Synthetic nucleic acids and cells for immune cell differentiation

Abstract

Provided herein is a synthetic nucleic acid encoding at least one promoter, a transgene, and at least one inhibitory nucleic acid sequence. Cells, stables cell lines, and viruses expressing the same are also provided herein.

Description

Attorney Docket No: 701039-000143WOPT SYNTHETIC NUCLEIC ACIDS AND CELLS FOR IMMUNE CELL DIFFERENTIATION CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is an International Application which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Applications No.63 / 553,436, filed on February 14, 2024, the contents of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The technology described herein relates to immune cell differentiation methods. BACKGROUND

[0003] Chimeric antigen receptor (CAR) T cell therapy has transformed cancer treatment, yet sustained remission, which correlates with long-term persistence of CAR T cells, is only seen in less than half of treated patients. In addition, the process of manufacturing CAR T cells is complex, costly, and time-consuming and wider use of this approach is limited by insufficient availability of suitable cGMP facilities. While human cord blood hematopoietic stem and progenitor cells and pluripotent stem (hPSC: human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs)) cells hold promise as a potentially unlimited source for mass-produced allogeneic T cells and so called “off the shelf CAR T cells”, existing in vitro differentiation methods suffer from low efficiency and significant variability depending on the specific donor or hPSC source used. SUMMARY

[0004] One aspect provided herein describes a synthetic nucleic acid that contains a transgene with at least one promoter, a protein-encoding open-reading frame, and at least one inhibitory nucleic acid sequence.

[0005] In one embodiment of this aspect, or any aspect herein, the transgene is operatively linked to an EF1a promoter, a U6 promoter, an H1 promoter, a PGK1 promoter, or Ubiquitin-C (UBC) promoter. For example, a promoter suitable for expressing an inhibitory nucleic acid sequence, such as aU6 or H1 promoter capable of directing RNA polymerase III mediated expression of an shRNA or an EF1alpha- (EEF1A1), PGK1-, or Ubiquitin-C (UBC) promoter capable of directing RNA polymerase II mediated expression of a miRNA or of an mRNA that encodes a dominant-negative protein, and at least one promoter is a promoter such as an EF1alpha- (EEF1A1), PGK1-, or Ubiquitin-C (UBC) promoter that is capable of mediating expression of an mRNA that encodes a functional protein.

[0006] In one embodiment of this aspect, or any aspect herein, the transgene encodes a functional BCL-XL protein. 1 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0007] In one embodiment of this aspect, or any aspect herein, the inhibitory nucleic acid sequence is a shRNA sequence, miRNA sequence, sgRNA sequence, antisense RNA sequence, or other type of trans-acting oligonucleotide sequence whose expression results in inhibition of the expression or activity of a target gene.

[0008] In one embodiment of this aspect, or any aspect herein, the inhibitory nucleic acid sequence inhibits a target gene selected from the group consisting of EZH1, TP53, or TET2.

[0009] In one embodiment of this aspect, or any aspect herein, each of the protein-encoding transgene sequence and at least one inhibitory nucleic acid sequence are operatively linked to the at least one promoter.

[0010] In one embodiment of this aspect, or any aspect herein, the at least one inhibitory nucleic acid sequence is operatively linked to a U6 promoter.

[0011] In one embodiment of this aspect, or any aspect herein, the synthetic nucleic acid further comprises a sequence encoding terminal repeats flanking the at least one promoter, the transgene, and the least one shRNA.

[0012] In one embodiment of this aspect, or any aspect herein, the synthetic nucleic acid comprises from 5’ to 3’, (a) a 5’ terminal repeat, (b) at least one inhibitory nucleic acid sequence operatively linked to a promoter, (c) the transgene operatively linked to a promoter, and (c) a 3’ terminal repeat.

[0013] In one embodiment of this aspect, or any aspect herein, the synthetic nucleic acid comprises from 5’ to 3’, (a) a 5’ terminal repeat, (b) at least one inhibitory nucleic acid sequence operatively linked to a promoter, (c) the BCL-XL operatively linked to the EF1a promoter, and (d) a 3’ terminal repeat.

[0014] In one embodiment of this aspect, or any aspect herein, the synthetic nucleic acid comprises from 5’ to 3’, (a) a 5’ terminal repeat, (b) at least one of EZH1 shRNA operatively linked to a U6 promoter, TP53 shRNA operatively linked to a U6 promoter, and TET2 shRNA operatively linked to a promoter, (c) the BCL-XL operatively linked to the EF1a promoter, and (d) a 3’ terminal repeat.

[0015] In one embodiment of this aspect, or any aspect herein, the synthetic nucleic acid comprises from 5’ to 3’, (a) a 5’ terminal repeat, (b) at least one inhibitory nucleic acid sequence operatively linked to a U6 promoter, TP53 shRNA operatively linked to a U6 promoter, and TET2 shRNA operatively linked to a promoter, (c) the BCL-XL operatively linked to the EF1a promoter, and (d) a 3’ terminal repeat.

[0016] In one embodiment of this aspect, or any aspect herein, the synthetic nucleic acid comprises from 5’ to 3’, (a) a 5’ terminal repeat, (b) at least one of TP53 shRNA and TET2 shRNA operatively linked to a U6 promoter, TP53 shRNA operatively linked to a U6 promoter, and TET2 shRNA operatively linked to a promoter, (c) the BCL-XL operatively linked to the EF1a promoter, and (d) a 3’ terminal repeat. 2 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0017] In one embodiment of this aspect, or any aspect herein, the synthetic nucleic acid further comprises a sequence encoding a marker gene.

[0018] In one embodiment of this aspect, or any aspect herein, the terminal repeat is a long terminal repeat (LTR).

[0019] In one embodiment of this aspect, or any aspect herein, the synthetic nucleic acid is a plasmid.

[0020] Another aspect provided herein describes a synthetic nucleic acid comprising from 5’ to 3’ a sequence encoding, (a) a 5’ terminal repeat, (b) at least one of EZH1 shRNA operatively linked to a U6 promoter, TP53 shRNA operatively linked to a U6 promoter, and TET2 shRNA operatively linked to a promoter, (c) the BCL-XL operatively linked to the EF1a promoter, and (d) a 3’ terminal repeat.

[0021] Another aspect provided herein describes a synthetic nucleic acid comprising from 5’ to 3’ a sequence encoding, (a) a 5’ terminal repeat, (b) a TET2 shRNA operatively linked to a promoter, (c) the BCL-XL operatively linked to the EF1a promoter, and (d) a 3’ terminal repeat.

[0022] Another aspect provided herein describes a virus expressing any of the synthetic nucleic acids described herein. In one embodiment of this aspect, or any aspect herein, the virus is selected from the group consisting of Retroviruses (alpha-retrovirus, gamma-retrovirus, or lentivirus), adeno-associated virus, paramyxovirus, alphanodavirus.

[0023] Another aspect provided herein describes a lentivirus expressing any of the synthetic nucleic acids described herein.

[0024] Another aspect provided herein describes a cell-free system expressing any of the synthetic nucleic acids described herein. Cell-free systems for viral vector production are further described in, for example, Cerqueira A., et al. Journal of Virology, 2016; Sheng J., et al. The Royal Society of Chemistry, 2017; and Svitkin Y.V., and Sonenberg N. Journal of Virology, 2003; the contents of which are incorporated herein by reference in their entireties.

[0025] Another aspect provided herein describes a host system expressing any of the synthetic nucleic acids described herein.

[0026] In one embodiment, the host system can be a host cell, such as an insect cell, a mammalian cell, a virus, or a bacterial packaging cell. For example, a host system for manufacturing an AAV vector could further comprise a baculovirus expression system, for example, if the host system is an insect cell. The baculovirus expression system is designed for efficient large-scale viral production and expression of recombinant proteins from baculovirus-infected insect cells. Baculovirus expression systems are further described in, e.g., U.S. Patent Numbers US6919085B2; US6225060B1; US5194376A; the contents of each are incorporated herein by reference in their entireties. Exemplary insect cells include but are not limited to Sf9, Sf21, Hi-5, and S2 insect cell lines.

[0027] In another embodiment, the host system is a cell-free system. For example, the vectors can be synthesized and assembled in an in vitro system. One can prepare cassettes that will express the necessary enzymatic protein, e.g., for lentivirus, pol; for AAV, Rep. In one embodiment, the cell-free 3 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT system comprises helper phage particles. Helper phage particles, for example, M13K07, provide the necessary gene products for particle formation when using phage vectors. Helper phage particles are further reviewed in, for example, in (2005) Helper Phage. In: Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine. Springer, Berlin, Heidelberg; the contents of which are incorporated herein by reference in its entirety.

[0028] Other cassettes can be assembled that will express the necessary structural proteins, e.g., for lentivirus, gag and env; for AAV, the cap gene that expresses VP1, VP2, and VP3. Another vector will be synthesized having a gene operably linked to the desired transgene, which is ultimately flanked between packaging sequences, such as a LTR or an ITR. Various methods to accomplish this are known in the art. Cell-free systems for vector production are further described in, for example, Cerqueira A., et al. Journal of Virology, 2016; Sheng J., et al. The Royal Society of Chemistry, 2017; and Svitkin Y.V., and Sonenberg N. Journal of Virology, 2003; the contents of which are incorporated herein by reference in their entireties.

[0029] Another aspect provided herein describes a cell comprising any of the synthetic nucleic acids described herein, any of the viruses of claims described herein, or any of the lentivirus described herein.

[0030] In one embodiment of this aspect, or any aspect herein, the cell is a human pluripotent stem cell.

[0031] In one embodiment of this aspect, or any aspect herein, the cell is an iPSC.

[0032] Another aspect provided herein describes a stable cell line comprising any of the synthetic nucleic acids described herein, any of the viruses described herein, or any of the lentivirus described herein.

[0033] In one embodiment of this aspect, or any aspect herein, the cell is a human pluripotent stem cell.

[0034] In one embodiment of this aspect, or any aspect herein, the cell is an iPSC.

[0035] Another aspect provided herein describes a method comprising differentiating a population of any of the cells or stable cell line described herein for a sufficient time to promote differentiation into a population of T cells.

[0036] Another aspect provided herein describes a method comprising: differentiating a population of any of the cells or stable cell lines described herein for a sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium; and differentiating the resultant population of CD34+ hemogenic endothelium in a CD3+-T-cell-differentiation media in the presence of a Notch ligand for a sufficient time to promote differentiation into a population of CD3+ T cells.

[0037] In one embodiment of this aspect, or any aspect herein, the Notch ligand is attached to a solid substrate. In one embodiment of this aspect, or any aspect herein, the Notch ligand is attached to a 4 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT cell culture dish. In one embodiment of this aspect, or any aspect herein, the Notch ligand is not derived from a stromal cell.

[0038] In one embodiment of this aspect, or any aspect herein, differentiating the hemogenic endothelium in the presence of a Notch ligand does not comprise co-culturing with a stromal cell expressing a Notch ligand.

[0039] In one embodiment of this aspect, or any aspect herein, differentiating the hemogenic endothelium in the presence of a Notch ligand does not comprise co-culturing with OP9-DL1 cells or OP9-DL4 cells.

[0040] In one embodiment of this aspect, or any aspect herein, the Notch ligand is selected from the group consisting of Delta-like-1 (DLL1), Delta-like-4 (DLL4), immobilized Delta1ext-IgG, and immobilized Delta4ext-IgG.

