bases can also be modified. Any modified base useful for inhibiting or interfering with the expression of a target sequence can be used. For example, halogenated bases such as 5-bromouracil and 5-iodouracil can be incorporated. The bases can also be alkylated; for example, 7-methylguanosine can be incorporated in place of a guanosine residue. Non-natural bases that result in successful inhibition can also be incorporated.Preferred siRNA modifications include RNA duplexes containing 2'-deoxy-2'-fluorouridine or locked nucleic acid (LAN) nucleotides and either phosphodiester or various numbers of phosphorothioate linkages. Such modifications are known to those skilled in the art and are described, for example, in Braasch et al., Biochemistry, 42:7967-7975, 2003. Most useful modifications to siRNA molecules can be introduced using chemistries established for antisense oligonucleotide technology. Preferably, modifications include minimal 2'-O-methyl modifications, and preferably exclude such modifications. Modifications also preferably exclude modifications of the free 5'-hydroxyl group of siRNA. The examples herein provide specific examples of RNA interference agents, such as shRNA molecules, that effectively target mRNA.

[0288] In one embodiment, the nucleic acid is a G9a / GLP or EZH1-specific RNA interfering agent or a vector encoding the RNA interfering agent. In one embodiment, the RNA interfering 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).

[0289] In some embodiments, the nucleic acid inhibitor is an EZH1-specific nucleic acid selected from the group consisting of an aptamer that binds to EZH1, an EZH1-specific RNA interfering agent, and a vector encoding an EZH1-specific RNA interfering agent, and the RNA interfering agent comprises one or more of the nucleotide sequences selected from SEQ ID NOs: 11-19.

[0290] In one embodiment, the multilineage hematopoietic progenitor cells are contacted with a viral vector or vectors carrying a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-19.

[0291] In one embodiment, contact with the histone methyltransferase inhibitor occurs two or more times. For example, after an initial first contact of the multilineage hematopoietic progenitor cells with a virus or vector carrying a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-19, or after contact with a small molecule inhibitor described herein, the contacted cells are washed to remove the virus or vector, and then the washed cells are contacted a second time with the same virus or vector used in the first contact.

[0292] It is contemplated herein that the Cas9 / CRISPR system of genome editing can be used with the methods, cells, and compositions described herein. Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) system is useful for RNA programmable genome editing (see, for example, Jinek, M. et al. Science (2012) 337 (6096): 816-821).

[0293] Transactivating crRNA (tracrRNA) is a small transcoding 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) constitutes an RNA-mediated defense system that protects against viruses and plasmids. This defense pathway has three steps. First, a copy of the invading nucleic acid is integrated into the CRISPR locus. Then, CRISPR RNA (crRNA) is transcribed from this CRISPR locus. The crRNA is then incorporated into an effector complex, where the crRNA guides the complex to the invading nucleic acid, and the Cas protein degrades the nucleic acid. (See, e.g., Terns MP and Terns RM (2011). Curr Opin Microbiol 14(3):321-7.) There are several pathways for CRISPR activation, one of which requires the tracrRNA, which plays a role in the maturation of the crRNA. The tracrRNA is complementary to and base-pairs with the pre-crRNA, forming an RNA duplex. This is cleaved by the RNA-specific ribonuclease RNase III to form a crRNA / tracrRNA hybrid. This hybrid serves 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.)

[0294] In some embodiments, the Cas9 / CRISPR guide RNA is designed to target exon 3 of the EZH1 gene, which is present in all known EZH1 transcripts. The exon 3 sequence is ATTACAGCAAGATGGAAATACCAAATCCCCCTACCTCCAAATGTATCACTTACTGGAAAAGAAAAGTGAAATCTGAATACATGCGACTTCGACAACTTAAACGGCTTCAGGCAAATATGGGTGCAAAG (SEQ ID NO: 20).

[0295] Non-limiting exemplary gRNAs targeting 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), CATTGGAGGTAGGGGGATT (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).

[0296] In another embodiment, the Cas9 / CRISPR guide RNA is designed to target exon 4 of the EZH1 gene, which is also present in all known EZH1 transcripts. The exon 4 sequence is GCTTTGTATGTGGCAAATTTTGCAAAGGTTCAAGAAAAAACCCAGATCCTCAATGAAGAATGGAAGAAGCTTCGTGTCCAACCTGTTCAGTCAATGAAGCCTGTGAGTGGACACCCTTTTCTCAAAAAG (SEQ ID NO: 33).

[0297] Non-limiting exemplary gRNAs targeting 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).

[0298] In one embodiment, a vector is used as a delivery vehicle to introduce any of the nucleic acid inhibitors of histone methyltransferases described herein into a target cell selected from a population of cells described herein (e.g., ESCs, PSCs, iPSCs, hemogenic endothelium, HSCs). In one embodiment, a vector is used as a delivery vehicle to introduce any of the nucleic acids described herein comprising a nucleic acid inhibitor of histone methyltransferase described herein into a target cell selected from a population of cells described herein (e.g., ESCs, PSCs, iPSCs, hemogenic endothelium, HSCs). In vivo expression of the nucleic acid inhibitor is to degrade the mRNA of the targeted histone methyltransferase, such as G9a / GLP or EZH1, so as to reduce and inhibit expression of the respective histone methyltransferase, with the goal of reducing histone H3 methylation in the transfected cell and relieving repression of gene expression therein.

[0299] In one embodiment, the host cell is an embryonic stem cell, a somatic stem cell, a progenitor cell, a bone marrow cell, a hematopoietic stem cell, a hematopoietic progenitor cell, an immune cell such as a T cell or a B cell, an erythrocyte, a fibroblast, a keratinocyte, or a myeloid progenitor cell. In one embodiment, the host cell is isolated from a subject. In one embodiment, the host cell is isolated from a subject diagnosed with a hematological disorder.

[0300] In one embodiment, the vector further comprises a spleen focus-forming virus promoter, a tetracycline-inducible promoter, a doxycycline (Dox)-inducible promoter, or a β-globin locus control region and a β-globin promoter. In one embodiment, the promoter provides targeted expression of the nucleic acid molecule therein. Other examples of promoters include, but are not limited to, the CMV promoter and the EF1-alpha promoter for various transgenes, and the U6 promoter for shRNAs targeting EZH1.

[0301] In one embodiment, the vector is a viral or non-viral vector. Non-limiting examples of viral vectors for gene delivery and expression in cells include retroviruses, adenoviruses (types 2 and 5), adeno-associated viruses (AAV), helper-dependent adenoviral vectors (HdAd), hybrid adenoviral vectors, herpesviruses, poxviruses, human foamy virus (HFV), and lentiviruses. Exemplary vectors useful in the inventions described herein include episomal vectors, integrating vectors, non-integrating vectors, and excisable vectors.

[0302] Stroma-free T cell differentiation In some embodiments, the differentiation method comprises differentiating the resulting population of CD34+ hemogenic endothelium in a CD3+ T cell differentiation medium in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of CD3+ T cells. The method described herein is a stromal-free T cell differentiation method. Compared to differentiation using stromal cells expressing Notch ligand, stromal-free differentiation unexpectedly results in an increased number of differentiated T cells, with a smaller proportion of these T cells being innate-like cells.

[0303] In one embodiment, the soluble Notch ligand is provided at a concentration of 1 nM to 100 nM, or at a concentration of 5 nM to 15 nM.

[0304] In one embodiment, 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 more.

[0305] In one embodiment, 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 more.

[0306] In one embodiment, the soluble Notch ligand is provided at a concentration of at least 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM or more.

[0307] In a preferred embodiment, the soluble Notch ligand is provided at a concentration of 10 nM.

[0308] In some embodiments, the cells are cultured 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 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 46 days, at least 47 days, at least 48 days, at least 49 days, at least 50 days, at least 51 days, at least 52 days, at least 53 days, at least 54 days, at least 55 days, at least 56 days, at least 57 days, at least 58 days, at least 59 days, at least 60 days, at least 61 days, at least 62 days, at least 63 days, at least 64 days, at least 65 days, at least 66 days, at least 67 days, at least 68 days, at least 69 days, at least 70 days, at least 71 days, at least 72 days, at least 73 days The cells are exposed to and cultured with soluble Notch ligand for 6 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 46 days, at least 47 days, at least 48 days, at least 49 days, at least 50 days, or more.

[0309] Differentiation without stroma The methods described herein are stromal-free T cell differentiation methods, i.e., methods that do not involve co-culturing with stromal cells or any other type of support cells. Co-culturing with stromal cells, such as mouse stromal cells, can limit the translational potential of iPSC-derived T cells and may result in, for example, transplant rejection due to the presence of stromal cells. Furthermore, T cells differentiated using stromal cells exhibit an innate-like phenotype (e.g., as measured by TCRgd expression, a marker for gamma delta T cells). Preferably, the T cells exhibit an adaptive phenotype, characterized, for example, by expression of TCRα and β. Furthermore, as described herein, stromal-free T cell differentiation methods result in increased numbers of CD3+ T cells (e.g., CD4+CD8+ cells) compared to differentiation methods that involve stromal co-culture.

[0310] Thus, using the stroma-free method, and in one embodiment, in combination with inhibition of epigenetic regulators (e.g., HMTs, e.g., EZH1, G9a / GLP), differentiated T cells exhibit at least the following unexpected advantages compared to the stroma co-culture method: (1) increased likelihood of engraftment in humans, (2) reduced numbers of innate-like T cells, (3) increased numbers and / or percentages of resulting 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) a gene expression profile most similar to alpha-beta T cells, (5) a more diverse TCR repertoire, and / or (6) increased TCR CDR length.

[0311] As used herein, the term "support or stromal cells," when used in the context of cell differentiation, refers to any cells that can create, promote, or support a microenvironment for the growth, proliferation, differentiation, or expansion of multipotent hematopoietic progenitor cells or T or B cells. Non-limiting examples of support cells that are not constituted by the differentiation methods described herein include, but are not limited to, stromal cells and fibroblasts.

[0312] Feeder cells previously used in co-culture for cell differentiation purposes are typically stromal cells. However, the methods described herein do not include co-cultures containing stromal cells. Examples of stromal cell lines not encompassed by the differentiation methods described herein include, but are not limited to, the mouse MS5 stromal cell line, mouse bone marrow-derived stromal cell lines such as S10, S17, OP9 (e.g., OP9-DL1 or OP9-DL4 cells), and the BMS2 cell line, human bone marrow stromal cell lines such as those described in U.S. Patent No. 5,879,940 (incorporated herein by reference in its entirety), or any other similar cells that express and present extracellular Notch ligands or secrete Notch ligands. OP9-DL1 cells are a bone marrow-derived stromal cell line that ectopically expresses the Notch ligand, Delta-like 1 (DLL1). Methods for differentiating pluripotent stem cells into T cells using OP9-Notch ligand-expressing cells are known in the art. See, e.g., U.S. Patent Nos. 7,575,925, 8,772,028, 8,871,510, and 9,206,394, and U.S. Patent Publication Nos. 2009 / 0217403, 2011 / 0123502, 2011 / 0052554, 2011 / 0027881, 2011 / 0236363, 2012 / 0149100, 2013 / 0281304, 2014 / 0322808, 2014 / 0248248, and 2014 / 0037599. These references are incorporated herein by reference in their entireties.

[0313] Described herein are methods for differentiating T cells from pluripotent stem cells, the methods not including co-culturing the cells with feeder or stromal cells. In some embodiments, the soluble Notch ligand used herein is not derived from stromal cells. In some embodiments, differentiating hemogenic endothelium in the presence of a soluble Notch ligand does not include co-culturing with stromal cells that express a Notch ligand. In some embodiments, differentiating hemogenic endothelium in the presence of a soluble Notch ligand does not include co-culturing with OP9-DL1 or OP9-DL4 cells.

[0314] T cell differentiation medium In some embodiments, the differentiation method comprises differentiating the resulting population of CD34+ hemogenic endothelium in CD3+ T cell differentiation medium for a time sufficient to promote differentiation into a population of CD3+ T cells. In some embodiments, the time sufficient 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. In some embodiments, the time sufficient to promote differentiation into a population of CD3+ T cells is at most 6 weeks.

[0315] In some embodiments, polypeptides (e.g., growth factors or differentiation factors) that can be expressed by support cells or stromal cells can be provided in the cell culture medium. Non-limiting examples of polypeptides that support T cell differentiation that can be included in the cell culture medium include IL-7, SCF, Flt3, and TPO. Interleukin-7 (IL-7) is a hematopoietic growth factor secreted by stromal cells in the bone marrow and thymus and is involved in the development of B cells and T cells. Stem cell factor (also known as SCF, KIT ligand, KL, or steel factor) is a cytokine that binds to the c-KIT receptor (CD117) and is involved in T cell differentiation. FLT3 (also called Flit3 or Fms-like tyrosine kinase 3) is a class III receptor tyrosine kinase that regulates hematopoiesis. Thrombopoietin (TPO or THPO) is a cytokine that is primarily involved in megakaryocytopoiesis but also plays a role in maintaining hematopoietic stem cells (HSCs). See, e.g., Wang et al., Distinct roles of IL-7 and stem cell factor in the OP9-DL1 T cell differentiation culture system. Exp Hematol. 2006 Dec;34(12):1730-40.

[0316] In some embodiments, the CD3+ T cell differentiation medium is serum-free. In some embodiments, the CD3+ T cell differentiation medium comprises at least one of SCF, FLT3, and / or IL7. In some embodiments, the CD3+ T cell differentiation medium comprises SCF, FLT3, and IL7. In some embodiments, the CD3+ T cell differentiation medium comprises 30 ng / ml SCF, 15 ng / ml FLT3, and 25 ng / ml IL7. In some embodiments, the CD3+ T cell differentiation medium comprises 100 ng / ml SCF, 100 ng / ml FLT3, and 50 ng / ml IL7. In some embodiments, the CD3+ T cell differentiation medium comprises FLT3 and IL7. In some embodiments, the CD3+ T cell differentiation medium comprises 15 ng / ml FLT3 and 25 ng / ml IL7. In some embodiments, the CD3+ T cell differentiation medium comprises 100 ng / ml FLT3 and 50 ng / ml IL7.