[0041] In one embodiment of this aspect, or any aspect herein, immobilized Delta1ext-IgG consists of an extracellular domain of human Delta-like-1 fused to the Fc domain of a human immunoglobulin gene or that of an immunoglobulin gene from another suitable species, such as mouse or rabbit.

[0042] In one embodiment of this aspect, or any aspect herein, the sufficient time to promote differentiation into a population of CD3+ T cells is at least 4 weeks.

[0043] In one embodiment of this aspect, or any aspect herein, the CD3+-T-cell-differentiation media is serum-free.

[0044] In one embodiment of this aspect, or any aspect herein, the cells are contacted with a lymphoid induction media comprising SCF, FLT3, TPO, IL-3, and IL-7.

[0045] In one embodiment of this aspect, or any aspect herein, the cells are contacted with a T-cell specification media comprising SCF, FLT3, TPO, and IL-7.

[0046] In one embodiment of this aspect, or any aspect herein, the cells are contacted with a T-cell maturation media comprising FLT3 and IL-7.

[0047] In one embodiment of this aspect, or any aspect herein, the cells are contacted with a SP T-cell induction media comprising FLT3, IL-7, CD3 / CD28 T cell activator, and IL-15.

[0048] In one embodiment of this aspect, or any aspect herein, the cells are contacted with the lymphoid induction media for at least 7 days, i.e., from day 0 to day 7 of method.

[0049] In one embodiment of this aspect, or any aspect herein, the cells are contacted with the T-cell specification media at least 7 days, i.e., from day 7 to day 14 of method.

[0050] In one embodiment of this aspect, or any aspect herein, the cells are contacted with the T-cell maturation media at least 20 days, i.e., from day 14 to day 35 of method.

[0051] In one embodiment of this aspect, or any aspect herein, the cells are contacted with the SP T- cell induction media at least 7 days, i.e., from day 35 to day 42 of method.

[0052] In one embodiment of this aspect, or any aspect herein, the population of CD34+ hemogenic endothelium is CD45 negative / low. 5 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0053] In one embodiment of this aspect, or any aspect herein, the population of CD34+ hemogenic endothelium is CD73 negative.

[0054] In one embodiment of this aspect, or any aspect herein, the population of CD34+ hemogenic endothelium has improved potential for T cell differentiation.

[0055] In one embodiment of this aspect, or any aspect herein, the resultant population of T cells are CD3+TCRa+b+ cells.

[0056] In one embodiment of this aspect, or any aspect herein, the method further comprises the step of genetically modifying the resultant population of CD34+ hemogenic endothelium or the resultant population of CD3+TCRa+b+ cells T cells, or any resultant cells that are more mature than CD34+ hemogenic endothelial cells or less mature than CD3+TCRa+b+ T cells.

[0057] In one embodiment of this aspect, or any aspect herein, the genetic modification is editing an endogenous HLA, removing an endogenous TCR, and / or expressing a chimeric antigen receptor (CAR).

[0058] In one embodiment of this aspect, or any aspect herein, the sufficient time to promote differentiation from a cell into a population of CD34+ hemogenic endothelium is about 8 days.

[0059] In one embodiment of this aspect, or any aspect herein, the sufficient time to promote differentiation from the population of CD34+ cells into a population of resultant T-cells is about 14 days.

[0060] In one embodiment of this aspect, or any aspect herein, the resultant T cells comprise a high percentage of CD3+TCRa+b+ cells.

[0061] Another aspect provided herein describes an immune cell produced by any of the methods described herein.

[0062] Another aspect provided herein describes a composition comprising any of the immune cell described herein or population thereof.

[0063] In one embodiment of this aspect, or any aspect herein, the composition further comprises a pharmaceutically acceptable carrier.

[0064] Another aspect provided herein describes a pharmaceutical composition comprising any of the immune cell described herein or population thereof, and a pharmaceutically acceptable carrier.

[0065] In one embodiment of this aspect, or any aspect herein, for use in cellular therapy in a subject.

[0066] Another aspect provided herein describes a method of cellular therapy, the method comprising administering any of the immune cell described herein or population thereof, or any of the compositions described herein, or any of the pharmaceutical compositions described herein to a recipient subject in need thereof.

[0067] In one embodiment of this aspect, or any aspect herein, the recipient subject has undergone chemotherapy and / or irradiation. 6 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0068] In one embodiment of this aspect, or any aspect herein, the recipient subject has deficiencies in immune function and / or lymphocyte reconstitution.

[0069] In one embodiment of this aspect, or any aspect herein, the recipient subject has cancer.

[0070] In one embodiment of this aspect, or any aspect herein, the recipient subject has an auto- immune disease.

[0071] In one embodiment of this aspect, or any aspect herein, the immune cell or population thereof is autologous to the recipient subject.

[0072] In one embodiment of this aspect, or any aspect herein, the immune cell or population thereof is HLA type matched with the recipient subject.

[0073] In one embodiment of this aspect, or any aspect herein, the immune cell or population thereof is not HLA type matched with the recipient subject.

[0074] Another aspect provided herein describes a use of any of the cells or stable cell lines described herein for differentiation into a population of CD34+ hemogenic endothelium.

[0075] Another aspect provided herein describes a use of any of the cells or stable cell line described herein for differentiation into a population of resultant T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Fig.1A and 1B present an exemplary N-BETT lentiviral plasmid. Fig 1A presents a schematic of the plasmid. Fig.1B presents a sequence of the plasmid in Fig.1A. The highlighted text in Fig.1B corresponds with the elements of Fig.1A. Fig.1B presents SEQ ID NO: 4.

[0077] Fig.2A and 2B present an exemplary N-BETT lentiviral plasmid. Fig 2A presents a schematic of the plasmid. Fig.2B presents a sequence of the plasmid in Fig.2A. The highlighted text in Fig.2B corresponds with the elements of Fig.2A. Fig.2B presents SEQ ID NO: 5.

[0078] Fig.3 presents a schematic of a T cell differentiation protocol, e.g., as described in Example 2. The addition of a CAR by lentiviral transfer is done during T-cell maturation, typically around Day 17-21, after the cells have recovered from the replating and before the frequent cell death and debris formation that occurs during SP T-cell induction.

[0079] Fig.4 presents a plasmid map for a lentiviral vector designed to overexpress mNeon and BCL-XL while simultaneously knocking down EZH1, TP53 and TET2 (N-BETT). (pLVX- mNeon-2a-BCL-XL-U6-shEZH1-shTP53-shTET2). BCL-XL: anti-apoptotic gene (PMID: 9310841, 7621080). Transgenic expression of a related gent (BCL2) has been shown to improve the persistence of CAR-T cells (PMID:33429845). shEZH1: reducing EZH1 expression promotes lymphopoiesis (PMID: 29342143) and the development of cyctotoxic T cells with enhanced antitumor activity (PMID: 35931029). shTP53: reducing expression of the TP53 tumor suppressor gene can protect thymocytes from apoptosis (PMID: 12637507). shTET2: TET2 inactivation can increase the 7 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT persistence and anti-tumor activity of CAR-T cells and tumor-infiltrating lymphocytes (PMID 35110735; 33589517).

[0080] Fig.5 presents bar graphs depicting flow cytometric analysis of indicated populations in hiPSC derived iT cells (WT vs N-BETT), mean+ / - SEM, N=3. ns: not significant, *: P ≤ 0.05, **:P ≤ 0.01, ***: P ≤ 0.001, ****: P ≤ 0.0001, multiple t-tests. This plot demonstrates the significant improvement of the N-BETT-hiPSCs compared to Wildtype (WT) in all tested parameters (viability, CD45+, CD3+, CD3+TCRab+, CD5+CD7+, CD8SP). Note that the cells should not be double positive for CD4+CD8+ at this point anymore, so no difference to WT is expected).

[0081] Fig.6 presents bar graphs depicting yield of CD3+TCRab+ iT cells per input CD34+ cells derived from WT (1157.2) vs N-BETT hiPSCs in indicated protocols. Yield of CD3+TCRab+ iT cells per input CD34+ cells derived from N-BETT hiPSCs in both protocols. Yield of CD3+TCRab+ iT cells calculated per input hiPSC. Wildtype hemogenic endothelial cells derived from hiPSCs with the simplified CD34+ derivation protocol fail to differentiate into mature CD3+TCRab+ cells. When N- BETT is introduced into hiPSC the derived CD34+ cells can overcome this limitation. For reference, wildtype hiPSC using the Keller EB method give rise to ~0.1 mature T cells. Compared to this N- BETT 2DK-derived CD34+ cells give rise to T cells at more than 3 orders of magnitude higher efficiencyMean + / - SEM, N=number of dots.

[0082] Fig.7 present representative flow plots of iT day 42 cells derived from hiPSCs carrying the N- BETT construct and differentiated into CD34+ cells via two different protocols (Keller EB vs 2DK protocol)

[0083] Fig.8 presents flow plots depicting CD3 (x-axis) vs TCRab (y-axis) staining in hiPSC-derived iT cells on day 42. hiPSCs are either WT 1157.2 or carry the N-BETT construct in all -1 versions. The cells have been pre-gated on single, live, CD45+.

[0084] Fig.9 presents flow plots depicting CD8 (x-axis) vs CD4 (y-axis) staining in hiPSC-derived iT cells on day 28. Pre-gated on single, live, CD45+ cells. The performance of N-BETT lacking either TP53 KD, or EZH1 KD or BCLXL OE was compared to hiPSCs just overexpressing BCLXL. Empty plots indicate no live cells, CD45+ cells at the time of analysis. BCLXL overexpression is an essential component in N-BETT; BCLXL OE alone is not sufficient to promote iT differentiation.

[0085] Fig.10 presents a schematic of the stroma free EZ-T cell platform for the derivation of allogenic CAR-T cells from human iPSCs. This platform can be further described in, e.g., Jing et al., 2022), which is incorporated herein by reference in its entirety.

[0086] Fig.11 presents data showing increased viability in N-BETT iT cells compared to WT at all stages of iT differentiation. hiPSCs have been stably transduced with lentivirus encoding the N-BETT construct. N-BETT positive hiPSCs and control untransduced hiPSCs have been differentiated into hematopoietic progenitors, following T cell differentiation utilizing the protocol 8 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT developed by Jing et al., 2022. (Bar graphs) Improved T cell marker expression in hiPSC-derived T cells stably expressing N-BETT.

[0087] Figs 12A and 12B present data showing N-BETT positive cells outcompete WT cells. (Fig. 12A) Flow cytometric assessment of mNeon fluorescence in hiPSC-derived CD34+ cells undergoing iT cell differentiation after low MOI (0.5) transduction with N-BETT lentivirus. By week 6 of iT cell differentiation the culture is almost entirely made up of mNeon+ cells indicating that N-BETT expressing cells outcomepete untransduced cells during iT cell differentiation. This effect has been shown in two unrelated hiPSC lines. (Fig.12B) Flow cytometric assessment of CD4+CD8+ double positive cells at day 28 of iT cell differentiation (same experimental setup as in panel A). The percentage of CD4+CD8+ double positive cells is significantly higher in the mNeon+ fraction of the culture, indicating improved T cell differentiation in cells expressing N-BETT. N=3 and N=5 in two unrelated hiPSC lines.

[0088] Fig.13 presents data showing hiPSC-derived N-BETT T cells transduced with CD19 CAR cells display potent anti-tumor activity. hiPSC-derived N-BETT iT cells have been transduced with anti CD19 CAR (Kymriah [tisagenlecleucel]) and co-cultured with a Burkitt’s lymphoma cell line (RAJI). CAR+ N-BETT iT cells efficiently reduce the number of CD19+ cells compared to N-BETT iT cells lacking a CAR.

[0089] Figs 14A and 14B present data showing BCL-XL OE and shTET2 are essential components of N-BETT. (Fig.14A) Flow cytometric assessment of mNeon+ vs mNeon- cell distribution within the CD4+CD8+ double positive iT cell fraction following low MOI transduction with either the full- length N-BETT lentiviral vector or N-1 iterations following two different hematopoietic progenitor differentiation protocols. In both protocols, omission of Bcl-xl from the vector completely attenuates the growth and differentiation advantage of N-BETT positive cells. Removal of TET2 shRNA from the construct has a negative impact on the phenotype, while TP53 and EZH1 shRNA can be omitted from the construct, without drastically reducing the percentage of mNeon+ cells. (Fig.15B) Impact of factor omission on CD3+TCR⍺β+ iT cell differentiation compared to full length N-BETT. DETAILED DESCRIPTION Synthetic Nucleic Acids

[0090] One aspect provided herein describes a synthetic nucleic acid encoding at least one promoter, a transgene, and at least one inhibitory nucleic acid sequence.

[0091] In one embodiment, the inhibitory nucleic acid sequence is an RNAi. The term “RNAi” as used herein refers to interfering RNA or RNA interference. RNAi refers to a means of selective post- transcriptional gene silencing by destruction of specific mRNA by molecules that bind and inhibit the processing of mRNA, for example inhibit mRNA translation or result in mRNA degradation. As used herein, the term "RNAi" refers to any type of interfering RNA, including but are not limited to, 9 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT siRNA, shRNA, endogenous microRNA and artificial microRNA. For instance, it includes sequences previously identified as siRNA, regardless of the mechanism of down-stream processing of the RNA (i.e., although siRNAs are believed to have a specific method of in vivo processing resulting in the cleavage of mRNA, such sequences can be incorporated into the vectors in the context of the flanking sequences described herein).

[0092] In one embodiment of this aspect, or any aspect herein, the inhibitory nucleic acid sequence inhibits a target gene selected from the group consisting of EZH1, TP53, or TET2.

[0093] Methods and compositions described herein require that the EZH1 is targeted. As used herein, “enhancer of zeste 1 polycomb repressive complex 2 subunit (EZH1)” refers to a component of a noncanonical Polycomb repressive complex-2 (PRC2) that mediates methylation of histone H3 lys27 (H3K27) and functions in the maintenance of embryonic stem cell pluripotency and plasticity. Sequences for EZH1, also known as KMT6B, are known for a number of species, e.g., human EZH1 (NCBI Gene ID: 2145) polypeptide (e.g., NCBI Ref Seq NP_ 001308008.1) and mRNA (e.g., NCBI Ref Seq NM_ 001321079.2). EZH1 can refer to human EZH1, including naturally occurring variants, molecules, and alleles thereof. EZH1 refers to the mammalian EZH1 of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like. The nucleic sequence of SEQ ID NO: 1 comprises the nucleic sequence which encodes EZH1. atggagg attacagcaa gatggaaata ccaaatcccc 61 ctacctccaa atgtatcact tactggaaaa gaaaagtgaa atctgaatac atgcgacttc 121 gacaacttaa acggcttcag gcaaatatgg gtgcaaaggc tttgtatgtg gcaaattttg 181 caaaggttca agaaaaaacc cagatcctca atgaagaatg gaagaagctt cgtgtccaac 241 ctgttcagtc aatgaagcct gtgagtggac acccttttct caaaaagtgt accatagaga 301 gcattttccc gggatttgca agccaacata tgttaatgag gtcactgaac acagttgcat 361 tggttcccat catgtattcc tggtcccctc tccaacagaa ctttatggta gaagatgaga 421 cggttttgtg caatattccc tacatgggag atgaagtgaa agaagaagat gagactttta 481 ttgaggagct gatcaataac tatgatggga aagtccatgg tgaagaagag atgatccctg 541 gatccgttct gattagtgat gctgtttttc tggagttggt cgatgccctg aatcagtact 601 cagatgagga ggaggaaggg cacaatgaca cctcagatgg aaagcaggat gacagcaaag 661 aagatctgcc agtaacaaga aagagaaagc gacatgctat tgaaggcaac aaaaagagtt 721 ccaagaaaca gttcccaaat gacatgatct tcagtgcaat tgcctcaatg ttccctgaga 781 atggtgtccc agatgacatg aaggagaggt atcgagaact aacagagatg tcagacccca 841 atgcacttcc ccctcagtgc acacccaaca tcgatggccc caatgccaag tctgtgcagc 901 gggagcaatc tctgcactcc ttccacacac ttttttgccg gcgctgcttt aaatacgact 961 gcttccttca cccttttcat gccaccccta atgtatataa acgcaagaat aaagaaatca 1021 agattgaacc agaaccatgt ggcacagact gcttcctttt gctggaagga gcaaaggagt 1081 atgccatgct ccacaacccc cgctccaagt gctctggtcg tcgccggaga aggcaccaca 1141 tagtcagtgc ttcctgctcc aatgcctcag cctctgctgt ggctgagact aaagaaggag 1201 acagtgacag ggacacaggc aatgactggg cctccagttc ttcagaggct aactctcgct 10 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 1261 gtcagactcc cacaaaacag aaggctagtc cagccccacc tcaactctgc gtagtggaag 1321 caccctcgga gcctgtggaa tggactgggg ctgaagaatc tctttttcga gtcttccatg 1381 gcacctactt caacaacttc tgttcaatag ccaggcttct ggggaccaag acgtgcaagc 1441 aggtctttca gtttgcagtc aaagaatcac ttatcctgaa gctgccaaca gatgagctca 1501 tgaacccctc acagaagaag aaaagaaagc acagattgtg ggctgcacac tgcaggaaga 1561 ttcagctgaa gaaagataac tcttccacac aagtgtacaa ctaccaaccc tgcgaccacc 1621 cagaccgccc ctgtgacagc acctgcccct gcatcatgac tcagaatttc tgtgagaagt 1681 tctgccagtg caacccagac tgtcagaatc gtttccctgg ctgtcgctgt aagacccagt 1741 gcaataccaa gcaatgtcct tgctatctgg cagtgcgaga atgtgaccct gacctgtgtc 1801 tcacctgtgg ggcctcagag cactgggact gcaaggtggt ttcctgtaaa aactgcagca 1861 tccagcgtgg acttaagaag cacctgctgc tggccccctc tgatgtggcc ggatggggca 1921 ccttcataaa ggagtctgtg cagaagaacg aattcatttc tgaatactgt ggtgagctca 1981 tctctcagga tgaggctgat cgacgcggaa aggtctatga caaatacatg tccagcttcc 2041 tcttcaacct caataatgat tttgtagtgg atgctactcg gaaaggaaac aaaattcgat 2101 ttgcaaatca ttcagtgaat cccaactgtt atgccaaagt ggtcatggtg aatggagacc 2161 atcggattgg gatctttgcc aagagggcaa ttcaagctgg cgaagagctc ttctttgatt 2221 acaggtacag ccaagctgat gctctcaagt acgtggggat cgagagggag accgacgtcc 2281 tttag (SEQ ID NO: 1)

[0094] Methods and compositions described herein require that the TP53 is targeted. As used herein, “tumor protein p53 (TP53)” refers to a tumor suppressor protein containing transcriptional activation, DNA binding, and oligomerization domains. Sequences for TP53, also known as P53, BCC7, LFS1, BMFS5, and TRP53, are known for a number of species, e.g., human TP53 (NCBI Gene ID: 7157) polypeptide (e.g., NCBI Ref Seq NP_ 000537.3) and mRNA (e.g., NCBI Ref Seq NM_ 000546.6). TP53 can refer to human TP53, including naturally occurring variants, molecules, and alleles thereof. TP53 refers to the mammalian TP53 of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like. The nucleic sequence of SEQ ID NO: 2 comprises the nucleic sequence which encodes TP53. atggagga gccgcagtca gatcctagcg tcgagccccc 181 tctgagtcag gaaacatttt cagacctatg gaaactactt cctgaaaaca acgttctgtc 241 ccccttgccg tcccaagcaa tggatgattt gatgctgtcc ccggacgata ttgaacaatg 301 gttcactgaa gacccaggtc cagatgaagc tcccagaatg ccagaggctg ctccccccgt 361 ggcccctgca ccagcagctc ctacaccggc ggcccctgca ccagccccct cctggcccct 421 gtcatcttct gtcccttccc agaaaaccta ccagggcagc tacggtttcc gtctgggctt 481 cttgcattct gggacagcca agtctgtgac ttgcacgtac tcccctgccc tcaacaagat 541 gttttgccaa ctggccaaga cctgccctgt gcagctgtgg gttgattcca cacccccgcc 601 cggcacccgc gtccgcgcca tggccatcta caagcagtca cagcacatga cggaggttgt 661 gaggcgctgc ccccaccatg agcgctgctc agatagcgat ggtctggccc ctcctcagca 721 tcttatccga gtggaaggaa atttgcgtgt ggagtatttg gatgacagaa acacttttcg 781 acatagtgtg gtggtgccct atgagccgcc tgaggttggc tctgactgta ccaccatcca 11 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 841 ctacaactac atgtgtaaca gttcctgcat gggcggcatg aaccggaggc ccatcctcac 901 catcatcaca ctggaagact ccagtggtaa tctactggga cggaacagct ttgaggtgcg 961 tgtttgtgcc tgtcctggga gagaccggcg cacagaggaa gagaatctcc gcaagaaagg 1021 ggagcctcac cacgagctgc ccccagggag cactaagcga gcactgccca acaacaccag 1081 ctcctctccc cagccaaaga agaaaccact ggatggagaa tatttcaccc ttcagatccg 1141 tgggcgtgag cgcttcgaga tgttccgaga gctgaatgag gccttggaac tcaaggatgc 1201 ccaggctggg aaggagccag gggggagcag ggctcactcc agccacctga agtccaaaaa 1261 gggtcagtct acctcccgcc ataaaaaact catgttcaag acagaagggc ctgactcaga 1321 ctga (SEQ ID NO: 2)