[0317] The concentrations of SCF, FLT3, and / or IL7 should be adjusted so 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. In some embodiments, the concentration of SCF (e.g., in CD3+ T cell differentiation medium) is 30 ng / mL. In some embodiments, the concentration of SCF (e.g., in CD3+ T cell differentiation medium) is 100 ng / mL. The concentration of FLT3 can range from 1 ng / mL to 200 ng / mL. In some embodiments, the concentration of FLT3 (e.g., in CD3+ T cell differentiation medium) is 15 ng / mL. In some embodiments, the concentration of FLT3 (e.g., in CD3+ T cell differentiation medium) is 100 ng / mL. The concentration of IL7 can range from 1 ng / mL to 200 ng / mL. In some embodiments, the concentration of IL7 (e.g., in CD3+ T cell differentiation medium) is 25 ng / mL. In some embodiments, the concentration of IL7 (e.g., in CD3+ T cell differentiation medium) is 50 ng / ml.

[0318] In some embodiments, the CD3+ T cell differentiation medium further comprises thrombopoietin (TPO) for at least the first two weeks of differentiation in the CD3+ T cell differentiation medium. By way of non-limiting example, the CD3+ T cell differentiation medium further comprises thrombopoietin (TPO) for at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days of differentiation in the CD3+ T cell differentiation medium. In some embodiments, the CD3+ T cell differentiation medium comprising TPO promotes differentiation into a population of CD5+CD7+ ProT cells. Such CD5+CD7+ ProT cells can be detected after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days of differentiation in CD3+ T cell differentiation medium. In some embodiments, CD5+CD7+ ProT cells can be detected after at least 2 weeks of differentiation in CD3+ T cell differentiation medium.

[0319] In some embodiments, a concentration of TPO should be used to promote differentiation of hemogenic endothelium into a population of CD3+ T cells. In some embodiments, the concentration of TPO can range from 1 ng / mL to 200 ng / mL. In some embodiments, the concentration of TPO (e.g., in CD3+ T cell differentiation medium) is 5 ng / mL. In some embodiments, the concentration of TPO (e.g., in CD3+ T cell differentiation medium) is 50 ng / mL.

[0320] In some embodiments, the CD3+ T cell differentiation medium (e.g., comprising IL-7 and / or FLT3) further comprises SCF for at least the first two weeks of differentiation in the CD3+ T cell differentiation medium. As a non-limiting example, the CD3+ T cell differentiation medium further comprises SCF for at least the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days of differentiation in the CD3+ T cell differentiation medium. In some embodiments, the CD3+ T cell differentiation medium comprising SCF promotes differentiation into a population of CD5+CD7+ ProT cells.

[0321] In some embodiments, SCF, FLT3, IL7, and / or TPO are present in the CD3+ T cell differentiation medium 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 ng / mL, at least 45 ng / mL, at least 50 ng / mL, at least 55 ng / mL, at least 60 ng / mL, at least 65 ng / mL, at least 70 ng / mL, at least 7 ... at least 65 ng / mL, at least 70 ng / mL, at least 75 ng / mL, at least 80 ng / mL, at least 85 ng / mL, at least 90 ng / mL, at least 95 ng / mL, at least 100 ng / mL, at least 105 ng / mL, at least 110 ng / mL, at least 115 ng / mL, at least 120 ng / mL, at least 125 ng / mL, at least 130 ng / mL, at least 135 ng / mL, at least 140 ng / mL, at least 145 ng / mL, at least 150 ng / mL, at least 155 ng / mL, at least 160 ng / mL, at least 165 ng / mL, at least 170 ng / mL, at least 175 ng / mL, at least 180 ng / mL, at least 185 ng / mL, at least 190 ng / mL, at least 195 ng / mL, or at least 200 ng / mL. The concentrations of SCF, FLT3, IL7, and / or TPO may be the same or different.

[0322] In some embodiments, CD3+ T cells can be detected after at least 5.0 weeks of differentiation in CD3+ T cell differentiation medium. In some embodiments, CD3+ T cells can be detected after at least 1.5, 2, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 weeks of differentiation in CD3+ T cell differentiation medium. 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, 2, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 weeks of differentiation in CD3+ T cell differentiation medium.

[0323] In some embodiments, the method further comprises differentiating the population of CD4+CD8+ T cells in single positive T cell differentiation medium for a time sufficient to promote differentiation into a population of CD4+ cells and a population of CD8+ cells. In some embodiments, the time sufficient to promote differentiation of 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. In some embodiments, the time sufficient to promote differentiation of 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.

[0324] In some embodiments, the single-positive T cell differentiation medium contains 10 ng / ml of IL-15 and a T cell activator. Interleukin-15 (IL-15), like IL-7, is a member of the interleukin-2 (IL-2) superfamily and shares many activities with IL-2, including the ability to stimulate lymphocytes. In some embodiments, various concentrations of IL-15 can be used as long as they still promote the differentiation of CD4+CD8+ T cells into single-positive CD4+ and CD8+ cells. In some embodiments, the concentration of IL-15 can range from 1 ng / ml to 200 ng / ml, with a preferred concentration being 10 ng / ml.

[0325] In some embodiments, the T cell activator comprises a component (e.g., a soluble tetrameric antibody complex) that binds to the CD3 and CD28 (and optionally CD2) cell surface ligands. Binding of the T cell activator results in cross-linking of the CD3 and CD28 (and optionally CD2) cell surface ligands, thereby providing the primary and costimulatory signals necessary for T cell activation.

[0326] In some embodiments, the T cell activator comprises a CD3 / CD28 T cell activator (e.g., at a concentration of 10 μl / ml). Such CD3 / CD28 T cell activators are commercially available (e.g., through StemCell Technology™, item #10970). In some embodiments, the concentration of CD3 / CD28 T cell activator should be used to promote differentiation of CD4+CD8+ T cells into single positive CD4+ and CD8+ cells. In some embodiments, the concentration can range from 1 μl / ml to 200 μl / ml, with a preferred concentration being 10 μl / ml.

[0327] In some embodiments, the T cell activator comprises CD3 / CD28 T cell activator Dynabeads (e.g., used at one bead per cell). Such CD3 / CD28 T cell activator Dynabeads are commercially available (e.g., via ThermoFisher™, #11132D). In some embodiments, a concentration of CD3 / CD28 T cell activator Dynabeads should be used to promote differentiation of CD4+CD8+ T cells into single positive CD4+ and CD8+ cells. In some embodiments, the concentration can range from 1 bead / cell to 20 beads / cell, with a preferred concentration being 1 bead / cell.

[0328] In some embodiments, the method further comprises a CD4+ cell enrichment and / or CD8+ cell enrichment step after at least one week (e.g., in single-positive T cell differentiation medium). In some embodiments, the CD4+ cell enrichment and / or CD8+ cell enrichment step may occur after 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 culture in single-positive T cell differentiation medium.

[0329] Methods for enriching CD4+ or CD8+ cells are known in the art. As non-limiting examples, CD4+ or CD8+ cells can be enriched using anti-CD4 or anti-CD8 antibodies using magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS).

[0330] In some embodiments, the entire T cell differentiation protocol described herein is performed in a stroma-free environment, e.g., cells are cultured exposed to a non-stroma-derived Notch ligand (e.g., a Notch ligand immobilized on a tissue culture plate). In some embodiments, at least a portion of the T cell differentiation protocol (e.g., CD3 +The differentiation of cells (including culture in T cell differentiation medium and single positive T cell differentiation medium) occurs in a stroma-free environment, e.g., the cells are cultured exposed to a non-stroma-derived Notch ligand (e.g., a Notch ligand immobilized on a tissue culture plate).

[0331] Induced T cell populations As described herein, using the stroma-free method, and in one embodiment, in combination with inhibition of epigenetic regulators (e.g., HMTs, e.g., EZH1, G9a / GLP), the induced population of T cells exhibits at least the following unexpected advantages compared to the stroma co-culture method: (1) increased likelihood of engraftment in humans, (2) reduced numbers of innate-like T cells, (3) increased numbers and / or percentages of resulting 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) a gene expression profile most similar to alpha-beta T cells, (5) a more diverse TCR repertoire, and / or (6) increased TCR CDR lengths.

[0332] In some embodiments, populations of T cells (e.g., CD3+ T cells, CD4+CD8+ T cells, CD4+ T cells, CD8+ T cells) induced using the stromal-free methods and / or inhibition of an epigenetic regulator (e.g., an HMT, e.g., EZH1, G9a / GLP) described herein exhibit a transplantation or engraftment rate that is at least 10% higher than populations of T cells induced using stromal methods. In some embodiments, populations of T cells induced using the stromal-free methods and / or inhibition of an epigenetic regulator described herein exhibit a transplantation or engraftment rate that is 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 60%, at least 75%, at least 80%, at least 95%, at least 100%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50 ... exhibiting 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 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold or more increased engraftment or survival rates.

[0333] In some embodiments, a minority of the populations of T cells (e.g., CD3+ T cells, CD4+CD8+ T cells, CD4+ T cells, CD8+ T cells) induced using the stromal-free methods and / or inhibition of epigenetic regulators described herein are TCRgd +Gamma delta T cells (γδ T cells) are T cells with a unique T cell receptor (TCR) on their surface. Most T cells are αβ (alpha beta) T cells, with a TCR composed of two glycoprotein chains called α (alpha) and β (beta) TCR chains. In contrast, gamma delta (γδ) T cells have a TCR composed of one γ (gamma) chain and one δ (delta) chain. Like other "non-conventional" T cell subsets with invariant TCRs, such as CD1d-restricted natural killer T cells, gamma delta T cells exhibit several properties that place them at the boundary between the more evolutionarily primitive innate immune system, which allows rapid, beneficial responses to a variety of foreign agents, and the adaptive immune system, in which B and T cells orchestrate a slower, highly antigen-specific immune response that leads to long-term memory against subsequent challenges with the same antigen. Gamma delta T cells can be considered a component of adaptive immunity in that they can rearrange TCR genes to generate junctional diversity and develop a memory phenotype. However, various subsets can also be considered part of innate immunity, where specific TCRs can function as pattern recognition receptors. See, e.g., Born WK, Reardon CL, O'Brien RL (February 2006). "The function of gamma delta T cells in innate immunity." Current Opinion in Immunology. 18(1):31-8.

[0334] In some embodiments, at most 10% of the population of T cells (e.g., CD3+ T cells, CD4+CD8+ T cells, CD4+ T cells, CD8+ T cells) induced using the stromal-free methods and / or inhibition of epigenetic regulators described herein are TCRgd +In some embodiments, 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 30%, at most 31%, at most 32%, at most 33%, at most 34%, at most 35%, at most 36%, at most 37%, at most 38%, at most 39%, at most 40%, at most 41%, at most 42%, at most 43%, at most 44%, at most 45%, at most 46%, at most 47%, at most 48%, at most 49%, at most 50%, at most 51%, at most 52%, at most 53%, at most 54%, at most 55%, at most 56%, at most 57%, at most 58%, at most 59%, at most 60%, at most 61%, at most 62%, at most 63%, at most 64%, at most 65%, at most 66%, at most 67%, at most 68%, at most 69%, at most 70%, at most 71%, at most 72%, at most 73%, at most 74%, at most 75%, at most 76%, at most 77%, at most 78%, at most 79%, at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, %, 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%, at most 36%, at most 37%, at most 38%, at most 39%, at most 40%, at most 41%, at most 42%, at most 43%, at most 44%, at most 45%, at most 46%, at most 47%, at most 48%, or at most 49% are TCRgd + is.

[0335] In some embodiments, a population of T cells (e.g., CD3+ T cells, CD4+CD8+ T cells, CD4+ T cells, CD8+ T cells) induced using the stromal-free methods and / or inhibition of epigenetic regulators described herein contains at least 10% more T cells than a population of T cells induced using the stromal methods or without inhibition of epigenetic regulators. In some embodiments, a population of T cells induced using the stromal-free methods and / or inhibition of epigenetic regulators described herein contains 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 5% more T cells than a population of T cells induced using the stromal-free methods or without inhibition of epigenetic regulators. 0%, 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 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 500x, 1,000x or more T cells.

[0336] In some embodiments, populations of T cells (e.g., CD3+ T cells, CD4+CD8+ T cells, CD4+ T cells, CD8+ T cells) induced using the stromal-free methods and / or inhibition of epigenetic regulators described herein exhibit a gene expression profile that is more similar to αβ T cells than to other cells (e.g., γδ T cells, NK cells, T cells derived from iPSCs using the OP9-DL4 co-culture system, T cells differentiated from umbilical cord blood CD34+ HSPCs), e.g., the gene profile of the induced T cells is at least 0.5% more similar to αβ T cells compared to another cell type. In one embodiment, populations of T cells induced using the stromal-free methods and / or inhibition of epigenetic regulators described herein exhibit a gene expression profile of T cell signature genes and / or αβ T cell signature genes that differs by at most 10% from the gene expression profile of αβ T cells. In one embodiment, a population of T cells derived using the stroma-free methods and / or inhibition of epigenetic regulators described herein exhibits a gene expression profile of T cell signature genes and / or αβT signature cell genes that differs by 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) from the gene expression profile of αβT cells. In one embodiment, a population of T cells induced using the stroma-free methods and / or inhibition of epigenetic regulators described herein exhibits a gene expression profile of T cell signature genes and / or αβ T cell signature genes that differs from the gene expression profile of αβ T cells by 1% to 5%, 2% to 6%, 3% to 7%, 4% to 8%, 5% to 9%, 5% to 10%, 5% to 15%, 10% to 15%, or 15% to 20%.

[0337] In one embodiment, a population of T cells derived using the stromal-free methods and / or inhibition of epigenetic regulators described herein exhibits a gene expression profile 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%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, The induced T cells exhibit gene expression profiles that are 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more similar. In one embodiment, the induced T cells have a greater percentage similarity to the gene expression profile of αβ T cells than to the gene profile of another cell type. One skilled in the art can determine the similarity of gene expression of T cells and αβ T cells derived from the stroma-free methods described herein using standard methods, for example, transcriptome sequencing (FACS-sorted cells) of a particular cell type.

[0338] In one embodiment, the population of T cells induced using the stroma-free methods and / or inhibition of epigenetic regulators described herein has a T cell population of 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, 0.835, 0.84, 0.845, 0.85, 0.855, 0.86, 0.865 , 0.87, 0.875, 0.88, 0.885, 0.89, 0.895, 0.9, 0.905, 0.91, 0.915, 0.92, 0.925, 0.93, 0.935, 0.94, 0.945, 0.95, 0.955, 0.96, 0.965, 0.97, 0.975, 0.98, 0.985, 0.99, 0.995, or 1.0.