[0095] Methods and compositions described herein require that the TET2 is targeted. As used herein, “tet methylcytosine dioxygenase 2 (TET2)” refers a methylcytosine dioxygenase that catalyzes the conversion of methylcytosine to 5-hydroxymethylcytosine. Sequences for TET2, also known as MDS, IMD75, and KIAA1546, are known for a number of species, e.g., human TET2 (NCBI Gene ID: 54790) polypeptide (e.g., NCBI Ref Seq NP_ 001120680.1) and mRNA (e.g., NCBI Ref Seq NM_ 001127208.3). TET2 can refer to human TET2, including naturally occurring variants, molecules, and alleles thereof. TET2 refers to the mammalian TET2 of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like. The nucleic sequence of SEQ ID NO: 3 comprises the nucleic sequence which encodes TET2. atgg 301 aacaggatag aaccaaccat gttgagggca acagactaag tccattcctg ataccatcac 361 ctcccatttg ccagacagaa cctctggcta caaagctcca gaatggaagc ccactgcctg 421 agagagctca tccagaagta aatggagaca ccaagtggca ctctttcaaa agttattatg 481 gaataccctg tatgaaggga agccagaata gtcgtgtgag tcctgacttt acacaagaaa 541 gtagagggta ttccaagtgt ttgcaaaatg gaggaataaa acgcacagtt agtgaacctt 601 ctctctctgg gctccttcag atcaagaaat tgaaacaaga ccaaaaggct aatggagaaa 661 gacgtaactt cggggtaagc caagaaagaa atccaggtga aagcagtcaa ccaaatgtct 721 ccgatttgag tgataagaaa gaatctgtga gttctgtagc ccaagaaaat gcagttaaag 781 atttcaccag tttttcaaca cataactgca gtgggcctga aaatccagag cttcagattc 841 tgaatgagca ggaggggaaa agtgctaatt accatgacaa gaacattgta ttacttaaaa 901 acaaggcagt gctaatgcct aatggtgcta cagtttctgc ctcttccgtg gaacacacac 961 atggtgaact cctggaaaaa acactgtctc aatattatcc agattgtgtt tccattgcgg 1021 tgcagaaaac cacatctcac ataaatgcca ttaacagtca ggctactaat gagttgtcct 1081 gtgagatcac tcacccatcg catacctcag ggcagatcaa ttccgcacag acctctaact 1141 ctgagctgcc tccaaagcca gctgcagtgg tgagtgaggc ctgtgatgct gatgatgctg 1201 ataatgccag taaactagct gcaatgctaa atacctgttc ctttcagaaa ccagaacaac 1261 tacaacaaca aaaatcagtt tttgagatat gcccatctcc tgcagaaaat aacatccagg 1321 gaaccacaaa gctagcgtct ggtgaagaat tctgttcagg ttccagcagc aatttgcaag 12 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 1381 ctcctggtgg cagctctgaa cggtatttaa aacaaaatga aatgaatggt gcttacttca 1441 agcaaagctc agtgttcact aaggattcct tttctgccac taccacacca ccaccaccat 1501 cacaattgct tctttctccc cctcctcctc ttccacaggt tcctcagctt ccttcagaag 1561 gaaaaagcac tctgaatggt ggagttttag aagaacacca ccactacccc aaccaaagta 1621 acacaacact tttaagggaa gtgaaaatag agggtaaacc tgaggcacca ccttcccaga 1681 gtcctaatcc atctacacat gtatgcagcc cttctccgat gctttctgaa aggcctcaga 1741 ataattgtgt gaacaggaat gacatacaga ctgcagggac aatgactgtt ccattgtgtt 1801 ctgagaaaac aagaccaatg tcagaacacc tcaagcataa cccaccaatt tttggtagca 1861 gtggagagct acaggacaac tgccagcagt tgatgagaaa caaagagcaa gagattctga 1921 agggtcgaga caaggagcaa acacgagatc ttgtgccccc aacacagcac tatctgaaac 1981 caggatggat tgaattgaag gcccctcgtt ttcaccaagc ggaatcccat ctaaaacgta 2041 atgaggcatc actgccatca attcttcagt atcaacccaa tctctccaat caaatgacct 2101 ccaaacaata cactggaaat tccaacatgc ctggggggct cccaaggcaa gcttacaccc 2161 agaaaacaac acagctggag cacaagtcac aaatgtacca agttgaaatg aatcaagggc 2221 agtcccaagg tacagtggac caacatctcc agttccaaaa accctcacac caggtgcact 2281 tctccaaaac agaccattta ccaaaagctc atgtgcagtc actgtgtggc actagatttc 2341 attttcaaca aagagcagat tcccaaactg aaaaacttat gtccccagtg ttgaaacagc 2401 acttgaatca acaggcttca gagactgagc cattttcaaa ctcacacctt ttgcaacata 2461 agcctcataa acaggcagca caaacacaac catcccagag ttcacatctc cctcaaaacc 2521 agcaacagca gcaaaaatta caaataaaga ataaagagga aatactccag acttttcctc 2581 acccccaaag caacaatgat cagcaaagag aaggatcatt ctttggccag actaaagtgg 2641 aagaatgttt tcatggtgaa aatcagtatt caaaatcaag cgagttcgag actcataatg 2701 tccaaatggg actggaggaa gtacagaata taaatcgtag aaattcccct tatagtcaga 2761 ccatgaaatc aagtgcatgc aaaatacagg tttcttgttc aaacaataca cacctagttt 2821 cagagaataa agaacagact acacatcctg aactttttgc aggaaacaag acccaaaact 2881 tgcatcacat gcaatatttt ccaaataatg tgatcccaaa gcaagatctt cttcacaggt 2941 gctttcaaga acaggagcag aagtcacaac aagcttcagt tctacaggga tataaaaata 3001 gaaaccaaga tatgtctggt caacaagctg cgcaacttgc tcagcaaagg tacttgatac 3061 ataaccatgc aaatgttttt cctgtgcctg accagggagg aagtcacact cagacccctc 3121 cccagaagga cactcaaaag catgctgctc taaggtggca tctcttacag aagcaagaac 3181 agcagcaaac acagcaaccc caaactgagt cttgccatag tcagatgcac aggccaatta 3241 aggtggaacc tggatgcaag ccacatgcct gtatgcacac agcaccacca gaaaacaaaa 3301 catggaaaaa ggtaactaag caagagaatc cacctgcaag ctgtgataat gtgcagcaaa 3361 agagcatcat tgagaccatg gagcagcatc tgaagcagtt tcacgccaag tcgttatttg 3421 accataaggc tcttactctc aaatcacaga agcaagtaaa agttgaaatg tcagggccag 3481 tcacagtttt gactagacaa accactgctg cagaacttga tagccacacc ccagctttag 3541 agcagcaaac aacttcttca gaaaagacac caaccaaaag aacagctgct tctgttctca 3601 ataattttat agagtcacct tccaaattac tagatactcc tataaaaaat ttattggata 3661 cacctgtcaa gactcaatat gatttcccat cttgcagatg tgtagagcaa attattgaaa 3721 aagatgaagg tcctttttat acccatctag gagcaggtcc taatgtggca gctattagag 13 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 3781 aaatcatgga agaaaggttt ggacagaagg gtaaagctat taggattgaa agagtcatct 3841 atactggtaa agaaggcaaa agttctcagg gatgtcctat tgctaagtgg gtggttcgca 3901 gaagcagcag tgaagagaag ctactgtgtt tggtgcggga gcgagctggc cacacctgtg 3961 aggctgcagt gattgtgatt ctcatcctgg tgtgggaagg aatcccgctg tctctggctg 4021 acaaactcta ctcggagctt accgagacgc tgaggaaata cggcacgctc accaatcgcc 4081 ggtgtgcctt gaatgaagag agaacttgcg cctgtcaggg gctggatcca gaaacctgtg 4141 gtgcctcctt ctcttttggt tgttcatgga gcatgtacta caatggatgt aagtttgcca 4201 gaagcaagat cccaaggaag tttaagctgc ttggggatga cccaaaagag gaagagaaac 4261 tggagtctca tttgcaaaac ctgtccactc ttatggcacc aacatataag aaacttgcac 4321 ctgatgcata taataatcag attgaatatg aacacagagc accagagtgc cgtctgggtc 4381 tgaaggaagg ccgtccattc tcaggggtca ctgcatgttt ggacttctgt gctcatgccc 4441 acagagactt gcacaacatg cagaatggca gcacattggt atgcactctc actagagaag 4501 acaatcgaga atttggagga aaacctgagg atgagcagct tcacgttctg cctttataca 4561 aagtctctga cgtggatgag tttgggagtg tggaagctca ggaggagaaa aaacggagtg 4621 gtgccattca ggtactgagt tcttttcggc gaaaagtcag gatgttagca gagccagtca 4681 agacttgccg acaaaggaaa ctagaagcca agaaagctgc agctgaaaag ctttcctccc 4741 tggagaacag ctcaaataaa aatgaaaagg aaaagtcagc cccatcacgt acaaaacaaa 4801 ctgaaaacgc aagccaggct aaacagttgg cagaactttt gcgactttca ggaccagtca 4861 tgcagcagtc ccagcagccc cagcctctac agaagcagcc accacagccc cagcagcagc 4921 agagacccca gcagcagcag ccacatcacc ctcagacaga gtctgtcaac tcttattctg 4981 cttctggatc caccaatcca tacatgagac ggcccaatcc agttagtcct tatccaaact 5041 cttcacacac ttcagatatc tatggaagca ccagccctat gaacttctat tccacctcat 5101 ctcaagctgc aggttcatat ttgaattctt ctaatcccat gaacccttac cctgggcttt 5161 tgaatcagaa tacccaatat ccatcatatc aatgcaatgg aaacctatca gtggacaact 5221 gctccccata tctgggttcc tattctcccc agtctcagcc gatggatctg tataggtatc 5281 caagccaaga ccctctgtct aagctcagtc taccacccat ccatacactt taccagccaa 5341 ggtttggaaa tagccagagt tttacatcta aatacttagg ttatggaaac caaaatatgc 5401 agggagatgg tttcagcagt tgtaccatta gaccaaatgt acatcatgta gggaaattgc 5461 ctccttatcc cactcatgag atggatggcc acttcatggg agccacctct agattaccac 5521 ccaatctgag caatccaaac atggactata aaaatggtga acatcattca ccttctcaca 5581 taatccataa ctacagtgca gctccgggca tgttcaacag ctctcttcat gccctgcatc 5641 tccaaaacaa ggagaatgac atgctttccc acacagctaa tgggttatca aagatgcttc 5701 cagctcttaa ccatgataga actgcttgtg tccaaggagg cttacacaaa ttaagtgatg 5761 ctaatggtca ggaaaagcag ccattggcac tagtccaggg tgtggcttct ggtgcagagg 5821 acaacgatga ggtctggtca gacagcgagc agagctttct ggatcctgac attgggggag 5881 tggccgtggc tccaactcat gggtcaattc tcattgagtg tgcaaagcgt gagctgcatg 5941 ccacaacccc tttaaagaat cccaatagga atcaccccac caggatctcc ctcgtctttt 6001 accagcataa gagcatgaat gagccaaaac atggcttggc tctttgggaa gccaaaatgg 6061 ctgaaaaagc ccgtgagaaa gaggaagagt gtgaaaagta tggcccagac tatgtgcctc 6121 agaaatccca tggcaaaaaa gtgaaacggg agcctgctga gccacatgaa acttcagagc 14 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 6181 ccacttacct gcgtttcatc aagtctcttg ccgaaaggac catgtccgtg accacagact 6241 ccacagtaac tacatctcca tatgccttca ctcgggtcac agggccttac aacagatata 6301 tatga (SEQ ID NO: 3)

[0096] In one embodiment, the synthetic nucleic acid comprises from 5’ to 3’, (a) a 5’ terminal repeat, (b) at least one inhibitory nucleic acid sequence operatively linked to a promoter, (c) the transgene operatively linked to a promoter, and (c) a 3’ terminal repeat.

[0097] In one embodiment, the synthetic nucleic acid comprises from 5’ to 3’, (a) a 5’ terminal repeat, (b) at least one inhibitory nucleic acid sequence operatively linked to a promoter, (c) the BCL-XL operatively linked to the EF1a promoter, and (d) a 3’ terminal repeat.

[0098] In one embodiment, the synthetic nucleic acid comprises from 5’ to 3’, (a) a 5’ terminal repeat, (b) at least one of EZH1 shRNA operatively linked to a U6 promoter, TP53 shRNA operatively linked to a U6 promoter, and TET2 shRNA operatively linked to a promoter, (c) the BCL-XL operatively linked to the EF1a promoter, and (d) a 3’ terminal repeat.

[0099] In one embodiment of this aspect, or any aspect herein, the synthetic nucleic acid comprises from 5’ to 3’, (a) a 5’ terminal repeat, (b) at least one inhibitory nucleic acid sequence operatively linked to a U6 promoter, TP53 shRNA operatively linked to a U6 promoter, and TET2 shRNA operatively linked to a promoter, (c) the BCL-XL operatively linked to the EF1a promoter, and (d) a 3’ terminal repeat.

[0100] In one embodiment of this aspect, or any aspect herein, the synthetic nucleic acid comprises from 5’ to 3’, (a) a 5’ terminal repeat, (b) at least one of TP53 shRNA and TET2 shRNA operatively linked to a U6 promoter, TP53 shRNA operatively linked to a U6 promoter, and TET2 shRNA operatively linked to a promoter, (c) the BCL-XL operatively linked to the EF1a promoter, and (d) a 3’ terminal repeat.

[0101] Another aspect provided herein describes a synthetic nucleic acid comprising from 5’ to 3’ a sequence encoding, (a) a 5’ terminal repeat, (b) at least one of EZH1 shRNA operatively linked to a U6 promoter, TP53 shRNA operatively linked to a U6 promoter, and TET2 shRNA operatively linked to a promoter, (c) the BCL-XL operatively linked to the EF1a promoter, and (d) a 3’ terminal repeat.

[0102] Another aspect provided herein describes a synthetic nucleic acid comprising from 5’ to 3’ a sequence encoding, (a) a 5’ terminal repeat, (b) a TET2 shRNA operatively linked to a promoter, (c) the BCL-XL operatively linked to the EF1a promoter, and (d) a 3’ terminal repeat.

[0103] In one embodiment, the synthetic nucleic acid comprises a sequence of SEQ ID NO: 4 or SEQ ID NO: 5 (see, e.g., Figs 1A-2B, and 4) In one embodiment, the synthetic nucleic acid comprises a sequence at is 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%, identity to SEQ ID NO: 4 or SEQ ID NO: 5. 15 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0104] In one embodiment of this aspect, the promoter is a EF1a promoter, a U6 promoter, an H1 promoter, a PGK1 promoter, or Ubiquitin-C (UBC) promoter. For example, a promoter suitable for expressing an inhibitory nucleic acid sequence, such as aU6 or H1 promoter capable of directing RNA polymerase III mediated expression of an shRNA or an EF1alpha- (EEF1A1), PGK1-, or Ubiquitin-C (UBC) promoter capable of directing RNA polymerase II mediated expression of a miRNA or of an mRNA that encodes a dominant-negative protein, and at least one promoter is a promoter such as an EF1alpha- (EEF1A1), PGK1-, or Ubiquitin-C (UBC) promoter that is capable of mediating expression of an mRNA that encodes a functional protein.

[0105] In one embodiment of this aspect, or any aspect herein, the transgene encodes a functional BCL-XL protein.

[0106] Methods and compositions described herein require that the BCL-XL is targeted. As used herein, “BCL2 like 1 (BCL-XL)” refers to a protein encoded by this gene belongs to the BCL-2 protein family, which form hetero- or homodimers and act as anti- or pro-apoptotic regulators that are involved in a wide variety of cellular activities. Sequences for BCL-XL, also known as BCLX; BCL2L; Bcl-X; PPP1R52; BCL-XL; and BCL2L1 are known for a number of species, e.g., human BCL-XL (NCBI Gene ID: 598), polypeptide (e.g., NCBI Ref Seq NP_001182.1) and mRNA (e.g., NCBI Ref Seq NM_001191.4). BCL-XL can refer to human BCL-XL, including naturally occurring variants, molecules, and alleles thereof. BCL-XL refers to the mammalian BCL-XL of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like. The nucleic sequence of SEQ ID NO: 4 comprises the nucleic sequence which encodes a codon-optimized BCL-XL. 1ATGAGCCAGT CCAACCGGGA ACTGGTGGTG GACTTCCTGT CATATAAGCT GAGCCAGAAA61 GGGTACAGCT GGTCCCAATT TAGCGACGTG GAGGAAAATA GAACAGAGGC CCCTGAAGGC 121 ACCGAGAGCG AGATGGAAAC CCCTAGCGCC ATCAACGGCA ATCCTAGCTG GCACCTGGCT 181 GACAGCCCCG CCGTGAACGG CGCCACGGCC CACTCTTCTA GCCTGGACGC CAGAGAGGTG 241 ATCCCTATGG CCGCCGTTAA GCAGGCCCTG AGAGAAGCCG GAGATGAGTT CGAACTGCGG 301 TACAGACGGG CCTTCAGCGA CCTGACCTCC CAGCTGCACA TTACACCTGG CACCGCCTAC 361 CAGAGCTTCG AGCAGGTGGT GAACGAGCTG TTTAGAGATG GCGTCAATTG GGGCAGAATC 421 GTGGCCTTCT TCAGCTTCGG CGGCGCCCTG TGCGTGGAAA GCGTGGATAA GGAAATGCAG 481 GTGCTGGTGA GCAGAATCGC CGCTTGGATG GCCACCTACC TGAACGACCA CCTGGAGCCC 541 TGGATCCAGG AGAACGGAGG CTGGGACACC TTCGTGGAAC TCTACGGCAA CAACGCCGCT 601 GCTGAAAGCA GAAAGGGCCA GGAGAGATTC AACAGATGGT TCCTGACCGG CATGACAGTG 661 GCCGGAGTGG TCCTTCTGGG CAGCCTCTTT AGCAGAAAGt ga (SEQ ID NO: 4

[0107] In one embodiment of this aspect, or any aspect herein, each of the transgene and at least one inhibitory nucleic acid sequence are operatively linked to the at least one promoter.

[0108] In one embodiment of this aspect, or any aspect herein, the transgene is operatively linked to a EF1a promoter. 16 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0109] In one embodiment of this aspect, or any aspect herein, the at least one inhibitory nucleic acid sequence is operatively linked to a U6 promoter.

[0110] In one embodiment of this aspect, or any aspect herein, the synthetic nucleic acid further comprises a sequence encoding terminal repeats flanking the at least one promoter, the transgene, and the least one inhibitory nucleic sequence.

[0111] In one embodiment of this aspect, or any aspect herein, the synthetic nucleic acid further comprises a sequence encoding a marker gene. Markers genes encode, for example, fluorescent proteins (e.g., green fluorescent protein (GFP), red fluorescent protein (RFP), etc.), nucleic acid tags (e.g., HA-tag, MYC-tag, etc.). Marker genes are useful in identifying cells expressing the nucleic acid encoding the marker gene, for example, by microscopy, Western-blotting, or PCR-based assays.

[0112] In one embodiment of this aspect, or any aspect herein, the terminal repeat is a long terminal repeat (LTR).

[0113] In one embodiment of this aspect, or any aspect herein, the synthetic nucleic acid is a plasmid.

[0114] The synthetic nucleic acid may be contained in and thus further include a vector. Many such vectors useful for transferring exogenous genes into target mammalian cells are available. The vectors may be episomal, e.g., plasmids, virus-derived vectors such as cytomegalovirus, adenovirus, etc., or may be integrated into the target cell genome, through homologous recombination or random integration, e.g., retrovirus-derived vectors such as MMLV, HIV-1, ALV, etc. In some embodiments, combinations of retroviruses and an appropriate packaging cell line may also find use, where the capsid proteins will be functional for infecting the target cells. Usually, the cells and virus will be incubated for at least about 24 hours in the culture medium. The cells are then allowed to grow in the culture medium for short intervals in some applications, e.g., 24-73 hours, or for at least two weeks, and may be allowed to grow for five weeks or more, before analysis. Commonly used retroviral vectors are "defective", i.e., unable to produce viral proteins required for productive infection. Replication of the vector requires growth in the packaging cell line.

[0115] The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, artificial chromosome, virus, virion, etc.

[0116] As used herein, the term "expression vector" refers to a vector that directs expression of an synthetic nucleic acid linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, 17 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification. The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g., 5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0117] Integrating vectors have their delivered RNA / DNA permanently incorporated into the host cell chromosomes. Non-integrating vectors remain episomal which means the nucleic acid contained therein is never integrated into the host cell chromosomes. Examples of integrating vectors include retroviral vectors, lentiviral vectors, hybrid adenoviral vectors, and herpes simplex viral vector.

[0118] One example of a non-integrative vector is a non-integrative viral vector. Non-integrative viral vectors eliminate the risks posed by integrative retroviruses, as they do not incorporate their genome into the host DNA. One example is the Epstein Barr oriP / Nuclear Antigen-1 (“EBNA1”) vector, which is capable of limited self-replication and known to function in mammalian cells. As containing two elements from Epstein-Barr virus, oriP and EBNA1, binding of the EBNA1 protein to the virus replicon region oriP maintains a relatively long-term episomal presence of plasmids in mammalian cells. This particular feature of the oriP / EBNA1 vector makes it ideal for generation of integration-free iPSCs. Another non-integrative viral vector is adenoviral vector and the adeno- associated viral (AAV) vector.

[0119] Another non-integrative viral vector is RNA Sendai viral vector, which can produce protein without entering the nucleus of an infected cell. The F-deficient Sendai virus vector remains in the cytoplasm of infected cells for a few passages, but is diluted out quickly and completely lost after several passages (e.g., 10 passages).

[0120] Another example of a non-integrative vector is a minicircle vector. Minicircle vectors are circularized vectors in which the plasmid backbone has been released leaving only the eukaryotic promoter and cDNA(s) that are to be expressed.

[0121] One aspect herein is a virus expressing any of the synthetic nucleic acids described herein.

[0122] In one embodiment of this aspect, the virus is selected from the group consisting of Retroviruses (alpha-retrovirus, gamma-retrovirus, or lentivirus), adeno-associated virus, paramyxovirus, or alphanodavirus. 18 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0123] Another aspect herein is a lentivirus expressing any of the synthetic nucleic acids described herein.

[0124] Another aspect provided herein describes a cell-free system expressing any of the synthetic nucleic acids described herein. Cell-free systems for viral vector production are further described in, for example, Cerqueira A., et al. Journal of Virology, 2016; Sheng J., et al. The Royal Society of Chemistry, 2017; and Svitkin Y.V., and Sonenberg N. Journal of Virology, 2003; the contents of which are incorporated herein by reference in their entireties.

[0125] Another aspect provided herein describes a host system expressing any of the synthetic nucleic acids described herein.