[0339] In some embodiments, 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 similar or substantially similar to alpha beta T cells. + The population of 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 that has a Pearson correlation coefficient compared to peripheral blood alpha beta T cells that is at least 0.85.

[0340] In some embodiments, immune cells induced using, for example, stroma-free and / or epigenetic regulator inhibition described herein, exhibit a gene expression profile most similar to alpha beta T cells. In some embodiments, the immune cells exhibit a gene expression profile similar or substantially similar to alpha beta T cells. In some embodiments, the immune cells exhibit 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 cells exhibit a gene expression profile that has a Pearson correlation coefficient compared to peripheral blood alpha beta T cells that is at least 0.85.

[0341] In some embodiments, the population of T cells induced using the stroma-free methods and / or inhibition of epigenetic regulators described herein is 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 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 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, At least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, 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, at least 100, at least 125,The induced T cells express at least 150 or more signature genes from αβ T cells. In one embodiment, the induced T cells express a greater number of signature genes from αβ T cells than from another cell type. As used herein, the term "signature gene" refers to a gene that exhibits a characteristic expression pattern in a particular cell type (e.g., T cells, αβ T cells), and signature genes may be required for the function of the particular cell type. Non-limiting examples of T cell signature genes and αβ T cell signature genes are further described herein. Particular cell types (e.g., T cells, αβ T cells) exhibit gene signatures or gene expression signatures that include a single or combined group of genes within the cell that have a unique, characteristic pattern of gene expression (i.e., signature genes).

[0342] In some embodiments, the population of T cells induced using the stroma-free methods and / or inhibition of epigenetic regulators described herein is 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 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 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 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, expressing genes from at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, 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, at least 100, at least 125, at least 150 or more αβ T cells.In one embodiment, the induced T cells express a greater number of genes from αβ T cells than signature genes from another cell type.

[0343] Non-limiting examples of 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), RAF1 (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), SHC1 (SHC (Src homology 2 domain-containing) adaptor protein 1), PRKCB (protein kinase C beta), MAP2K4 (map mitogen-activated protein kinase kinase 4), MAP2K1 (mitogen-activated protein kinase kinase 1), RAC1 (Rac family small GTPase 1), FYN (Fyn proto-oncogene, Src family tyrosine kinase), RELA (RELA proto-oncogene, NF-KB subunit, v-rel avian reticuloendotheliosis virus oncogene homolog A), LCK (Lck proto-oncogene, Src family tyrosine kinase), CALM2 (calmodulin 2), CD3D (CD3 antigen, delta subunit), CALM1 (calmodulin 1), CD247 (T cell surface glycoprotein CD3 zeta chain), CD3E (T cell surface glycoprotein CD3 epsilon chain), CD3G (T cell surface glycoprotein CD3 gamma chain), FOS (Fos proto-oncogene, AP-1 transcription factor subunit), PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), PLCG1 (phospholipase C gamma 1), SOS1 (son of sevenless homolog 1, SOS Ras / Rac guanine nucleotide exchange factor 1), ELK1 (ETS transcription factor ELK1), PPP3CC (protein phosphatase 3 catalytic subunit gamma), MAP3K1 (mitogen-activated protein kinase kinase kinase 1), PPP3CA (protein phosphatase 3 catalytic subunit alpha), NFKB1 (nuclear factor kappa B subunit 1), NFATC2 (nuclear factor of activated T cells 2), NFATC1 (nuclear factor of activated T cells 1, AP-1 transcription factor subunit), JUN (Jun proto-oncogene), MAPK8 (mitogen-activated protein kinase 8), RASA1 (RAS These include P21 protein activator 1, PPP3CB (protein phosphatase 3 catalytic subunit beta), PRKCA (protein kinase C alpha), MAPK3 (mitogen-activated protein kinase 3), and NFATC4 (nuclear factor of activated T cells 4).

[0344] Non-limiting examples of αβ T cell signature genes include ATP11B (ATPase phospholipid transport 11B), PPP4R3A (protein phosphatase 4 regulatory subunit 3A), CAB39 (calcium-binding protein 39), GLS (glutaminase), UBE2Z (ubiquitin-conjugating enzyme E2Z), INPP4A (inositol polyphosphate-4-phosphatase type IA), RAB22A (Ras-related protein Rab-22A, member of the Ras oncogene family), SMARCD2 (SWI / SNF (SWItch / sucrose non-fermenting)-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), ESYT2 (extended synaptotagmin 2), RAC1 (Rac1, family small GTPase 1), EIF3B (eukaryotic translation initiation factor 3 subunit B), NEK7 (NIMA (never in mitosis gene A)-related kinase 7), MDFIC (MyoD (myoblast determining protein 1) family inhibitor domain containing), YWHAH (tyrosine 3-monooxygenase / tryptophan 5-monooxygenase-activating protein eta), MCMBP (minichromosome maintenance complex-associated protein), GOLPH3 (golgi phosphoprotein 3), PTGER4 (prostaglandin E receptor 4), B3GNT2 (UDP-GlcNAc:beta-Gal beta-1,3-N-acetylglucosaminyltransferase 2, galactosyltransferase 7), PITPNC1 (phosphatidylinositol transfer protein cytoplasmic 1), ARAP2 (ArfGAP with RhoGAP domain, ankyrin repeats, and PH domain 2, Arf and Rho GAP adaptor protein 2), ZFP36L2 (zinc finger protein 36, C3H1-type like 2), EFHD2 (EF-hand domain family member D2, swiplosin-1), CPD (carboxypeptidase D), KLRB1 (killer cell lectin-like receptor B1), DUSP1 (dual specificity phosphatase 1), CMPK1 (cytidine / uridine monophosphate kinase 1), RASGRP1 (Ras guanyl-releasing protein 1),TM9SF3 (Transmembrane 9 Superfamily Member 3), MAPK1 (Mitogen-Activated Protein Kinase 1), GSPT1 (G1 to S Phase Transition 1), PNRC1 (Proline-Rich Nuclear Receptor Coactivator 1), TMEM248 (Transmembrane Protein 248), STT3B (STT3 (staurosporine and temperature-sensitive) oligosaccharyltransferase complex catalytic subunit B), KHDRBS1 (KH (K Homology) RNA-Binding Domain-Containing, Signal Transduction Associated 1), GNPTAB (N-Acetylglucosamine-1-phosphate dehydrogenase) transferase subunits alpha and beta), GRSF1 (G-rich RNA sequence-binding factor 1), TARP (TCR gamma alternative reading frame protein, T cell receptor gamma chain), ZBTB16 (zinc finger and BTB (for BR-C, ttk, bab) domain-containing 16, zinc finger protein 145 (Kruppel-like, expressed in promyelocytic leukemia)), TGFBR1 (transforming growth factor beta receptor 1), LGALS3BP (galectin 3-binding protein), CD5 (T cell surface glycoprotein CD5), CD 4 (T cell surface glycoprotein CD4), LRRN3 (leucine-rich repeat neuronal 3), SLC40A1 (solute carrier family 40 member 1), CYSLTR1 (cysteinyl leukotriene receptor 1), H4C3 (H4 clustered histone 3), CISH (cytokine-inducible SH2 (Src homology 2)-containing protein), CD8B (T cell surface glycoprotein CD8 beta chain), MAL (Mal, T cell differentiation protein, myelin and lymphocyte protein), SUN2 (Sad1 and Unc84 domain-containing 2, Rab5 interacting protein) protein), CCR7 (CC motif chemokine receptor 7), GNLY (granulysin), ANKLE2 (ankyrin repeat and LEM (LAP2, emerin, MAN1) domain containing 2), PSIP1 (PC4 (positive cofactor 4) and SFRS1 (serine and arginine-rich splicing factor 1) interacting protein 1, lens epithelium-derived growth factor), PITPNA (phosphatidylinositol transfer protein alpha), RBM15B (RNA-binding motif protein 15B), PTPRA (protein tyrosine phosphatase receptor type A),MARK2 (microtubule affinity-regulating kinase 2), BLOC1S4 (biogenesis subunit of lysosomal organelle complex 1 4), SIAH2 (Siah E3 ubiquitin protein ligase 2), MXD4 (maximal dimerization protein 4), SRM (spermidine synthase), SESN1 (sestrin 1), SSBP4 (single-stranded DNA-binding protein 4), TAF10 (TATA box-binding protein-associated factor 10), DUSP2 (dual specificity phosphatase 2), LPCAT1 (lysophosphatidylcholine acyltransferase 1), RASAL3 (Ras protein activator-like 3), TRIM65 (tripartite motif-containing 65), FAM50A (with sequence similarity 50 Family member A with sequence similarity to 89), PIM3 (Pim-3 proto-oncogene, serine / threonine kinase), SIPA1 (signal-induced proliferation associated 1), FAM89B (family member B with sequence similarity to 89), ZBTB7A (zinc finger and BTB (for BR-C, ttk, bab) domain-containing 7A, short transcript inducer-binding factor protein 1), NIN (nine in), NR1D2 (nuclear receptor subfamily 1 group D member 2), SIK3 (salt-inducible kinase 3), ARHGAP26 (Rho GTPase-activating protein 26), IL18RAP (interleukin-18 receptor accessory protein), CNR2 (cannabinoid receptor 2), EOMES (eomesodermin), KLRC1 (killer cell lectin-like receptor C1), SEL1L3 (suppressor of phospho-12-like protein 3), IL12RB2 (interleukin-12 receptor subunit beta 2), COTL1 (coactosin-like F-actin-binding protein 1), PIK3AP1 (phosphoinositide-3-kinase adapter) Adapter protein 1), TBX21 (T-box transcription factor 21), FAM43A (family member A with sequence similarity 43), KLRD1 (killer cell lectin-like receptor D1), SLAMF7 (signaling lymphocyte activation molecule (SLAM) family member 7), S1PR5 (sphingosine-1-phosphate receptor 5), LAG3 (lymphocyte activation 3), ABCG1 (ATP-binding cassette subfamily G member 1), S100B (S100 calcium-binding protein, beta),These include CCL22 (CC motif chemokine ligand 22), CEBPD (CCAAT box enhancer binding protein delta), IL17F (interleukin 17F), and CEACAM1 (CEA cell adhesion molecule 1).

[0345] In some embodiments, populations of T cells induced using the stromal-free methods and / or inhibition of epigenetic regulators described herein exhibit a more diverse TCR repertoire compared to T cells not induced using such stromal-free methods or without inhibition of epigenetic regulators. In some embodiments, populations of T cells induced using the stromal-free methods and / or inhibition of epigenetic regulators described herein exhibit a Productivity-Simpson clonality value of about 0.000 to 0.025. Values ​​closer to 0 represent a higher level of diversity relative to clonality. Values ​​closer to 1 represent a higher level of clonality relative to diversity. In some embodiments, populations of T cells induced using the stroma-free methods and / or inhibition of epigenetic regulators described herein exhibit a productivity 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. In some embodiments, populations of T cells induced using the stroma-free methods and / or inhibition of epigenetic regulators described herein exhibit a productivity Simpson clonality value of about 0.025.

[0346] The variable domains of both the T cell receptor (TCR) α and β chains each have three hypervariable or complementarity determining regions (CDRs, e.g., CDR1, CDR2, CDR3). In some embodiments, populations of T cells induced using the stromal-free methods and / or inhibition of epigenetic regulators described herein exhibit increased CDR (e.g., CDR1, CDR2, CDR3) lengths compared to T cells induced using the stromal methods or without inhibition of epigenetic regulators. In some embodiments, populations of T cells induced using the stromal-free methods and / or inhibition of epigenetic regulators described herein exhibit CDR (e.g., CDR1, CDR2, CDR3) lengths that are, on average, about 3 nucleotides (nt), 6 nt, 9 nt, or 12 nt or more longer than the CDRs of T cells induced using the stromal methods or without inhibition of epigenetic regulators. In some embodiments, populations of T cells induced using the stroma-free methods and / or inhibition of epigenetic regulators described herein exhibit CDR (e.g., CDR1, CDR2, CDR3) lengths that 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 more. In some embodiments, populations of T cells induced using the stroma-free methods and / or inhibition of epigenetic regulators described herein exhibit CDR3 lengths that average about 42 nt in length, compared to an average of 39 nt for control iPSC-derived T cells or an average of 45 for peripheral blood mononuclear cell (PBMC)-derived T cells.

[0347] Genetic modification of T cells In some embodiments, the resulting population of CD34+ hemogenic endothelium or another population described herein (e.g., ESCs, iPSCs, HSCs, CD5+CD7+ ProT cells, CD3+ T cells, CD4+CD8+ T cells, CD4+ T cells, CD8+ T cells) is genetically modified. In some embodiments, native T cell receptor loci can be removed and / or replaced to enhance targeted specificity. In some embodiments, endogenous HLA (e.g., class I and / or class II major histocompatibility complex) can be edited or removed. In some embodiments, genetic modification can include the 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 immunotherapy.

[0348] In some embodiments, the genetic modification comprises expressing a chimeric antigen receptor (CAR). Chimeric antigen receptors (also known as CARs, chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors) are receptor proteins engineered to confer new capabilities to T cells to target specific proteins. These receptors are chimeric because they combine both antigen binding and T cell activation functions in a single receptor. Methods for engineering chimeric antigen receptor T cells (also known as CAR T cells) are known in the art. For example, U.S. Patent Nos. 7,446,190, 8,399,645, 8,822,647, 9,212,229, 9,273,283, 9,447,194, 9,587,020, 9,932,405, 10,125,193, 10,221,245, 10,273,300, 10,287,354, U.S. Patent Publication No. 2016 / 0152723, PCT Publication No. 2009 / 091826, PCT Publication No. 2012 / 079000, PCT Publication No. 2014 / 165707 , 2015 / 164740, 2016 / 168595(A1), 2017 / 040945, 2017 / 100428, 2017 / 117112, 2017 / 149515, 2018 / 067992, 2018 / 102787, 2018 / 102786, 2018 / 165228, and 2019 / 084288, the contents of each of which are incorporated herein by reference in their entirety.

[0349] In some embodiments, methods of genetically modifying cells to express a CAR can include, but are not limited to, transfection or electroporation of cells with a vector encoding a CAR, transduction with a viral vector encoding a CAR (e.g., retrovirus, lentivirus), gene editing using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganuclease-TALENs, or CRISPR-Cas, or any other method known in the art for genetically modifying cells to express a CAR.

[0350] Preferably, a population of cells at an early stage of differentiation (e.g., ESCs, PSCs, iPSCs, hemogenic endothelium, HSCs) is genetically modified with a CAR.

[0351] In some embodiments, 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, an autoimmune disease, or a heart disease (e.g., cardiac fibrosis). As used herein, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide uncontrollably and can invade nearby tissues. Cancer cells can also spread to other parts of the body via the blood and lymphatic system. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or 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 tissues. Leukemia is a cancer that begins in blood-forming tissues, 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 cells of the immune system. Central nervous system cancer is a cancer that begins in the tissues of the brain and spinal cord.

[0352] In some embodiments, the cancer is a primary cancer. In some embodiments, the cancer is a malignant cancer. As used herein, the term "malignant" refers to a cancer in which a group of tumor cells exhibit one or more of the following: uncontrolled growth (i.e., dividing beyond normal limits), invasion (i.e., invading and destroying adjacent tissue), and metastasis (i.e., spreading to other parts of the body via the lymph or blood). As used herein, the term "metastasis" refers to the spread of cancer from one part of the body to another. Tumors formed by spread cells are called "metastatic tumors" or "metastases." Metastatic tumors contain cells similar to those in the original (primary) tumor. As used herein, the terms "benign" or "non-malignant" refer 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.

[0353] "Cancer cell" or "tumor cell" refers to an individual cell of a cancerous growth or tissue. A tumor generally refers to a swelling or lesion formed by the abnormal proliferation 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.

[0354] As used herein, the term "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 normal tissue. Thus, a tumor can be benign, premalignant, or malignant.

[0355] A subject with cancer or tumor is one who has objectively measurable cancer cells in his or her body. This definition includes malignant, actively growing cancers as well as potentially dormant tumors or micrometastases. Cancers that migrate from their original location and disseminate to other vital organs can ultimately lead to the death of the subject through functional deterioration of the affected organ.

[0356] Examples of cancer include carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma (GBM), hepatocellular carcinoma, hepatoma, intraepithelial neoplasia, kidney cancer, and laryngeal cancer. cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), lymphoma including Hodgkin's lymphoma and non-Hodgkin's lymphoma, melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer Cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer, and other carcinomas and sarcomas, as well as B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, post-transplant lymphoproliferative disorder (PTLD), and abnormal blood vessel proliferation associated with phacomatosis, edema (such as that associated with brain tumors), and Meigs' syndrome. Preferably, for CAR T therapy, the cancer is a blood cancer such as leukemia or lymphoma.

[0357] Immunotherapy with chimeric antigen receptor (CAR) T cells offers a promising method for improving cure rates and reducing morbidity in patients with cancer. In this regard, CD19-specific CAR T cell therapy has achieved dramatic objective responses in a high percentage of patients with CD19-positive leukemia or lymphoma. Thus, in some embodiments, the antigen-binding region of the CAR is directed against CD19; see, e.g., U.S. Pat. Nos. 10,221,245, 10,357,514, U.S. Patent Publication No. 2016 / 0152723, and PCT Publication No. 2016 / 033570, the contents of each of which are incorporated herein by reference in their entirety.

[0358] Tumor antigens are proteins produced by tumor cells that induce immune responses, particularly T cell-mediated immune responses. The selection of the antigen-binding domain of the present invention depends on the specific type of cancer to be treated. Tumor antigens are known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), EGFRvIII, IL-11Ra, IL-13Ra, EGFR, B7H3, Kit, CA-IX, CS-1, MUC1, BCMA, bcr-abl, HER2, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), ALK, CD19, CD123, cyclin B1, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RU1, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAX5, SART3, CLL-1, fucosyl-GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, polysialic acid, PLAC1, RU1, RU2 (AS), intestinal carboxylesterase, Lewis Y, sLe, LY6K, mutated hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYP1B1, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-1a, LMP2, NCAM, p53, p53 mutant, Ras mutant, gp100, prostein, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, legumain, HPV E6, E7, survivin and telomerase, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate cancer tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAD-CT-1, MAD-CT-2, MelanA / MART1, XAGE1, ELF2M, ERG (TMPRSS2ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoint, NY-BR-1, ephrin B2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. In preferred embodiments, 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., U.S. Patent Publication Nos. 2017 / 0209492 and 2018 / 0022795, the contents of each of which are incorporated by reference in their entirety.

[0359] cell replacement therapy In one embodiment, provided herein is a population of engineered immune cells produced by the methods described herein, wherein the population of T cells is produced using the stromal-free differentiation methods described herein. In some embodiments, the population of engineered immune cells comprises immune cells differentiated using the methods described herein, including, but not limited to, PSCs, iPSCs, hemogenic endothelial cells, HSCs, CD5+CD7+ProT cells, CD3+ T cells, CD4+CD8+ T cells, CD4+ T cells, and CD8+ T cells. In some embodiments, the immune cells exhibit a gene expression profile most similar to alpha-beta T cells.

[0360] In one embodiment, the population of cells further comprises a pharmaceutically acceptable carrier. These engineered immune cells can be cultured and expanded to increase the number of cells used.

[0361] The engineered immune cells described herein are useful in laboratories for biological research. For example, these cells can be derived from individuals with a genetic disease or deficiency and used in laboratories to study the biology of the disease or deficiency and to screen and test potential remedies for the disease or deficiency.

[0362] Alternatively, the engineered immune cells described herein are useful for cell replacement therapy and other medical treatments in subjects in need, such as patients undergoing chemotherapy or radiation or both, who have significant deficiencies in immune function and / or lymphocyte reconstitution, or cancer immunotherapy.

[0363] In various embodiments, the engineered immune cells described herein are administered (i.e., implanted or transplanted) to a subject in need of cell replacement therapy.

[0364] In one embodiment, provided herein is a method of cell replacement therapy or a method for treating cancer, an autoimmune disorder, a hematological disease, or other genetic diseases and disorders in a subject, the method comprising: (a) providing somatic cells from a donor subject; (b) generating multilineage hematopoietic progenitor cells (e.g., hemogenic endothelial, HSPC) from somatic cell-derived pluripotent stem cells as described in any of the preceding paragraphs; (c) optionally inhibiting histone methyltransferase in the resulting population of multilineage hematopoietic progenitor cells as described in any of the preceding paragraphs; (d) differentiating the resulting population of multilineage hematopoietic progenitor cells in the presence of a Notch ligand to promote differentiation into lymphoid lineages (e.g., T cells) as described in any of the preceding paragraphs; and (e) transplanting or administering the resulting differentiated lymphoid cells to a recipient subject.

[0365] In one embodiment, the host subject and recipient subject are the same individual. Alternatively, the host subject and recipient subject are not the same individual, but are at least HLA-compatible.

[0366] Hematological disorders are diseases that primarily affect the blood. Non-limiting examples of such diseases or disorders include bone marrow-derived disorders such as hemoglobinopathies (inborn errors of the hemoglobin molecule or the rate of hemoglobin synthesis), e.g., sickle cell disease, thalassemia, and methemoglobinemia; anemia (a deficiency of red blood cells or hemoglobin), pernicious anemia; disorders resulting in decreased cell counts, such as myelodysplastic syndrome, neutropenia (decreased neutrophil count), and thrombotic thrombocytopenic purpura (TTP), thrombocytosis, and hematopoietic malignancies, such as lymphoma, myeloma, and leukemia. 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's macroglobulinemia, and plasmacytoma; leukemias with an increased number of defective WBCs such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid 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), T-cell large granular lymphocyte leukemia (T-LGL), and aggressive NK cell leukemia.

[0367] Provided herein is a method for treating an autoimmune disease, comprising administering an effective amount of immune cells or a population thereof, or a composition or pharmaceutical composition described herein, to a patient in need thereof. "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 react with endogenous self-peptides, causing tissue destruction. Thus, the immune response is mounted against the subject's own antigens, referred to as autoantigens. "Autoantigen," as used herein, refers to antigens of normal host tissue. Normal host tissue does not contain tumor cells.

[0368] Non-limiting examples of treatable autoimmune diseases 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). Additional non-limiting autoimmune diseases include autoimmune thrombocytopenia, immune neutropenia, antihemophilic factor VIII inhibitors, antiphospholipid syndrome, Kawasaki syndrome, ANCA-associated disease, polymyositis, bullous pemphigoid, multiple sclerosis (MS), Guillain-Barré syndrome, chronic polyneuropathy, ulcerative colitis, diabetes, autoimmune thyroiditis, Graves' ophthalmopathy, rheumatoid arthritis, ulcerative colitis, primary sclerosing cholangitis, systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, Hashimoto's thyroiditis, Goodpasture's syndrome, autoimmune hemolytic anemia, scleroderma with anti-collagen antibodies, mixed connective tissue disease, pernicious anemia, idiopathic Addison's disease, autoimmune-related infertility, glomerulonephritis (e.g., crescentic glomerulonephritis, proliferative glomerulonephritis), insulin resistance, and autoimmune diabetes (type 1 diabetes, insulin-dependent diabetes mellitus). Autoimmune diseases are also recognized to include atherosclerosis and Alzheimer's disease. In another embodiment, 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 autonomic neuropathy, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, autoimmune leukemia ... Autoimmune urticarial neuropathy, autoimmune axonal neuropathy, Baro's disease, Behçet's disease, Castleman's disease, celiac disease, Chagas' disease, chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid, benign mucous membrane pemphigoid, Cogan's syndrome, cold agglutinin disease, Coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), dilated cardiomyopathy, discoid lupus, Dressler's syndrome, endometriosis,Eosinophilic angiocentric fibrosis, eosinophilic fasciitis, erythema nodosum, Evans syndrome, fibrosing alveolitis, giant cell arteritis (temporal arteritis), Hashimoto's encephalitis, Henoch-Schönlein purpura, herpes gestationis, idiopathic hypocomplementemic tubulointerstitial nephritis, multiple myeloma, multifocal motor neuropathy, NMDA receptor antibody encephalitis, IgG4-related disease, IgG4-related sclerosing disease, inflammatory aortic aneurysm, inflammatory pseudotumor, inclusion body myositis, interstitial cystitis, juvenile arthritis, Kuttner's tumor, Lambert-Eaton syndrome group, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear IgA disease (LAD), Lyme disease, chronic, mediastinal fibrosis, Meniere's disease, microscopic polyangiitis, Mikulicz syndrome, Mooren's ulcer, Much-Habermann disease, multifocal fibrosclerosis, narcolepsy, optic neuritis, Ormond's disease (retroperitoneal fibrosis), relapsing rheumatoid arthritis, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus), paraneoplastic cerebellar degeneration, polyneuropathy Disorders, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsonage-Turner syndrome, periaortitis, periarteritis, peripheral neuropathy, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, autoimmune polyglandular syndrome types I, II, and III, polymyalgia rheumatica, postpericardiotomy syndrome, progestational dermatitis, primary biliary cirrhosis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reflex sympathetic dystrophy These include: Reiter's syndrome, relapsing polychondritis, restless legs syndrome, rheumatic fever, Leede's thyroiditis, sarcoidosis, Schmidt's syndrome, scleritis, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-body syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, Tolosa-Hunt syndrome, transverse myelitis, undifferentiated connective tissue disease (UCTD), bullous dermatoses, vitiligo, Rasmussen's encephalitis, and Waldenström's macroglobulinemia.

[0369] As used herein, the terms "administering," "introducing," and "transplanting" are used interchangeably in the context of placing the described cells, e.g., hematopoietic progenitor cells, into a subject by a method or route that results in at least partial localization of the introduced cells at a desired site, such as a site of injury or repair, so that a desired effect occurs. Cells, e.g., hematopoietic progenitor cells, or their differentiated progeny (e.g., T cells), can be administered by any suitable route that results in delivery to a desired location in a subject, where at least a portion of the transplanted cells or components of the cells remain viable.

[0370] In various embodiments, the engineered immune cells described herein are optionally expanded ex vivo before administration to a subject. In other embodiments, the engineered immune cells are optionally cryopreserved for a period of time and then thawed before administration to a subject.

[0371] Engineered immune cells used in cell replacement therapy can be autologous / self-produced ("autologous") or non-autologous ("non-autologous", e.g., allogeneic, syngeneic, or xenogeneic) with respect to the recipient of the cells. "Autologous," as used herein, refers to cells from the same subject. "Allogeneic," as used herein, refers to cells of the same species that are genetically different from the comparative cell. "Syngeneic," as used herein, refers to cells of a different subject that are genetically identical to the comparative cell. "Xenogeneic," as used herein, refers to cells of a different species from the comparative cell. In a preferred embodiment, the cells of the invention are allogeneic.

[0372] In various embodiments, the engineered immune cells described herein that are transplanted into a subject in need thereof are autologous or allogeneic to the subject.

[0373] In various embodiments, the engineered immune cells described herein may be derived from one or more donors or may 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.

[0374] In various embodiments, the engineered immune cells described herein may be derived from one or more donors or may be obtained from an autologous source.

[0375] In various embodiments, prior to transplantation, the recipient subject is treated with chemotherapy and / or radiation.

[0376] In one embodiment, chemotherapy and / or radiation is to deplete endogenous stem cells and promote engraftment of transplanted cells.

[0377] In various embodiments, prior to transplantation, engineered immune cells or histone methyltransferase-inhibited multilineage hematopoietic progenitor cells or T cells differentiated using the stromal-free methods described herein are treated ex vivo with prostaglandin E2 and / or the antioxidant N-acetyl-L-cysteine ​​(NAC) to promote subsequent engraftment in the recipient subject.

[0378] In various embodiments, the recipient subject is a human.

[0379] In various embodiments, the subject has previously been diagnosed with HIV or other viral disease, a blood disorder, or has undergone cancer treatment.

[0380] In one embodiment, a subject is selected to provide somatic cells that can be used to generate iPSCs and engineered immune cells as described herein, hi one embodiment, the selected subject has a genetic disease or deficiency.

[0381] In various embodiments, the donor subject is a human, a non-human animal, a rodent, or a non-rodent. For example, the subject can be any mammal, such as a human, other primate, pig, rodent such as a mouse or rat, rabbit, guinea pig, hamster, cow, horse, cat, dog, sheep or goat, or non-mammal, such as a bird.