[0126] In one embodiment, the host system can be a virus-free delivery system. Exemplary virus- free delivery systems are well established in the art and can be adapted by a skilled person for the purpose of delivering any of the synthetic nucleic acids described herein. Examples of suitable virus- free methods include, but are not limited to, transposon systems such as the PiggyBac or the Sleeping Beauty systems, RNA replicon systems, such as the Venezuelan Equine Encephalitis virus derived RNA replicon system, or lipid nanoparticle-based RNA transfection methods.

[0127] As used herein, the term “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain a nucleic acid encoding a polypeptide as described herein in place of non- essential viral genes. The vector and / or particle may be utilized for the purpose of transferring nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0128] In one embodiment, the vector is a virus or a non-viral vector. Non-limiting examples of viral vectors for gene delivery and expressions in cells are retrovirus, adenovirus (types 2 and 5), adeno- associated virus (AAV), Helper-dependent adenoviral vector (HdAd), hybrid adenoviral vectors, herpes virus, pox virus, human foamy virus (HFV), and lentivirus. Exemplary vectors useful in the invention described herein include episomal vectors, integrating vectors, non-integrating vectors, and excisable vectors.

[0129] In one embodiment, a vector is used as a transport vehicle to introduce any of the herein described synthetic nucleic acid into the target cells selected from the cell populations as described herein (e.g., ESCs; PSCs; iPSCs; hemogenic endothelium; HSCs). In one embodiment, a vector is used as a transport vehicle to introduce any of the herein described nucleic acid comprising the described nucleic acid inhibitors of a histone methyltransferase into the target cells selected from the cell populations as described herein (e.g., ESCs; PSCs; iPSCs; hemogenic endothelium; HSCs).

[0130] 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 B cell, an erythrocyte, a fibroblast, a keratinocyte, or a myeloid progenitor cell. In 19 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT one embodiment, the host cell is isolated from a subject. In one embodiment, the host cell is isolated from a subject who has been diagnosed with a hematological disease.

[0131] In one embodiment, the vector further comprises a spleen focus-forming virus promoter, a tetracycline-inducible promoter, a Doxycycline (Dox)-inducible, or a β-globin locus control region and a β-globin promoter. In one embodiment, the promoter provides for targeted expression of the nucleic acid molecule therein. Other examples of promoters include but are not limited to the CMV promoter and EF1-alpha promoters for the various transgenes, and U6 promoter for shRNAs targeting EZH1.

[0132] Another aspect herein is a cell comprising any of the synthetic nucleic acids described herein, any of the viruses of claims described herein, or any of the lentivirus described herein.

[0133] In one embodiment, the cell is a human pluripotent stem cell.

[0134] In one embodiment, the cell is an iPSC . In one embodiment, the cell is cord blood cells.

[0135] Another aspect provided herein describes a stable cell line comprising any of the synthetic nucleic acids described herein, any of the viruses described herein, or any of the lentivirus described herein. A stable cell line is a cell line that expresses any of the synthetic nucleic acids such that their progeny additionally expresses any of the synthetic nucleic acids. For example, the synthetic nucleic acids are integrated into the genome of the cell.

[0136] In one embodiment of this aspect, or any aspect herein, the cell is a human pluripotent stem cell. In one embodiment of this aspect, or any aspect herein, the cell is an iPSC. In one embodiment of this aspect, or any aspect herein, the cell is cord blood. Inhibitory Nucleic Acid

[0137] In one embodiment, the inhibitory nucleic acid sequence is an antisense oligonucleotide. As used herein, an “antisense oligonucleotide” refers to a synthesized nucleic acid sequence that is complementary to a DNA or mRNA sequence, such as that of a microRNA. Antisense oligonucleotides are typically designed to block expression of a DNA or RNA target by binding to the target and halting expression at the level of transcription, translation, or splicing. Antisense oligonucleotides of the present invention are complementary nucleic acid sequences designed to hybridize under cellular conditions to a gene, e.g., EZH1, TP53, or TET2. Thus, oligonucleotides are chosen that are sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient specificity in the context of the cellular environment, to give the desired effect. For example, an antisense oligonucleotide that inhibits EZH1, TP53, or TET2 may comprise at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, or more bases complementary to a portion of the coding sequence of the human EZH1, TP53, or TET2gene (e.g., SEQ ID NO: 1-3, respectively), respectively. 20 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0138] In some embodiments of any of the aspects, the inhibitory RNA (i.e., RNAi) is a dsRNA that inhibits EZH1, TP53, or TET2. A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of the target. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions.

[0139] In one embodiment, the iRNA is miRNA that inhibits EZH1, TP53, or TET2. microRNAs are small non-coding RNAs with an average length of 22 nucleotides. These molecules act by binding to complementary sequences within mRNA molecules, usually in the 3′ untranslated (3′UTR) region, thereby promoting target mRNA degradation or inhibited mRNA translation. The interaction between microRNA and mRNAs is mediated by what is known as the “seed sequence”, a 6–8-nucleotide region of the microRNA that directs sequence-specific binding to the mRNA through imperfect Watson–Crick base pairing. More than 900 microRNAs are known to be expressed in mammals. Many of these can be grouped into families on the basis of their seed sequence, thereby identifying a “cluster” of similar microRNAs. A miRNA can be expressed in a cell, e.g., as naked DNA. A miRNA can be encoded by a nucleic acid that is expressed in the cell, e.g., as naked DNA or can be encoded by a nucleic acid that is contained within a vector.

[0140] The inhibitory nucleic acid sequence results in gene silencing of the target gene (e.g., EZH1, TP53, or TET2), such as with an RNAi molecule (e.g., siRNA or miRNA). This entails a decrease in the mRNA level in a cell for a target by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100% of the mRNA level found in the cell without the presence of the inhibitory nucleic acid sequence. In one preferred embodiment, the mRNA levels are decreased by at least about 70%, about 80%, about 90%, about 95%, about 99%, about 100%. One skilled in the art will be able to readily assess whether the siRNA, shRNA, or miRNA effective target e.g., EZH1, TP53, or TET2, for its downregulation, for example by transfecting the siRNA, shRNA, or miRNA into cells and detecting the levels of a gene (e.g., EZH1, TP53, or TET2) found within the cell via Western-blotting.

[0141] In one embodiment, inhibitory nucleic acid sequence inhibits EZH1 mRNA at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100% of the mRNA level found in the cell without the presence of the inhibitory nucleic acid sequence. In one preferred embodiment, the EZH1 mRNA levels are decreased by at least about 70%, about 80%, about 90%, about 95%, about 99%, about 100%. 21 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0142] In one embodiment, inhibitory nucleic acid sequence inhibits TP53 mRNA at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100% of the mRNA level found in the cell without the presence of the inhibitory nucleic acid sequence. In one preferred embodiment, the TP53 mRNA levels are decreased by at least about 70%, about 80%, about 90%, about 95%, about 99%, about 100%.

[0143] In one embodiment, inhibitory nucleic acid sequence inhibits TET2 mRNA at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100% of the mRNA level found in the cell without the presence of the inhibitory nucleic acid sequence. In one preferred embodiment, the TET2 mRNA levels are decreased by at least about 70%, about 80%, about 90%, about 95%, about 99%, about 100%.

[0144] The RNA of an iRNA can be chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention may be synthesized and / or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference.

[0145] RNA interference (RNAi) mediated by short interfering RNAs (siRNA) or microRNAs (miRNA) is a powerful method for post-transcriptional regulation of gene expression. RNAi has been extensively used for the study of biological processes in mammalian cells and could constitute a therapeutic approach to human diseases in which selective modulation of gene expression would be desirable. Depending on the degree of complementarity between miRNA and target mRNA sequences, loss of gene expression occurs by inducing degradation of the cognate mRNA or by translational attenuation. Endogenous miRNAs are transcribed as primary transcripts and subsequently processed by the RNAse III enzyme Drosha to create a stem loop structure. Nuclear export and cleavage by Dicer generates a mature short double stranded molecule (siRNA) that is separated into guide and passenger strands. The guide strand is loaded into the RNA induced silencing complex (RISC), the effector complex mediating cleavage of target mRNAs with the functional guide strand binding to RISC proteins while the passenger strand is degraded. The loading of guide versus passenger strands into RISC largely depends on the 5’ end stability of the siRNA, with the less stable strand preferentially incorporated into RISC, although the exact regulation in mammalian cells is incompletely understood. The 5’ end of the guide strand contains the “seed region,” which is critical for target identification. Precise cleavage by Drosha and Dicer is critical for the generation of guide RNAs with defined seed regions that mediate efficient binding to the appropriate target mRNAs. Inaccurate processing results in binding to off-target molecules but a shift in cleavage sites also alters the nucleotide composition of duplex ends, which may have a profound effect on strand loading into RISC. 22 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0146] The inhibiting the expression of selected target polypeptides is through the use of RNA interference agents. RNA interference (RNAi) uses small interfering RNA (siRNA) duplexes that target the 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 evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target gene results in the sequence specific degradation or specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene (see e.g., Coburn, G. and Cullen, B. (2002) J. Virology 76(18):9225), thereby inhibiting 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 processive cleavage of long dsRNA into double-stranded fragments termed siRNAs. siRNAs are incorporated into a protein complex (termed “RNA induced silencing complex,” or “RISC”) that recognizes and cleaves target mRNAs. RNAi can also be initiated by introducing nucleic acid molecules, e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silence the expression of target genes. As used herein, “inhibition of target gene expression” includes any decrease in expression or protein activity or level of the target gene or protein encoded by the target gene as compared to a situation wherein no RNA interference has been induced. The decrease will be of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more as compared to the expression of a target gene or the activity or level of the protein encoded by a target gene which has not been targeted by an RNA interfering agent.

[0147] One skilled in the art would be able to design siRNA, shRNA, or miRNA to target EZH1, TP53, or TET2, e.g., using publicly available design tools. siRNA, shRNA, or miRNA is commonly made using companies such as Dharmacon (Layfayette, CO) or Sigma Aldrich (St. Louis, MO).

[0148] In one embodiment, the inhibitory nucleic acid sequence is a genome editing system including, but not limited to, zinc finger nucleases, TALENS, meganucleases, and CRISPR / Cas systems. In one embodiment, the genomic editing system used to incorporate the synthetic nucleic acids into the cell’s genome is not a CRISPR / Cas system; this can prevent undesirable cell death in cells that retain a small amount of Cas enzyme / protein. It is also contemplated herein that either the Cas enzyme or the sgRNAs are each expressed under the control of a different inducible promoter, thereby allowing temporal expression of each to prevent such interference.