[0382] In various embodiments, the donor has previously been diagnosed with HIV, a blood disorder, or cancer.

[0383] In one embodiment, the biological sample, population of embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells is obtained from a donor subject.

[0384] In various embodiments, the populations of biological samples, embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells described herein may be derived from one or more donors or may be obtained from autologous sources.

[0385] In one embodiment, embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells are isolated from a donor subject, transfected, optionally cultured, and then transplanted back into the same subject, i.e., an autologous cell transplant. Here, the donor and recipient subject are the same individual. In another embodiment, embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells are isolated from a donor that is HLA-type matched to the subject (recipient). Donor-recipient antigen type matching is well known in the art. HLA types include HLA-A, HLA-B, HLA-C, and HLA-D. These represent the minimum number of cell surface antigen matches required for transplantation. That is, the transfected cells are transplanted allogeneically into a different subject, i.e., the recipient host subject. Donor or subject 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 a nucleic acid molecule described herein, and the transfected cells are cultured, inhibited, and differentiated as disclosed, optionally expanded, and then transplanted into a recipient subject. In one embodiment, the transplanted engineered immune cells engraft the recipient subject. In one embodiment, the transplanted engineered immune cells reconstitute the immune system in the recipient subject. The transfected cells can be cryopreserved and stored after transfection, or cryopreserved and stored after cell expansion.

[0386] Engineered immune cells or histone methyltransferase-inhibited multilineage hematopoietic progenitor cells or T cells differentiated using the stromal-free methods described herein can be administered as part of a bone marrow or umbilical cord blood transplant in individuals who have or have not undergone myeloablative therapy. In one embodiment, the genetically modified cells contemplated herein are administered in a bone marrow transplant to an individual who has undergone chemoablative or radiation ablative bone marrow therapy.

[0387] In one embodiment, the dose of cells is delivered to the subject intravenously. In one embodiment, the cells are administered to the subject intravenously.

[0388] In certain embodiments, patients receive a dose of the modified cells described herein, for example, about 1 x 10 in one single intravenous dose. 5 cells / kg, approximately 5×10 5 cells / kg, approximately 1×10 6 cells / kg, approximately 2×10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 1×10 8 Receive cells / kg or more of engineered immune cells or histone methyltransferase-inhibited multilineage hematopoietic progenitor cells or T cells differentiated using the stroma-free methods described herein.

[0389] In certain embodiments, a patient receives a dose of the modified cells described herein, e.g., at least 1 x 10 in one single intravenous dose. 5 cells / kg, at least 5 × 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 × 10 6 cells / kg, at least 5 × 10 6 cells / kg, at least 6 × 10 6 cells / kg, at least 7 × 10 6 cells / kg, at least 8 × 10 6 cells / kg, at least 9 × 10 6 cells / kg, at least 1 x 10 7 cells / kg, at least 5 × 107 cells / kg, at least 1 x 10 8 Receive cells / kg or more of engineered immune cells or histone methyltransferase-inhibited multilineage hematopoietic progenitor cells or T cells differentiated using the stroma-free methods described herein.

[0390] In additional embodiments, the patient receives a dose of the modified cells described herein, e.g., about 1 x 10 5 cells / kg ~ approx. 1×10 8 cells / kg, approximately 1×10 6 cells / kg ~ approx. 1×10 8 cells / kg, approximately 1×10 6 cells / kg ~ approx. 9×10 6 cells / kg, approximately 2×10 6 cells / kg ~ approx. 8×10 6 cells / kg, approximately 2×10 6 cells / kg ~ approx. 8×10 6 cells / kg, approximately 2×10 6 cells / kg ~ approx. 5×10 6 cells / kg, approximately 3×10 6 cells / kg ~ approx. 5×10 6 cells / kg, approximately 3×10 6 cells / kg ~ approx. 4×10 8 Receive 100 cells / kg or any dose of cells / kg of engineered immune cells or histone methyltransferase-inhibited multilineage hematopoietic progenitor cells or T cells differentiated using the stromal-free methods described herein.

[0391] Generally, the engineered immune cells or histone methyltransferase-inhibited multilineage hematopoietic progenitor cells described herein, or T cells differentiated using the stromal-free method described herein, are administered as a suspension in a pharmaceutically acceptable carrier, e.g., as a therapeutic composition. A therapeutic composition contains a cell composition together with a physiologically tolerable carrier and, optionally, at least one additional bioactive agent described herein dissolved or dispersed therein as an active ingredient. In a preferred embodiment, a therapeutic composition is substantially non-immunogenic when administered to a mammal or human patient for therapeutic purposes, unless desired. Those skilled in the art will recognize that pharmaceutically acceptable carriers used in cell compositions do not contain amounts of buffers, compounds, cryopreservatives, preservatives, or other agents that substantially interfere with the viability of the cells delivered to a subject. Cell-containing formulations can include, for example, an osmotic buffer that allows for maintaining cell membrane integrity, and optionally, nutrients to maintain cell viability or enhance engraftment upon administration. Such formulations and suspensions will be known to those of skill in the art and / or can be adapted for use with the cells described herein using routine experimentation.

[0392] As used herein, the terms "pharmaceutically acceptable" and "physiologically tolerable," and their grammatical variations, are used interchangeably when referring to compositions, carriers, diluents, and reagents, and indicate that the material can be administered to or on a mammal without producing undesired physiological effects, such as nausea, dizziness, or upset stomach. A pharmaceutically acceptable carrier does not promote an immune response to the agent with which it is mixed, unless desired. The preparation of pharmacological compositions containing active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared to be injectable, either as liquid solutions or suspensions; however, solid forms suitable for solution or suspension in liquid prior to use may also be prepared. The preparation may also be emulsified or presented as a liposomal composition. The active ingredient may be mixed with excipients in amounts that are pharmaceutically acceptable, compatible with the active ingredient, and suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. Additionally, if desired, the composition may 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 compositions of the present invention may include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxides, as well as organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. Physiologically tolerable carriers are well known in the art. An exemplary liquid carrier is a sterile aqueous solution containing no materials in addition to the active ingredient and water, or containing a buffer such as sodium phosphate, physiological saline, or both at a physiological pH value, such as phosphate-buffered saline.Additionally, aqueous carriers may contain two or more buffer salts, as well as salts such as sodium and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions may also contain liquid phases in addition to and excluding water. Examples of such additional liquid phases include glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active agent used in the methods described herein that will be effective in treating 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 the art. For example, a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of the active ingredient in 0.9% sodium chloride solution.

[0393] In one embodiment, a "pharmaceutically acceptable" carrier does not include in vitro cell culture media.

[0394] In some embodiments, the described engineered immune cell compositions further comprise a pharmaceutically acceptable carrier.

[0395] In various embodiments, at least a second or subsequent dose of cells is administered to the recipient subject. For example, the second dose can be administered between about 1 day and 30 weeks after the previous dose. If necessary, for example, a total of two, three, four, or more subsequent doses can be delivered to the individual, as determined by a skilled clinician.

[0396] The cell composition may be administered by any suitable route that results in an effective cell 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 is delivered, i.e., at least 1×10 4The cells are delivered to the desired site over a period of time. Modes of administration include injection, infusion, or drip, where "injection" includes, but is not limited to, intravenous, intraarterial, intracerebroventricular, and intracardiac injection and infusion. Administration by injection or infusion is generally preferred for cell delivery.

[0397] Efficacy testing can be performed during the course of treatment using the methods described herein. Measurement of the severity of certain symptoms associated with a particular disease is noted before the start of treatment and then at a specific time period after the start of treatment. In some embodiments, pharmaceutical compositions comprising the immune or populations thereof described herein can be used for cell replacement therapy in subjects.

[0398] Accordingly, a subject of the present disclosure also provides compositions of modified (also called engineered) cells for use in in vivo cell replacement therapy, medical therapy such as cancer immunotherapy, and in disease modeling, drug screening, and in vitro study of hematological disorders.

[0399] An advantage of the disclosed protocol is that it is a method that allows for the semi-permanent bulk generation of desired immune cells or other types of hematopoietic cells (i.e., cells differentiated from multipotent HSCs) from a variety of cell sources from stem cells, hematopoietic progenitor cells, and mature, differentiated somatic cells, all of which can be easily harvested from the patient's body.

[0400] Generated engineered immune cells or engineered histone methyltransferase-inhibited CD34 + / CD38 lo / -Hematopoietic progenitor cells (e.g., hemogenic endothelial) or T cells differentiated using the stromal-free methods described herein can be transplanted into patients 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). One additional advantage is that if the donor of the source cells and the recipient of the engineered immune cells are the same person, the engineered immune cells generated will have an HLA that is identical to that of the recipient, which avoids host graft immune rejection after transplantation. In the case of a recipient patient who is HLA-allogeneic to the donor individual of the source cells, host graft immune rejection is greatly reduced.

[0401] The engineered immune cells or engineered histone methyltransferase-inhibited cells generated, CD34+ / CD38- hematopoietic progenitor cells, or T cells differentiated using the stroma-free methods described herein can also be cryopreserved until future need.

[0402] Currently, bone marrow transplantation is the most established cell replacement therapy for various blood disorders. The functional unit of bone marrow transplantation is the hematopoietic stem cell (HSC), which resides at the apex of a complex cellular hierarchy and replenishes blood development throughout life. The scarcity of HLA-matched HSCs significantly limits the ability to perform transplantation, disease modeling, and drug screening. Therefore, many studies aim to generate HSCs from alternative sources. Advances in reprogramming induced pluripotent stem cells (iPSCs) have provided access to a wide range of patient-specific pluripotent cells, which are promising sources for disease modeling, drug screening, and cell therapy. However, the inability to obtain engrafting hematopoietic stem and progenitor cells from human pluripotent stem cells (hPSCs) limits the characterization of blood disorders to in vitro assays. Generating HSCs by direct differentiation remains challenging, and novel approaches to this problem are needed.

[0403] Thus, in one aspect, described herein is a method of cell replacement therapy, the method comprising administering to a recipient subject in need thereof an immune cell or population thereof, or a composition comprising said immune cell or population thereof, or a pharmaceutical composition comprising said immune cell or population thereof, as described herein.

[0404] In some embodiments, the recipient subject has undergone chemotherapy and / or radiation therapy. In some embodiments, the recipient subject has a deficiency in immune function and / or lymphocyte reconstitution. In some embodiments, prior to transplantation, immune cells or populations thereof are treated ex vivo with prostaglandin E2 and / or the antioxidant N-acetyl-L-cysteine ​​(NAC) to promote subsequent engraftment in the recipient subject.

[0405] kit Another aspect of the technology described herein relates to, inter alia, kits for differentiating T cells using the stromal-free methods described herein. Described herein are kit components that may be included in one or more of the kits described herein.

[0406] In some embodiments, the kit includes an effective amount of a CD3+ T cell differentiation factor (e.g., IL-7, SCF, FLT3, and / or TPO), or an effective amount of an iPSC differentiation factor (e.g., OCT4, SOX2, KLF4, c-MYC, nanog, and / or LIN28), or an effective amount of a hemogenic endothelial differentiation factor (e.g., BMP4, SB-431542, CHIR99021, bFGF, VEGF, IL-6, IL-11, IGF-1, SCF, and EPO), or an effective amount of a single positive T cell differentiation factor (e.g., IL-15 and / or CD3 / CD28 The T cell differentiation factor may comprise a T cell activator, such as a T cell activator, or an effective amount of an inhibitor of an epigenetic regulator (e.g., MC1568, CAY10591, UNC0224, UNC0638, A366, BRD4770, BIX01294, UNC0642, UNC0631, UNC0646, UNC0321, E72, BIX-01338, BRD9539, chaetocin, or DCG066, e.g., an EZH1 RNA interference agent). As will be understood by those skilled in the art, such cell differentiation factors may be provided in a lyophilized or concentrated form that can be diluted before use in cultured cells. Preferred formulations include those that are non-toxic to cells and / or do not affect proliferation rate, viability, etc. The T cell differentiation factor may be provided in aliquots or unit doses.

[0407] In some embodiments, the kit comprises a soluble Notch ligand or a composition thereof. In some embodiments, the kit does not comprise a stromal cell as described herein.

[0408] In some embodiments, the kit further comprises a vector comprising a nucleic acid encoding the CAR.

[0409] In some embodiments, the components described herein may be provided as a kit, either singly or in any combination. The kit may include the components described herein, e.g., a composition comprising a soluble Notch ligand, a composition comprising a differentiation factor, or a composition described throughout this specification, e.g., a composition comprising a vector comprising a CAR. Such kits may optionally include one or more agents that enable detection of a marker for T cell maturation (e.g., CD5, CD7, CD3, CD4, CD8, TCRgd, TCR alpha or beta, etc.) or a set thereof. Such kits may optionally include one or more agents that enable detection of a marker for T cell activation (e.g., CD107a, CD69, CD25, HLA-DR, IFNg, TNFa, etc.) or a set thereof. Such kits may optionally include one or more agents that enable detection of a marker for hemogenic endothelium (e.g., CD34, CD38, CD45, KDR, CD235, CD43, etc.). Additionally, the kits optionally include informational material. The kit may also contain a substrate for coating the culture dish, such as laminin, fibronectin, poly-L-lysine, or methylcellulose.

[0410] In some embodiments, the compositions in the kit may be provided in a watertight or airtight container that is substantially free of other components of the kit. For example, a cell differentiation reagent may be provided in two or more containers, e.g., it may be provided in a container having sufficient reagents for a predetermined number of differentiation assays, e.g., one, two, three, or more. One or more components described herein may be provided in any form, e.g., liquid, dried, or lyophilized. Components described herein are preferably substantially pure and / or sterile. When components described herein are provided in a liquid solution, the liquid solution is preferably an aqueous solution, with a sterile aqueous solution being preferred.

[0411] The informational material may be descriptive, instructional, marketing, or other material related to the methods described herein. The informational material of the kit is not limited in form. In one embodiment, the informational material may include information regarding the generation of soluble Notch ligands, or information regarding the generation of differentiated T cells using the stromal-free methods described herein, or the concentration, expiration date, batch or production location information, etc., of reagents used herein, such as cell differentiation factors. In one embodiment, the informational material relates to methods for using or administering the components of the kit.