[0149] Similarly, CRISPR guide RNA can be designed using the Broad Institute (MIT) CRISPR software (available on the world-wide web at, for example, portals.broadinstitute.org / gpp / public / analysis-tools / sgrna-design), dna20, Clontech, AddGene, e-crisp, and Innovative Genomic using the mRNA or genomic gene of EZH1 as the target. 23 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0150] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas9-mediated gene disruption has been widely used in generating loss-of-function mutations in diverse organisms including mammals (Cong et al., 2013, Science, 339(6121):819-23; reviewed in Hsu et al., 2014, Cell, 157(6):1262-78)). Cas9-based knockout screens have been applied in identifying essential genes and genes involved in drug resistance in various cell lines. With respect to general information on CRISPR-Cas Systems, components thereof, and delivery of such components, including methods, materials, delivery vehicles, vectors, particles, AAV, and making and using thereof, including as to amounts and formulations, all useful in the practice of the instant invention, reference is made to: US Patents 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; US Patent Publications US 2014- 0310830, US 2014-0287938, US 2014-0273234, US2014-0273232, US 2014-0273231, US 2014- 0256046, US 2014-0248702, US 2014-0242700, US 2014-0242699, US 2014-0242664, US 2014- 0234972, US 2014-0227787, US 2014-0189896, US 2014-0186958, US 2014-0186919, US 2014- 0186843, US 2014-0179770 and US 2014-0179006, US 2014-0170753; European Patents EP 2784 162 B1 and EP 2771468 B1; European Patent Applications EP 2771468 (EP13818570.7), EP 2764 103 (EP13824232.6), and EP 2784162 (EP14170383.5); and International Application No. WO 2014 / 093661, all of which are incorporated herein by reference in their entirety.

[0151] The CRISPR / Cas system envisaged for use in the context of the invention can make use of 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 may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell.

[0152] As described herein, the CRISPR / Cas system is used to specifically target a multitude of sequences within the continuous genomic region of interest. The targeting typically comprises introducing into each cell of a population of cells a vector system of one or more vectors comprising an engineered, non-naturally occurring CRISPR-Cas system comprising: at least one Cas protein, and one or more guide RNAs of the guide RNA library described herein.

[0153] In these methods, the Cas protein and the one or more guide RNAs may be on the same or on different vectors of the system and are integrated into each cell, whereby each guide sequence targets a sequence within the continuous genomic region in each cell in the population of cells. The Cas protein is operably linked to a regulatory element to ensure expression in said cell, more particularly a promoter suitable for expression in the cell of the cell population. In particular embodiments, the promoter is an inducible promoter, such as a doxycycline inducible promoter. When transcribed within the cells of the cell population, the guide RNA comprising the guide sequence directs 24 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT sequence-specific binding of a CRISPR-Cas system to a target sequence in the continuous genomic region. Typically binding of the CRISPR-Cas system induces cleavage of the continuous genomic region by the Cas protein.

[0154] When a nucleic acid encoding one or more sgRNAs and a nucleic acid encoding an RNA- guided endonuclease each need to be administered in vivo, the use of an adenovirus associated vector (AAV) is specifically contemplated. Other vectors for simultaneously delivering nucleic acids to both components of the genome editing / fragmentation system (e.g., sgRNAs, RNA-guided endonuclease) include lentiviral vectors, such as Epstein Barr, Human immunodeficiency virus (HIV), and hepatitis B virus (HBV). Each of the components of the RNA-guided genome editing system (e.g., sgRNA and endonuclease) can be delivered in a separate vector as known in the art or as described herein.

[0155] In one embodiment, the inhibitory nucleic acid sequence is an inhibitory RNA (iRNA). The RNAi can be single stranded or double stranded. The iRNA can be siRNA, shRNA, endogenous microRNA (miRNA), or artificial miRNA. In one embodiment, an iRNA as described herein effects inhibition of the expression and / or activity of a target, e.g., EZH1, TP53, or TET2. In some embodiments of any of the aspects, the inhibitory nucleic acid sequence is siRNA that inhibits EZH1, TP53, or TET2. In some embodiments of any of the aspects, the inhibitory nucleic acid sequence is shRNA that inhibits EZH1, TP53, or TET2. In some embodiments of any of the aspects, the inhibitory nucleic acid sequence is miRNA that inhibits EZH1, TP53, or TET2. Differentiation Methods

[0156] In one aspect, described herein is a method comprising: (a) differentiating a population of pluripotent stem cells for a sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium; and (b) differentiating the resultant population of CD34+ hemogenic endothelium in a CD3+-T-cell differentiation media in the presence of a Notch ligand for a sufficient time to promote differentiation into a population of CD3+ T cells.

[0157] In some embodiments, the method further comprises inhibiting a histone methyltransferase in the resultant population of CD34+ hemogenic endothelium. Such an inhibition can increase the efficiency of differentiation into T cells. Accordingly, in one aspect, described herein is a method comprising: (a) differentiating a population of pluripotent stem cells for a sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium; (b) inhibiting a histone methyltransferase in the resultant population of CD34+ hemogenic endothelium; and (c) differentiating the resultant population of CD34+ hemogenic endothelium in a CD3+-T-cell differentiation media in the presence of a Notch ligand for a sufficient time to promote differentiation into a population of CD3+ T cells.

[0158] In some embodiments, the method further comprises inhibiting a histone methyltransferase in the resultant population of CD34+ hemogenic endothelium. Such an inhibition can increase the 25 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT efficiency of differentiation into T cells. Accordingly, in one aspect, described herein is a method comprising: (a) differentiating a population of pluripotent stem cells for a sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium; and (b) differentiating the resultant population of CD34+ hemogenic endothelium in a CD3+-T-cell differentiation media in the presence of a Notch ligand for a sufficient time to promote differentiation into a population of CD3+ T cells.

[0159] In one embodiment, the CD34+hemogenic endothelium population is cultured into a CD3+- T-cell-differentiation media comprising 100 ng / ml SCF, 100 ng / ml FLT3, and 50 ng / ml IL7 in the presence of 10 µg / mL Notch ligand for at least 4 weeks to promote differentiation into a population of CD3+T cells.

[0160] In one embodiment, the CD34+hemogenic endothelium population is cultured into a CD3+- T-cell-differentiation media comprising 100 ng / ml FLT3 and 50 ng / ml IL7 in the presence of 10 µg / mL Notch ligand for at least 4 weeks to promote differentiation into a population of CD3+T cells.

[0161] In one embodiment, the CD34+hemogenic endothelium population is cultured into a CD3+- T-cell-differentiation media comprising 30 ng / ml SCF, 15 ng / ml FLT3, and 25 ng / ml IL7 in the presence of 10 µg / mL Notch ligand for at least 4 weeks to promote differentiation into a population of CD3+T cells.

[0162] In one embodiment, the CD34+hemogenic endothelium population is cultured into a CD3+- T-cell-differentiation media comprising 15 ng / ml FLT3 and 25 ng / ml IL7 in the presence of 10 µg / mL Notch ligand for at least 4 weeks to promote differentiation into a population of CD3+T cells.

[0163] In one embodiment, the T cells are differentiated using any known method in the art. Exemplary T cell differentiation methods are described in, e.g., Nat Methods.2017 May;14(5):531- 538. doi: 10.1038 / nmeth.4258. Epub 2017 Apr 10.; Nature.2018 Jan 25;553(7689):506-510. doi: 10.1038 / nature25435. Epub 2018 Jan 17.; Int Immunol.2013 Oct;25(10):601-11. doi: 10.1093 / intimm / dxt027. Epub 2013 Aug 29.; and Nat Commun.2018 May 8;9(1):1828. doi: 10.1038 / s41467-018-04134-7.; Cell Stem Cell.2022 Aug 4;29(8):1181-1196.e6. doi: 10.1016 / j.stem.2022.06.014; or US Patent Application No.17 / 794,747, the contents of which are incorporated herein by reference in their entirety. Pluripotent Stem Cells

[0164] In some embodiments, the stroma-free T cell differentiation method comprises differentiating a population of pluripotent stem cells. Pluripotent stem cells (PSCs) have the potential to give rise to all the somatic tissues. In one embodiment of any method, cells, or composition described herein, the population of pluripotent stem cells is induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESC). IPSC and ESC can be produced by any method known in the art. In some embodiments, the 26 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT population of pluripotent stem cells comprises embryonic stem cells (ESC). Embryonic stem cells (ESCs) are stem cells derived from the undifferentiated inner mass cells of a human embryo.

[0165] Directed differentiation of PSCs aims to recapitulate embryonic development to generate patient-matched tissues by specifying the three germ layers. A common theme in directed differentiation across all germ layers is the propensity of PSCs to give rise to embryonic- and fetal- like cell types, which poses a problem for integration and function in an adult recipient. This distinction is particularly striking in the hematopoietic system, which emerges in temporally and spatially separated waves at during ontogeny. The earliest “primitive” progenitors emerge in the yolk sac at 8.5 dpc and give rise to a limited repertoire of macrophages, megakaryocytes and nucleated erythrocytes. These early embryonic-like progenitors are generally myeloid-based and cannot functionally repopulate the bone marrow of adult recipients. By contrast, “definitive” cells with hematopoietic stem cell (HSC) potential emerge later in arterial endothelium within the aorta-gonad- mesonephros (AGM) and other anatomical sites. Directed differentiation of PSCs gives rise to hematopoietic progenitors, which resemble those found in the yolk sac of the early embryo and those found in the first intra-embryonic hematopoietic sites. These progenitors lack functional reconstitution potential and their potential is biased to myeloid lineages and erythroid cells that express primarily embryonic or fetal globins. Thus, understanding key fate determining mechanisms that promote development of either primitive or definitive lineages is critical for specifying HSCs, and other adult- like cell types (e.g., red blood cells) from PSCs.

[0166] 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 produced by introducing only reprogramming factors OCT4, SOX2, KLF4 and optionally c-MYC or NANOG and LIN28A into mature cells. In some embodiments, the induced pluripotent stem cells are produced by introducing the reprogramming factors two or more times into the mature cells.

[0167] 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 to which the eventual immune cells would be reintroduced. That is, a somatic cell can be obtained from a subject, reprogrammed to an induced pluripotent stem cell, and then transfected and differentiated into a modified immune cell to be administered to the subject (e.g., autologous cells). Since the progenitors are essentially derived from an autologous source, the risk of engraftment rejection or allergic responses is reduced compared to the use of cells from another subject or group of subjects. In some embodiments, the cells for generating iPSCs are derived from non-autologous sources. In addition, the use of iPSCs negates the need for cells obtained from an embryonic source. Thus, in one embodiment, the PSCs used in the disclosed methods are not embryonic stem cells. 27 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0168] Although differentiation is generally irreversible under physiological contexts, several methods have been recently developed to reprogram somatic cells to induced pluripotent stem cells. Exemplary methods are known to those of skill in the art and are described briefly herein below.

[0169] As used herein, the term “reprogramming” refers to a process that alters or reverses the differentiation state of a differentiated cell (e.g., a somatic cell). Stated another way, reprogramming refers to a process of driving the differentiation of a cell backwards to a more undifferentiated or more primitive type of cell. It should be noted that placing many primary cells in culture can lead to some loss of fully differentiated characteristics. Thus, simply culturing such cells included in the term differentiated cells does not render these cells non-differentiated cells (e.g., undifferentiated cells) or pluripotent cells. The transition of a differentiated cell to pluripotency requires a reprogramming stimulus beyond the stimuli that lead to partial loss of differentiated character in culture. Reprogrammed cells also have the characteristic of the capacity of extended passaging without loss of growth potential, relative to primary cell parents, which generally have capacity for only a limited number of divisions in culture.