[0412] The kit may include components for detecting markers for cell differentiation. In addition, the kit may include one or more antibodies that bind to cell markers, or primers for RT-PCR or PCR reactions, for example, semi-quantitative or quantitative RT-PCR or PCR reactions. Such components can be used to evaluate the activation of cell maturation markers or the loss of undifferentiated or immature cell markers. When the detection reagent is an antibody, it can be supplied in a dry preparation, for example, lyophilized, or in solution. The antibody or other detection reagent can be linked to a label for use in detection, for example, a radioactive, fluorescent (e.g., GFP), or colorimetric label. When the detection reagent is a primer, it can be supplied in a dry preparation, for example, lyophilized, or in solution.

[0413] The kit is typically provided with its various elements contained in one package, e.g., a fiber-based, e.g., cardboard, or polymer, e.g., Styrofoam, box. The enclosure may be configured to maintain a temperature differential between the interior and exterior, e.g., to provide insulating properties to maintain the reagents at a preselected temperature for a preselected time.

[0414] definition For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise specified or implied from context, the following terms and phrases include the meanings provided below. The definitions are provided to help describe particular embodiments and are not intended to limit the claimed invention, since the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided herein shall control.

[0415] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0416] As used herein, the term "monomer" refers to one of the basic structural units of the oligomers described herein, ie, a single DLL4 ligand.

[0417] As used herein, the terms "oligomer" or "oligomeric complex" are used interchangeably herein and refer to a molecule having at least two monomers. The architecture of the oligomer may vary. Particular oligomers for use in the present invention include dimers, trimers, tetramers, pentamers, hexamers, octamers, icosahedrons, or any other higher order oligomeric state.

[0418] In the case of homo-oligomers, a single repeating structural unit forms the oligomer (i.e., only DLL4 monomers). In the case of hetero-oligomers, two or more structural units are repeated in a pattern or randomly to form the oligomer (i.e., at least DLL1 and DLL4).

[0419] As used herein, "scaffold" refers to a structure, either naturally occurring or synthetically produced, that can be immobilized to at least monomers to form an oligomeric complex.

[0420] As used herein, the term "cell" refers to a single cell as well as a population (i.e., more than one) of cells. A population may be a pure population containing one cell type, such as a population of pluripotent stem cells or a population of differentiated T cells. As used herein, the term "population" refers to a pure population or a population containing a majority of one cell type (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%). Alternatively, a population may contain more than one cell type, e.g., a mixed cell population. This is not meant to limit the number of cells in the population; for example, a mixed cell population may contain at least one differentiated cell. In the present invention, there is no limit to the number of cell types that a mixed cell population may contain.

[0421] As used herein, in one embodiment, the term "hematopoietic stem cell" or "HSC" refers to a stem cell that has the capacity for self-renewal and gives rise to all blood cell types of the three hematopoietic lineages: erythroid, lymphoid, and myeloid. These cell types include the myeloid lineage (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and the lymphoid lineage (T cells, B cells, NK cells). Human HSCs express the CD34 + , CD59 + , CD90 / Thy1 + , CD38 低 / - , c-kit / CD117 - / 低 , and Lin - Mouse HSCs are CD34 低 / - , SCA-1 + , CD90 / Thy1 + / 低 , CD38 + , c-Kit / CD117 + , and Lin -Detecting the expression of this panel of markers allows for the isolation of specific populations of cells via techniques such as fluorescence-activated cell sorting (FACS). In one embodiment, the term "hematopoietic stem cells" or "HSC" refers to stem cells that have the capacity for self-renewal and possess the following cell surface markers: CD34+, CD59+, Thy1 / CD90 + , CD38 lo / - , CD133+, c-Kit / CD117 - / lo , and Lin - In one embodiment, the term "hematopoietic stem cell" or "HSC" refers to a stem cell that is at least CD34+. In one embodiment, the term "hematopoietic stem cell" or "HSC" refers to a stem cell that has the capacity for self-renewal and is at least CD34 + and c-kit / CD117 lo / - In one embodiment, the term "hematopoietic stem cell" or "HSC" refers to a stem cell that has the capacity for self-renewal and is at least CD38 低 / - , c-kit / CD117 - / 低 The term HSC may be used interchangeably with the term "hematopoietic stem and progenitor cells" (HSPCs).

[0422] As used herein, the terms "iPS cells," "iPSCs," and "induced pluripotent stem cells" are used interchangeably and refer to pluripotent cells artificially derived from differentiated cells, e.g., somatic cells, by transfection with the following reprogramming factors: OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28. Alternative combinations of reprogramming factors include OCT4, SOX2, NANOG, and LIN28. The term hPSCs refers to human pluripotent stem cells.

[0423] As used herein, the term "lineage," when used in the context of stem and progenitor cell differentiation and development, refers to the cell differentiation and developmental pathways that a cell can take to become a fully differentiated cell. For example, HSCs have three hematopoietic lineages: erythroid, lymphoid, and myeloid. HSCs have the potential, i.e., the capacity, to differentiate and develop into the terminally differentiated cell types known for all three lineages. When the term "multilineage" is used, it means that the cell can differentiate and develop into the terminally differentiated cell types known for more than one lineage. For example, HSCs have multilineage potential. When the term "lineage-restricted" is used, it means that the cell can differentiate and develop into the terminally differentiated cell types known for one lineage. For example, common myeloid progenitor cells (CMPs) or megakaryocyte-erythroid progenitors (MEPs) are lineage-restricted because they can differentiate and develop only into the terminally differentiated cell types of the myeloid lineage, but not into cells of the lymphoid lineage. Terminally differentiated cells of the myeloid lineage include erythrocytes, monocytes, macrophages, megakaryocytes, myeloblasts, dendritic cells, and granulocytes (basophils, neutrophils, eosinophils, and mast cells), while terminally differentiated cells of the lymphoid lineage include T lymphocytes / T cells, B lymphocytes / B cells, dendritic cells, and natural killer cells.

[0424] As used herein, the term "progenitor cell" refers to an immature or undifferentiated cell that has the potential to subsequently mature (differentiate) into a specific cell type (fully differentiated or terminally differentiated cell), e.g., a blood cell, skin cell, bone cell, or hair cell. Progenitor cells have a more primitive cellular phenotype (e.g., earlier along a developmental pathway or progression than a fully differentiated cell) relative to cells that can arise through differentiation. In many cases, progenitor cells also have significant or very high proliferative potential. Progenitor cells can give rise to multiple different differentiated cell types or a single differentiated cell type, depending on the developmental pathway and the environment in which the cell develops and differentiates. Progenitor cells can also proliferate to create more progenitor cells that are similarly immature or undifferentiated.

[0425] The term "differentiated cell" refers to any primary cell that, in its native form, is not pluripotent as that term is defined herein. The term "differentiated cell" also encompasses partially differentiated cells, such as multipotent cells (e.g., adult somatic stem cells). In some embodiments, the term "differentiated cell" also refers to a cell of a more specialized cell type that is derived from a cell of a less specialized cell type (e.g., an undifferentiated cell or a reprogrammed cell) where the cell has undergone a cell differentiation process.

[0426] In the context of cellular ontogeny, the terms "differentiate" or "differentiating" are relative terms referring to a "differentiated cell," a cell that has progressed further down the developmental pathway than its progenitor. Thus, in some embodiments, a reprogrammed cell, as this term is defined herein, can differentiate into a lineage-restricted progenitor cell (such as a mesodermal stem cell or an endodermal stem cell), which can then differentiate into other types of progenitor cells further down the pathway (e.g., tissue-specific progenitor cells, such as cardiomyocyte precursors or pancreatic precursors), which can then differentiate into terminally differentiated cells that play characteristic roles in specific tissue types, and may or may not retain the ability to proliferate further.

[0427] The term "pluripotency," when used in reference to "multipotent cells," refers to cells that can differentiate into some, but not all, of the cells derived from all three germ layers. Thus, multipotent cells are partially differentiated cells. Multipotent cells are well known in the art, and examples of multipotent cells include, for example, hematopoietic stem cells and adult somatic stem cells such as neural stem cells, hair follicle stem cells, and liver stem cells. Multipotency means that a stem cell can form many types of cells in a given lineage, but cannot form cells of other lineages. For example, multipotent blood stem cells can form many different types of blood cells (red blood cells, white blood cells, platelets, etc.) but are unable to form cardiovascular progenitor cells (MICPs) differentiate into neurons, specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types, and pancreatic-derived multipotent progenitor (PMP) colonies produce cell types of the pancreatic lineage (cells that produce insulin, glucagon, amylase, or somatostatin) and neural lineage (morphologically neuron-, astrocyte-, or oligodendrocyte-like cells).

[0428] As used herein, the term "reprogramming gene" refers to a gene whose expression contributes to the reprogramming of differentiated cells, e.g., somatic cells, into undifferentiated cells (e.g., cells in a pluripotent state, a partially pluripotent state, or a multipotent state). Reprogramming genes can be, for example, genes encoding master transcription factors such as Sox2, Oct3 / 4, Klf4, Nanog, Lin-28, c-myc, etc. The term "reprogramming factor" refers to a protein encoded by a reprogramming gene.

[0429] The term "exogenous" refers to a substance present in a cell other than its natural source. As used herein, the term "exogenous" 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, etc.) or 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 lower amounts. A substance (e.g., a nucleic acid encoding a sox2 transcription factor, or a protein, e.g., a SOX2 polypeptide) is considered exogenous if it is introduced into a cell or an ancestor of a cell that inherits the substance.

[0430] As used herein, the term "isolated" means that the cells are placed in a condition other than their natural environment. The term "isolated" does not preclude the subsequent use of these cells in combination or mixture with other cells.

[0431] As used herein, the term "expanding" refers to increasing the number of similar cells through cell division (mitosis). The terms "proliferating" and "expanding" are used interchangeably.

[0432] As used herein, "cell surface marker" refers to any molecule expressed on the surface of a cell. Cell surface expression usually requires that the molecule have a transmembrane domain. Some molecules not normally found on the cell surface can be engineered by recombinant technology to be expressed on the surface of a cell. Many naturally occurring cell surface markers are referred to as "CD" or "cluster of differentiation" molecules. Cell surface markers often provide antigenic determinants to which antibodies can bind. A cell surface marker particularly relevant to the methods described herein is CD34. Useful hematopoietic progenitor cells (e.g., hemogenic endothelium) according to the present disclosure preferably express CD34, or in other words, are CD34 positive.

[0433] Cells can be designated as "positive" or "negative" for any cell surface marker, and both such designations are useful in practicing the methods described herein. A cell is considered "positive" for a cell surface marker if it expresses the marker on its cell surface in a sufficient amount to be detected using methods known to those of skill in the art, such as contacting the cell with an antibody that specifically binds to that marker, followed by flow cytometric analysis of the contacted cells to determine whether the antibody binds to the cell. It should be understood that while a cell may express messenger RNA for a cell surface marker, the cell must express it on its surface to be considered positive in the methods described herein. Similarly, a cell is considered "negative" or "negative / low" (abbreviated as "- / lo" or "lo / -") for a cell surface marker if it does not express the marker on its cell surface in a sufficient amount to be detected using methods known to those of skill in the art, such as contacting the cell with an antibody that specifically binds to that marker, followed by flow cytometric analysis of the contacted cells to determine whether the antibody binds to the cell. In some embodiments, for cell surface lineage marker-specific agents used, the agents can all contain the same label or tag, such as a fluorescent tag, and thus exclude or remove all cells positive for that label or tag, leaving uncontacted hematopoietic stem or progenitor cells for use in the methods described herein.

[0434] As used herein, the term "histone methyltransferase inhibitor" or "inhibitor" refers to any molecule that inhibits the expression of a histone methyltransferase (e.g., G9a, GLP, EZH1) or inhibits the catalytic activity of the enzyme toward methylated lysines present on substrate histone proteins. For example, a histone methyltransferase inhibitor can be an siRNA or dsRNA that inhibits the expression of G9a, GLP, or EZH1 in inhibited cells, or a gRNA that promotes the degradation of G9a, GLP, or EZH1 mRNA in inhibited cells. For example, a histone methyltransferase inhibitor is a small molecule that antagonizes enzyme activity. Examples include, but are not limited to, the small molecules described herein: AMI-1, A-366, BIX-01294, BIX01338, BRD4770, chaetocin, UNC0224, UNC0631, UNC0638, UNC0642, UNC0646, EPZ5676, EPZ005687, GSK343, EPZ-6438, 3-deazaneplanocin A (DZNeP)HCl, UNC1999, MM-102, SGC0946, entacapone, EPZ015666, UNC0379, EI1, MI-2 (menin-MLL inhibitor), MI-3 (menin-MLL inhibitor), PFI-2, GSK126, EPZ004777, BRD4770, and EPZ-6438.

[0435] As used herein, the term "small molecule" refers to chemical agents, including, but not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds having a molecular weight of less than about 10,000 grams per mole (i.e., including heteroorganic and organometallic compounds), organic or inorganic compounds having a molecular weight of less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. In some embodiments, the small molecule is a heteroorganic compound or an organometallic compound.

[0436] The term "inhibitory RNA" is meant to include nucleic acid molecules containing a sequence 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. Non-limiting examples of inhibitory RNAs include interfering RNA, shRNA, siRNA, ribozymes, antagomirs, and antisense oligonucleotides. Methods for producing inhibitory RNAs are described herein. Additional methods for producing inhibitory RNAs are known in the art. In one embodiment, 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.

[0437] As used herein, "interfering RNA" refers to any double-stranded or single-stranded RNA sequence that can either directly or indirectly (i.e., upon translation) inhibit or down-regulate gene expression by mediating RNA interference. Interfering RNAs include, but are not limited to, small interfering RNAs ("siRNAs") and small hairpin RNAs ("shRNAs"). "RNA interference" refers to the selective degradation of sequence-compatible messenger RNA transcripts.

[0438] As used herein, "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 double-stranded stem. Following post-transcriptional processing, the small hairpin RNA is converted into small interfering RNA by a cleavage event mediated by the enzyme Dicer, a member of the RNase III family. As used herein, the phrase "post-transcriptional processing" refers to mRNA processing that occurs after transcription and is mediated, for example, by the enzyme Dicer and / or Drosha.

[0439] As used herein, "small interfering RNA" or "siRNA" refers to any small RNA molecule that can inhibit or downregulate gene expression by mediating RNA interference in a sequence-specific manner. Small RNAs can be, for example, approximately 18-21 nucleotides in length. Each siRNA duplex is formed by a guide strand and a passenger strand. The endonuclease Argonaute 2 (Ago2) catalyzes the unwinding of the siRNA duplex. Upon unwinding, 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 mRNAs with complementary sequences, which results in endonucleolytic cleavage of the target mRNA.

[0440] Retroviruses are RNA viruses that utilize reverse transcriptase during their replication cycle. The term "retrovirus" refers to any known retrovirus (e.g., C-type retroviruses such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), and spumavirus.

[0441] Retroviral genomic RNA is converted into double-stranded DNA by reverse transcriptase. This double-stranded DNA form of the virus can integrate into the chromosomes of infected cells, whereupon it is called a "provirus." The provirus serves as a template for RNA polymerase II, directing the expression of RNA molecules that encode the structural proteins and enzymes necessary to produce new viral particles.

[0442] At each end of the provirus is a structure called a "long terminal repeat" or "LTR." The term "long terminal repeat (LTR)" refers to a domain of base pairs located at the end of retroviral DNA, which, in the context of their native sequence, are direct repeats and contain the U3, R, and U5 regions. LTRs generally provide essential functions for retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. LTRs contain multiple regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences required for viral genome replication and integration. Viral LTRs are divided into three regions, designated U3, R, and U5. The U3 region contains enhancer and promoter elements. The U5 region is a sequence between the primer binding site and the R region and contains a polyadenylation sequence. The R (repeat) region flanks the U3 and U5 regions. LTRs, consisting of the U3, R, and U5 regions, appear at both the 5' and 3' ends of the viral genome. In one embodiment of the present invention, the promoter in the LTR, including the 5'LTR, is replaced with a heterologous promoter. Examples of heterologous promoters that can be used include the spleen focus-forming virus (SFFV) promoter, the tetracycline-inducible (TET) promoter, the β-globin locus control region and β-globin promoter (LCR), and the cytomegalovirus (CMV) promoter.

[0443] The term "lentivirus" refers to a group (or genus) of retroviruses that cause slowly developing diseases. Viruses within this group include HIV (human immunodeficiency virus, including HIV types 1 and 2), the etiological agent of human acquired immunodeficiency syndrome (AIDS); Visna-Maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep; Caprine Arthritis-Encephalitis Virus, which causes immunodeficiency, arthritis, and encephalopathy in goats; Equine Infectious Anemia Virus, which causes autoimmune hemolytic anemia and encephalopathy in horses; Feline Immunodeficiency Virus (FIV), which causes immunodeficiency in cats; Bovine Immunodeficiency Virus (BIV), which causes lymph node tumors, lymphocytosis, and possibly central nervous system infection in cattle; and Simian Immunodeficiency Virus (SIV), which causes immunodeficiency and encephalopathy in subhuman primates. Diseases caused by these viruses are characterized by long incubation periods and protracted course. Typically, viruses latently infect monocytes and macrophages, from which they spread to other cells. HIV, FIV, and SIV also readily infect T lymphocytes, or T cells.

[0444] The term "R region" refers to the region within a retroviral LTR that begins at the beginning of the capping sequence (i.e., at the start of transcription) and ends just before the start of the polyA tract. The R region is also defined as flanking the U3 and U5 regions. The R region plays an important role during reverse transcription, allowing the transfer of nascent DNA from one end of the genome to the other.

[0445] The term "promoter / enhancer" refers to a DNA segment containing sequences capable of providing both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. Enhancers / promoters can be "endogenous," "exogenous," or "heterologous." An "endogenous" enhancer / promoter is one that is naturally linked to a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that is placed in juxtaposition with a gene by genetic engineering (i.e., molecular biological techniques) such that transcription of the gene is directed by the linked enhancer / promoter.

[0446] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. A nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one embodiment, a nucleic acid can be DNA. In another embodiment, a nucleic acid can be RNA. Suitable DNA can include, for example, genomic DNA or cDNA. Suitable RNA can include, for example, mRNA, iRNA, miRNA, siRNA, etc.

[0447] The nucleic acid may be selected from the group including, for example, a nucleic acid encoding a protein of interest, an oligonucleotide, a nucleic acid analogue, such as peptide nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), and locked nucleic acid (LNA). For example, such nucleic acid sequences include, but are not limited to, nucleic acid sequences encoding proteins that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, such as RNAi, shRNAi, siRNA, microRNAi (miRNA), and antisense oligonucleotides.

[0448] As used herein, the term "engraftment" in reference to a recipient host refers to the onset of new blood-forming cells, generating healthy blood stem cells derived from the transplanted cells, that appear in the recipient's blood after a minimum period of 10 days post-transplant. Engraftment can occur as early as 10 days post-transplant, but is more common around 14-20 days.

[0449] As used herein, the term "reconstitution" with respect to the immune or hematologic systems in a recipient host refers to the reconstitution of an innate reservoir or operative system, or a portion thereof, in the body of the recipient host to a native or functional state, such as in a post-chemotherapy bone marrow that has depleted bone marrow stem cells.

[0450] The terms "reduce," "reduce," "reduction," or "inhibit" are all used herein to mean a statistically significant amount of reduction. In some embodiments, "reduce," "reduction," or "reduce," or "inhibit" typically refers to a decrease of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and can include, for example, a decrease of 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. As used herein, "reduction" or "inhibition" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. The decrease may preferably be to a level that is accepted as within the normal range for individuals without the given disorder.

[0451] The terms "increased," "increase," "enhance," or "activate" are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms "increased," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a reference level, including, for example, 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 a 100% increase, or any increase between 10-100% compared to a reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold or more increase compared to a reference level. In the context of a marker or symptom, an "increase" is a statistically significant increase in such level.

[0452] As used herein, "subject" refers to a human or an animal. Typically, an animal is a vertebrate such as a primate, rodent, livestock, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cats, canine species, e.g., dogs, foxes, wolves, avian species, e.g., chickens, emus, ostriches, and fish, e.g., trout, catfish, and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0453] Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can be advantageously used as subjects that represent animal models for cell replacement therapy. The subject may be male or female.

[0454] The subject may be a subject who has previously been diagnosed or identified as suffering from or having a condition requiring treatment (e.g., a blood disorder, cancer, etc.) or one or more complications associated with such a condition, and optionally has already received treatment for the blood disorder or one or more complications associated with the blood disorder. Alternatively, the subject may be a subject who has not previously been diagnosed with a blood disorder or one or more complications associated with the blood disorder. For example, the subject may be a subject who exhibits one or more risk factors for a blood disorder or one or more complications associated with a blood disorder, or a subject who does not exhibit risk factors.

[0455] A "subject in need" of treatment for a particular condition can be a subject who has the condition, a subject who has been diagnosed with the condition, or a subject who is at risk of developing the condition.

[0456] 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 variant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0457] Alterations in the native amino acid sequence can be achieved by any of several techniques known to those skilled in the art. Mutations can be introduced at specific loci, for example, by synthesizing oligonucleotides containing mutant sequences flanked by restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog with the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be used to provide altered nucleotide sequences with specific codons altered according to the required substitution, deletion, or insertion. Techniques for making such alterations are very well established and include, for example, those disclosed by Walder et al. (Gene 42:133, 1986), Bauer et al. (Gene 37:73, 1985), Craik (BioTechniques, January 1985, 12-19), Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981), and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entireties. Any cysteine ​​residue not involved in maintaining the proper conformation of the polypeptide can also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bond(s) can be added to a polypeptide to improve its stability or promote oligomerization.

[0458] The term "expression" refers to the cellular processes involved in the production of RNA and protein, and optionally, protein secretion, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, as applicable. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragments of the invention, and / or the translation of mRNA into polypeptides.

[0459] In some embodiments, expression of a biomarker, target, or gene / polypeptide described herein is tissue-specific. In some embodiments, expression of a biomarker, target, or gene / polypeptide described herein is systemic. In some embodiments, expression of a biomarker, target, or gene / polypeptide described herein is systemic.

[0460] "Expression products" include RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" refers to a nucleic acid sequence that is transcribed into RNA (DNA) in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" sequences and 3'UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0461] In some embodiments, the polypeptides, nucleic acids, or cells described herein may be engineered. As used herein, "engineered" refers to aspects that have been manipulated by the hand of man. For example, a polypeptide is considered "engineered" if at least one aspect of the polypeptide, such as its sequence, has been manipulated by the hand of man so that it differs from a naturally occurring aspect. As is common practice and understood by those skilled in the art, the progeny of an engineered cell are typically still referred to as "engineered," regardless of the actual manipulation performed in the previous entity.

[0462] In some embodiments, the differentiated and / or engineered T cells described herein are exogenous. In some embodiments, the differentiated and / or engineered T cells described herein are ectopic. In some embodiments, the differentiated and / or engineered T cells described herein are not endogenous.

[0463] The term "exogenous" refers to a substance present within a cell other than its natural source. As used herein, the term "exogenous" can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide not normally found and introduced by a process involving the hand of man into a biological system, such as a cell or organism, where one desires to introduce the nucleic acid or polypeptide into such cell or organism. Alternatively, "exogenous" can refer to a nucleic acid or polypeptide that is found in relatively low amounts and introduced by a process involving the hand of man into a biological system, such as a cell or organism, where one desires to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to a biological system or cell. As used herein, "ectopic" refers to a substance found in an abnormal location and / or amount. Ectopic material may be one that is normally found in a given cell, but is found in much lower amounts and / or at a different time. Ectopic also includes substances, such as polypeptides or nucleic acids, that are not naturally found or expressed in a given cell in its natural environment.

[0464] Nucleic acids encoding polypeptides described herein (e.g., CAR polypeptides) can be comprised by vectors. As used herein, the term "vector" refers to a nucleic acid construct designed for delivery to a host cell or transfer between different host cells. As used herein, a vector can be viral or non-viral. The term "vector" encompasses any genetic element that, when associated with the appropriate control elements, is capable of replicating and transferring genetic sequences into a cell. Vectors can include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, chromosomes, viruses, virions, and the like.

[0465] A vector can be recombinant, e.g., contain sequences from at least two different sources. In some embodiments, a vector contains sequences from at least two different species. In some embodiments, a vector contains sequences from at least two different genes, e.g., contains a nucleic acid encoding a fusion protein or expression product operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., promoter, suppressor, activator, enhancer, response element, etc.).

[0466] In some embodiments, the vectors or nucleic acids described herein are codon-optimized, e.g., the native or wild-type sequence of a nucleic acid sequence has been altered or engineered to include alternative codons such that the altered or engineered nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence but is transcribed and / or translated with improved efficiency in a desired expression system. In some embodiments, the expression system is an organism (or cells derived from such an organism) other than the source of the native / wild-type sequence. In some embodiments, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, mouse cell, or human cell. In some embodiments, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in a human cell. In some embodiments, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in a yeast or yeast cell. In some embodiments, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in a bacterial cell. In some embodiments, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in an E. coli cell.

[0467] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or polypeptides from sequences linked to transcriptional regulatory sequences on the vector. The expressed sequences are often, but not necessarily, heterologous to the cell. An expression vector may contain additional elements; for example, an expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example, human cells for expression and prokaryotic hosts for cloning and amplificatio...

Claims

1. A soluble Notch ligand oligomeric complex comprising at least two Notch ligand monomers and a scaffold.

2. 2. The oligomeric complex of claim 1, wherein 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).

3. 2. The oligomeric complex of claim 1, wherein the Notch ligand monomer is a DLL4 ligand.

4. 2. The oligomeric complex of claim 1, wherein the complex comprises a single type of Notch ligand monomer.

5. 2. The oligomeric complex of claim 1, wherein the complex comprises at least two types of Notch ligand monomers.

6. The oligomeric complex of claim 1 , wherein the scaffold is a naturally occurring scaffold or a synthetic scaffold.

7. 10. The oligomeric complex of claim 1, wherein the complex is a dimer, trimer, tetramer, pentamer, hexamer, octamer, icosahedron, or any other higher order oligomeric state.

8. 2. The oligomeric conjugate of claim 1, wherein the conjugate is a 60-mer or a 120-mer.

9. The oligomeric complex of claim 1 , wherein the complex is a trimer.

10. 2. The oligomeric complex of claim 1, wherein the DLL4 ligand monomer comprises a sequence selected from SEQ ID NOs: 6-9.

11. A composition comprising the oligomeric conjugate of any one of claims 1 to 9.

12. A method of making a soluble Notch ligand oligomeric complex, said method comprising contacting a plurality of Notch ligand monomers with a population of scaffolds for a time sufficient to promote the formation of the complex.

13. 13. The method of claim 12, wherein said plurality of Notch ligand monomers further comprises a GS linker.

14. 14. The method of claims 12 and 13, wherein the plurality of Notch ligand monomers are fused to SpyTag via the GS linker.

15. 13. The method of claim 12, wherein the population of scaffolds further comprises a GGSGGS linker.

16. The method of claims 12 and 15, wherein the population of scaffolds is fused to SpyCatcher via a GGSGGS linker.

17. A soluble Notch ligand oligomeric complex produced by the method of any one of claims 12 to 16.

18. 18. A composition of soluble Notch ligand oligomers according to claim 17.

19. 19. A method of activating Notch signalling in a population of cells, said method comprising contacting the population of cells with a soluble Notch ligand oligomeric complex according to any one of claims 1 to 9 or 17, or a composition according to claim 10 or 18.

20. 20. The method of claim 19, wherein the population of cells is not adhered to a substrate.

21. 20. The method of claim 19, wherein the population of cells is adhered to a substrate.

22. 22. The method of any one of claims 19 to 21, wherein 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.

23. The method of any one of claims 19 to 22, wherein the cells are human cells.

24. The method of any one of claims 19 to 22, wherein the cell is a non-human cell.

25. 1. A method comprising: a) A population of pluripotent stem cells was isolated by transfection with CD34 ＋ allowing differentiation in aggregation medium for a time sufficient to promote differentiation into a hemogenic endothelial population; b) The obtained CD34 ＋ Inhibiting histone methyltransferases in hemogenic endothelial populations; c) the obtained CD34 ＋ The hemogenic endothelial population was identified as CD3 ＋ In the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of CD3 T cells. ＋ differentiating in a T cell differentiation medium.

26. 1. A method comprising: a) A population of pluripotent stem cells was isolated by transfection with CD34 ＋ allowing differentiation in aggregation medium for a time sufficient to promote differentiation into a hemogenic endothelial population; b) The obtained CD34 ＋ Inhibiting epigenetic regulators in the hemogenic endothelial population and c) the obtained CD34 ＋ The hemogenic endothelial population was identified as CD3 ＋ In the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of CD3 T cells. ＋ differentiating in a T cell differentiation medium.

27. 1. A method comprising: a) A population of pluripotent stem cells was isolated by transfection with CD34 ＋ allowing differentiation in aggregation medium for a time sufficient to promote differentiation into a hemogenic endothelial population; b) The obtained CD34 ＋ Inhibiting G9a and / or GLP in the hemogenic endothelial population; c) the obtained CD34 ＋ The hemogenic endothelial population was identified as CD3 ＋ In the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of CD3 T cells. ＋ differentiating in a T cell differentiation medium.

28. 1. A method comprising: a) A population of pluripotent stem cells was isolated by transfection with CD34 ＋ allowing differentiation in aggregation medium for a time sufficient to promote differentiation into a hemogenic endothelial population; b) The obtained CD34 ＋ The hemogenic endothelial population was identified as CD3 ＋ In the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of CD3 T cells. ＋ differentiating in a T cell differentiation medium.

29. 29. The method of any one of claims 25 to 28, wherein the soluble Notch ligand is soluble Delta-like-1 (DLL1), Delta-like-3 (DLL3), Delta-like-4 (DLL4), Jagged 1 (JAG1), or Jagged 2 (JAG2).

30. 30. The method of any one of claims 25 to 29, wherein the soluble Notch ligand is soluble DLL4.

31. The method of any one of claims 25 to 30, wherein differentiating the hemogenic endothelium in the presence of a soluble Notch ligand does not include co-culturing with stromal cells that express a Notch ligand.

32. The method of any one of claims 25 to 31, wherein 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.

33. The CD3 ＋ 33. The method of any one of claims 25 to 32, wherein the time sufficient to promote differentiation into a population of T cells is at least 4 weeks.

34. The CD3 ＋ The method of any one of claims 25 to 33, wherein the T cell differentiation medium is serum-free.

35. The CD3 ＋ The method of any one of claims 25 to 34, wherein the T cell differentiation medium comprises FLT3 and IL7.

36. The CD3 ＋ The method of any one of claims 25 to 35, wherein the T cell differentiation medium comprises 15 ng / ml FLT3 and 25 ng / ml IL7.

37. The CD3 ＋ The T cell differentiation medium comprises the CD3 ＋ 37. The method of any one of claims 25 to 36, further comprising 5 ng / mL thrombopoietin (TPO) and / or 30 ng / ml SCF for at least the first two weeks of differentiation in T cell differentiation medium.

38. CD3 containing TPO ＋ T cell differentiation medium is CD5 ＋ CD7 ＋ The method of any one of claims 25 to 37, which promotes differentiation into a population of ProT cells.

39. The CD3 ＋ A population of T cells is CD4 ＋ CD8 ＋ The method of any one of claims 25 to 28, comprising a population of T cells.

40. CD4 ＋ Cell populations and CD8 ＋ and in a single positive T cell differentiation medium for a time sufficient to promote differentiation into a population of CD4 ＋ CD8 ＋ 40. The method of claim 39, further comprising differentiating the population of T cells.

41. The CD4 ＋ CD8 ＋ CD4 from the T cell population ＋ T cell populations and CD8 ＋ 41. The method of claim 40, wherein the time sufficient to promote differentiation into a population of cells is at least one week.

42. CD34 ＋ CD4 from the hemogenic endothelial population ＋ T cell populations and CD8 ＋ 41. The method of claim 40, wherein the time sufficient to promote differentiation into a population of cells is at least 5 weeks.

43. 41. The method of claim 40, wherein the single positive T cell differentiation medium comprises 10 ng / mL IL-15 and a T cell activator.

44. 44. The method of claim 43, wherein the T cell activator comprises 10 ul / ml of CD3 / CD28 T cell activator.

45. 44. The method of claim 43, wherein the T cell activator comprises one CD3 / CD28 T cell activator Dynabead per cell.

46. At least one week later, CD4 ＋ Cell enrichment and / or CD8 ＋ 46. ​​The method of any one of claims 40 to 45, further comprising a step of cell enrichment.

47. The method of any one of claims 25 to 28, wherein the population of pluripotent stem cells comprises induced pluripotent stem cells (iPS cells) or embryonic stem cells (ESCs).

48. 48. The method of claim 47, wherein the induced pluripotent stem cells are generated by introducing only the reprogramming factors OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28 into mature cells.

49. 48. The method of claim 47, wherein the induced pluripotent stem cells are generated by introducing reprogramming factors into mature cells two or more times.

50. The population of pluripotent stem cells is cultured using embryoid bodies or 2D adherent cultures, and is then transfected with CD34 ＋ The method of any one of claims 25 to 28, wherein the cells are differentiated into a population of hemogenic endothelium.

51. CD34 ＋ 29. The method of any one of claims 25 to 28, wherein the time sufficient to promote differentiation into a population of hemogenic endothelium is at least 8 days.

52. 29. The method of any one of claims 25 to 28, wherein the aggregation medium comprises BMP4, SB-431542, CHIR99021, bFGF, VEGF, IL-6, IL-11, IGF-1, SCF, and EPO.

53. 29. The method of any one of claims 25 to 28, wherein the aggregation medium 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.

54. CD34 ＋ The expression of surface markers on a population of hemogenic endothelium was used to identify the CD34 ＋ 44. The method of any one of claims 40 to 43, further comprising selecting or isolating a population of hemogenic endothelium.

55. CD34 ＋ The method of any one of claims 40 to 44, wherein the population of hemogenic endothelium is CD45 negative / low.

56. CD34 ＋ The method of any one of claims 40 to 45, wherein the population of hemogenic endothelium is CD38 negative / low.

57. The obtained CD34 ＋ A population of hemogenic endothelial cells or the CD3 ＋ 29. The method of any one of claims 25 to 28, further comprising genetically modifying the population of T cells.

58. 58. The method of claim 57, wherein the genetic modification is to edit endogenous HLA, to ablate endogenous TCR, and / or to express a chimeric antigen receptor (CAR).

59. 26. The method of claim 25, wherein the histone methyltransferase catalyzes the addition of a methyl group to histone 3 lysine residue 9 (H3K9) and / or histone 3 lysine residue 27 (H3K27).

60. 26. The method of claim 25, wherein histone methyltransferases H3K9 and / or H3K27 are inhibited by small molecule inhibitors or nucleic acid inhibitors.

61. 61. The method of claim 60, wherein the histone methyltransferase H3K9 and / or H3K27 small molecule inhibitor is a heteroorganic or organometallic compound.

62. 61. The method of claim 60, 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), EI1, GSK343, GSK126, and UNC1999.

63. 61. The method of claim 60, wherein the nucleic acid inhibitor is a nucleic acid that targets the expression of a histone methyltransferase.

64. 61. The method of claim 60, wherein the nucleic acid inhibitor is an RNA interference inhibitor or drug.

65. The method of claim 60, wherein the nucleic acid inhibitor is an EZH1-specific nucleic acid selected from the group consisting of an aptamer that binds to EZH1, an EZH1-specific RNA interfering agent, and a vector encoding an EZH1-specific RNA interfering agent, and the RNA interfering agent comprises one or more of the nucleotide sequences selected from SEQ ID NOs: 11 to 19.

66. 27. The method of claim 26, wherein the epigenetic regulator is a DNA methyltransferase (DNMT), a methyl-CpG binding domain (MBD) protein, a DNA demethylase, a histone methyltransferase (HMT), a methyl-histone binding protein, a histone demethylase, a histone acetyltransferase (HAT), an acetyl-binding protein, or a histone deacetylase (HDAC).

67. 67. The method of claim 66, wherein the inhibitor of an epigenetic regulator is selected from the group consisting of UNC0224, MC1568, and CAY10591.

68. 68. The method of any one of claims 66 to 67, wherein the inhibitor of an epigenetic regulator is provided at a concentration of at least 500 nM.

69. 68. The method of any one of claims 66 to 67, wherein the time sufficient to promote differentiation of the population of CD34+ cells into a population of CD5+CD7+ proT cells is about 14 days.

70. 28. The method of claim 27, wherein the 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.

71. 71. The method of claim 70, wherein the G9a and / or GLP inhibitor is UNC0224.

72. 72. The method of any one of claims 70 to 71, wherein the G9a and / or GLP inhibitor is provided at a concentration of 300 nM to 5 uM.

73. 73. The method of any one of claims 70 to 72, wherein the time sufficient to promote differentiation of the population of CD34+ cells into a population of CD5+CD7+ proT cells is about 14 days.

74. 1. A method comprising: a) A population of pluripotent stem cells was isolated by transfection with CD34 ＋ allowing differentiation in aggregation medium for a time sufficient to promote differentiation into a hemogenic endothelial population; b) The obtained CD34 ＋ A population of hemogenic endothelium was cultured for at least 4 weeks in the presence of soluble Notch ligand, followed by CD3+ / CD ... ＋ Differentiate in T cell differentiation medium to obtain CD3 ＋ promoting differentiation of the T cell population into a T cell population; The CD3 ＋ The method, wherein the T cell differentiation medium further comprises 5 ng / mL TPO and 30 ng / ml SCF for at least the first two weeks.

75. 1. A method comprising: a) A population of pluripotent stem cells was isolated by transfection with CD34 ＋ allowing differentiation in aggregation medium for a time sufficient to promote differentiation into a hemogenic endothelial population; b) The obtained CD34 ＋ A population of hemogenic endothelium was cultured for at least 4 weeks in the presence of soluble Notch ligand, followed by CD3+ / CD ... ＋ Differentiate in T cell differentiation medium to obtain CD3 ＋ promoting differentiation of the T cell population into a T cell population; The CD3 ＋ The method, wherein the T cell differentiation medium further comprises, for at least the first two weeks, 5 ng / mL TPO, 30 ng / ml SCF, and a G9a / GLP inhibitor.

76. The CD3 ＋ 76. The method of any one of claims 25 to 75, wherein the population of T cells exhibits a gene expression profile most similar to alpha beta T cells.

77. The CD3 ＋ 77. The method of any one of claims 25 to 76, wherein the population of T cells exhibits a gene expression profile that is at least 10%, 20%, 30%, 40% or more similar to alpha beta T cells.

78. 78. The method of any one of claims 25-77, wherein said population of CD3+ T cells exhibits a gene expression profile having a Pearson correlation coefficient compared to peripheral blood alpha beta T cells of at least 0.

85.

79. The CD3 ＋ 79. The method of any one of claims 25 to 78, wherein the population of T cells exhibits a Productivity Simpson Clonality Value of about 0.

025.

80. The CD3 ＋ 80. The method of any one of claims 25-79, wherein the population of T cells exhibits T cell receptor (TCR) complementarity determining regions (CDRs) that are at least 3 nucleotides longer than immune cells differentiated without methyltransferase inhibition or using stromal cells.

81. 81. The method of any one of claims 25 to 80, wherein the soluble Notch ligand is used at a concentration of 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM or more.

82. An immune cell produced by the method of any one of claims 25 to 80.

83. 83. The immune cell of claim 82, wherein said immune cell exhibits a gene expression profile at least 10%, 20%, 30%, 40% or more similar to an alpha beta T cell.

84. 84. The immune cell of any one of claims 82 to 83, wherein said immune cell exhibits a gene expression profile having a Pearson correlation coefficient compared to peripheral blood alpha beta T cells of at least 0.

85.

85. 85. The immune cell of any one of claims 82 to 84, wherein the immune cell exhibits a productivity Simpson clonality value of about 0.

025.

86. 86. The immune cell of any one of claims 82-85, wherein the immune cell exhibits a T cell receptor (TCR) complementarity determining region (CDR) that is at least 3 nucleotides longer than immune cells differentiated using stromal cells without methyltransferase inhibition.

87. A composition comprising an immune cell or population thereof according to any one of claims 82 to 86.

88. 88. The composition of claim 87, further comprising a pharmaceutically acceptable carrier.

89. A pharmaceutical composition comprising the immune cell or population thereof according to any one of claims 82 to 86 and a pharmaceutically acceptable carrier.

90. 90. The pharmaceutical composition of claim 89 for use in cell replacement therapy in a subject.

91. 91. A method of cell replacement therapy, said method comprising administering to a recipient subject in need thereof an immune cell or population thereof according to any one of claims 82 to 86, or a composition according to claims 87 to 88, or a pharmaceutical composition according to claims 89 to 90.

92. 92. The cell replacement therapy method of claim 91, wherein the recipient subject is undergoing chemotherapy and / or radiation.

93. 92. The cell replacement therapy method of claim 91, wherein the recipient subject has a deficiency in immune function and / or lymphocyte reconstitution.

94. 94. The cell replacement therapy method of any one of claims 91 to 93, wherein prior to transplantation, the immune cells or populations thereof are treated ex vivo with prostaglandin E2 and / or the antioxidant N-acetyl-L-cysteine ​​(NAC) to promote subsequent engraftment in the recipient subject.

95. 95. The cell replacement therapy method of any one of claims 91 to 94, wherein the immune cells or population thereof are autologous to the recipient subject.

96. 96. The cell replacement therapy method of any one of claims 91 to 95, wherein the immune cells or population thereof are HLA type matched to the recipient subject.

97. A cell having activated Notch expression produced by the method of any one of claims 19 to 24.

98. 98. The cell of claim 97, wherein the cell has increased Notch expression compared to a suitable control.

99. 99. The cell of claim 97 or 98, wherein Notch expression in the cell is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more compared to a suitable control.

100. A composition comprising a cell or a population thereof according to any one of claims 97 to 99.

101. A pharmaceutical composition comprising the cell or population thereof according to any one of claims 97 to 99 and a pharmaceutically acceptable carrier.

102. 1. A method comprising: a) CD34 ＋ Inhibiting histone methyltransferases in hemogenic endothelial populations; b) the CD34 ＋ The hemogenic endothelial population was identified by CD56 ＋ natural killer (NK) cells in the presence of a soluble Notch ligand for a time sufficient to promote differentiation into a population of NK cells. differentiating in a differentiation medium.

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