[0170] The cell to be reprogrammed can be either partially or terminally differentiated prior to reprogramming. In some embodiments, reprogramming encompasses complete reversion of the differentiation state of a differentiated cell (e.g., a somatic cell) to a pluripotent state or a multipotent state. In some embodiments, reprogramming encompasses complete or partial reversion of the differentiation state of a differentiated cell (e.g., a somatic cell) to an undifferentiated cell (e.g., an embryonic-like cell). Reprogramming can result in expression of particular genes by the cells, the expression of which further contributes to reprogramming. In certain embodiments described herein, reprogramming of a differentiated cell (e.g., a somatic cell) causes the differentiated cell to assume an undifferentiated state (e.g., is an undifferentiated cell). The resulting cells are referred to as “reprogrammed cells,” or “induced pluripotent stem cells (iPSCs or iPS cells).”

[0171] Reprogramming can involve alteration, e.g., reversal, of at least some of the heritable patterns of nucleic acid modification (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., that occur during cellular differentiation. Reprogramming is distinct from simply maintaining the existing undifferentiated state of a cell that is already pluripotent or maintaining the existing less than fully differentiated state of a cell that is already a multipotent cell (e.g., a common myeloid stem cell). Reprogramming is also distinct from promoting the self-renewal or proliferation of cells that are already pluripotent or multipotent, although the compositions and methods described herein can also be of use for such purposes, in some embodiments.

[0172] The specific approach or method used to generate pluripotent stem cells from somatic cells (broadly referred to as “reprogramming”) is not necessarily critical to the methods described. Thus, any method that re-programs a somatic cell to the pluripotent phenotype would be appropriate for use in the methods described herein. 28 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0173] 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 to ES cell-like cells with expanded developmental potential by the direct transduction of Oct4, Sox2, Klf4, and optionally c-Myc. See US Patent Nos: 8058065 and 9045738 to Yamanaka and Takahashi. iPSCs resemble ES cells as they restore the pluripotency-associated transcriptional circuitry and much of the epigenetic landscape. In addition, mouse iPSCs satisfy all the standard assays for pluripotency: specifically, in vitro differentiation into cell types of the three germ layers, teratoma formation, contribution to chimeras, germline transmission, and tetraploid complementation.

[0174] Subsequent studies have shown that human iPS cells can be obtained using similar transduction methods, and the transcription factor trio, OCT4, SOX2, and NANOG, has been established as the core set of transcription factors that govern pluripotency. The production of iPS cells can be achieved by the introduction of nucleic acid sequences encoding stem cell-associated genes into an adult, somatic cell, using viral vectors.

[0175] OCT4, SOX2, KLF4 and c-MYC are the original four transcription factors identified to reprogram mouse fibroblasts into iPSCs. These same four factors were also sufficient to generate human iPSCs. OCT3 / 4 and SOX2 function as core transcription factors of the pluripotency network by regulating the expression of pluripotency-associated genes. Krüppel-like factor 4 (KLF4) is a downstream target of LIF-STAT3 signaling in mouse ES cells and regulates self-renewal. Human iPSCs can also be generated using four alternative factors; OCT4 and SOX2 are required but KLF4 and c-MYC could be replaced with NANOG, a homeobox protein important for the maintenance of pluripotency in both ES cells and early embryos, and LIN28, an RNA binding protein. The combination of OCT4, SOX2, NANOG and LIN28 reprogramming factors have been reported to be also sufficient to generate human iPSCs.

[0176] In one embodiment of any method, cells, or composition described herein, the iPSCs are produced, for example, by introducing exogenous copies of only three reprogramming factors OCT4, SOX2, and KLF4 into mature or somatic cells. In one embodiment of any method, cells, or composition described herein, c-MYC, or nanog and / or LIN28 are further introduced to iPSCs having exogenous gene coding copies of OCT4, SOX2, and KLF4 to differentiate into mature or somatic cells. In one embodiment of any method, cells, or composition described herein, the iPSCs are produced by introducing exogenous copies of reprogramming factors OCT4, SOX2, and KLF4, and optionally with c-MYC or nanog and / or LIN28 to differentiate into mature or somatic cells.

[0177] In one embodiment of any method, cells, or composition described herein, the iPSCs are produced by contacting mature cells with at least one vector, wherein the at least one vector carries an exogenous gene coding copy of reprogramming factors OCT4, SOX2, and KLF4, and optionally with c-MYC, or nanog and / or LIN28 to differentiate into mature or somatic cells, and wherein the 29 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT reprogramming factors are expressed in vivo in the contacted mature or somatic cells. The contacting is in vitro or ex vivo. The reprogramming factors needed for differentiation can all be expressed by one vector (e.g., a vector that carries an exogenous gene coding copy of OCT4, SOX2, KLF4, and c- MYC). Alternatively, the reprogramming factors can be expressed in more than one vector that is each used to contact the iPSCs. For example, an iPSCs can be contacted by a first vector that carries an exogenous gene coding copy of OCT4, SOX2, and a second vector that carries an exogenous gene coding copy KLF4 and c-MYC.

[0178] In one embodiment of any disclosed methods, the iPS cell comprises at least an exogenous copy of a nucleic acid sequence encoding a reprogramming factor selected from the group consisting of genes Oct4 (Pou5f1), Sox2, cMyc, Klf4, Nanog, Lin 28 and Glis1. In some embodiments, combinations of reprogramming factors are used. For example, a combination of four reprogramming factors consisting of Oct4, Sox2, cMyc, and Klf4, or a combination of four reprogramming factors consisting of Oct4, Sox2, Nanog, and Lin 28.

[0179] In one embodiment of any method, cells, or composition described herein, the iPSCs are produced by introducing the disclosed reprogramming factors, or any combination of the reprograming factors two or more times into the mature or somatic cells. In one embodiment, the combination of reprograming factors is different when a combination is introduced to the iPSC more than once, for example, the combination of Oct4 (Pou5f1), Sox2, cMyc, Klf4, Nanog is first introduced to the iPSCs, and the combination of Oct4 (Pou5f1), Sox2, cMyc is subsequently introduced to the iPSCs. In one embodiment of any method, cells, or composition described herein, the iPSCs are produced by contacting mature cells with the disclosed vector(s) factors two or more times into the mature / somatic cells.

[0180] In some embodiments, the population of pluripotent stem cells (e.g., iPSCs) are not differentiated in the presence of a Notch ligand. In some embodiments, the aggregation media used to promote the differentiation of the population of pluripotent stem cells (e.g., iPSCs) into a population of CD34+ hemogenic endothelium does not comprise a Notch ligand. In some embodiments, the cell culture vessel used during the differentiation of the population of pluripotent stem cells (e.g., iPSCs) into the population of CD34+ hemogenic endothelium does not comprise a Notch ligand.

[0181] iPS cells can be generated or derived from terminally differentiated somatic cells, as well as from adult stem cells, or somatic stem cells. That is, a non-pluripotent progenitor cell can be rendered pluripotent or multipotent by reprogramming. In such instances, it may not be necessary to include as many reprogramming factors as required to reprogram a terminally differentiated cell. Further, reprogramming can be induced by the non-viral introduction of reprogramming factors, e.g., by introducing the proteins themselves, or by introducing nucleic acids that encode the reprogramming factors, or by introducing messenger RNAs that upon translation produce the reprogramming factors (see e.g., Warren et al., Cell Stem Cell, 2010 Nov 5;7(5):618-30, this reference is incorporated herein 30 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 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, Sox 15, Sox 18, 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 comprise 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 to a somatic cell. In one embodiment, the methods and compositions described herein further comprise introducing one or more of each of Oct 4, Sox2, Nanog, c-MYC and Klf4 for reprogramming. As noted above, the exact method used for reprogramming is not necessarily critical to the methods and compositions described herein. However, where cells differentiated from the reprogrammed cells are to be used in, e.g., human therapy, in one embodiment the reprogramming is not effected by a method that alters the genome. Thus, in such embodiments, reprogramming is achieved, e.g., without the use of viral or plasmid vectors.

[0182] The efficiency of reprogramming (i.e., the number of reprogrammed cells) derived from a population of starting cells can be enhanced by the addition of various small molecules as shown by Shi, Y., et al (2008) Cell-Stem Cell 2:525-528, Huangfu, D., et al (2008) Nature Biotechnology 26(7):795-797, and Marson, A., et al (2008) Cell-Stem Cell 3:132-135, the contents of each of which are incorporated herein by reference in its entirety. Thus, an agent or combination of agents that enhance the efficiency or rate of induced pluripotent stem cell production can be used in the production of patient-specific or disease-specific iPSCs. Some non-limiting examples of agents that enhance reprogramming efficiency include soluble Wnt, Wnt conditioned media, BIX-01294 (a G9a histone methyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5'-azacytidine, dexamethasone, suberoylanilide hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), among others.

[0183] Other non-limiting examples of reprogramming enhancing agents include: Suberoylanilide Hydroxamic Acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, Depudecin (e.g., (-)-Depudecin), HC Toxin, Nullscript (4-(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- acetyl dinaline) and MS-27–275), MGCD0103, NVP-LAQ-824, CBHA (m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, Tubacin, 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 CHAP 50. Other reprogramming enhancing agents include, for example, dominant negative forms of the HDACs (e.g., catalytically inactive forms), siRNA inhibitors of the HDACs, 31 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT and antibodies that specifically bind to the HDACs. Such inhibitors are available, e.g., from BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Aton Pharma, Titan Pharmaceuticals, Schering AG, Pharmion, MethylGene, and Sigma Aldrich.

[0184] To confirm the induction of pluripotent stem cells for use with the methods described herein, isolated clones can be tested for the expression of a stem cell marker. Such expression in a cell derived from a somatic cell identifies the cells as induced pluripotent stem cells. Stem cell markers can 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, a cell that expresses Oct4 or Nanog is identified as pluripotent. Methods for detecting the expression of such markers can include, for example, RT-PCR and immunological methods that detect the presence of the encoded polypeptides, such as Western blots or flow cytometric analyses. In some embodiments, detection does not involve only RT-PCR, but also includes detection of protein markers. Intracellular markers may be best identified via RT-PCR, while cell surface markers are readily identified, e.g., by immunocytochemistry.

[0185] The pluripotent stem cell character of isolated cells can be confirmed by tests evaluating the ability of the iPSCs to differentiate to cells of each of the three germ layers. As one example, teratoma formation in nude mice can be used to evaluate the pluripotent character of the isolated clones. The cells are introduced to nude mice and histology and / or immunohistochemistry is performed on a tumor arising from the cells. The growth of a tumor comprising cells from all three germ layers, for example, further indicates that the cells are pluripotent stem cells.

[0186] Many US Patents and Patent Application Publications teach and describe methods of generating iPSCs and related subject matter. For examples, US Patent Nos: 8058065, 9347044, 9347042 , 9347045, 9340775, 9341625, 9340772, 9250230, 9132152, 9045738, 9005975, 9005976, 8927277, 8993329, 8900871, 8852941, 8802438, 8691574, 8735150, 8765470, 8058065, 8048675, and US Patent Publication Nos: 20090227032, 20100210014, 20110250692, 20110201110, 20110200568, 20110223669, 20110306516, 20100021437, 20110256626, 20110044961, 20120276070, 20120214243, 20120263689, 20120128655, 20120100568, 20130295064, 20130029866, 20130059386, 20130183759, 20130189786, 20130295579, 20130130387, 20130157365, 20140234973, 20140227736, 20140093486, 20140301988, 20140170746, 20140178989, 20140349401, 20140065227, and 20150140662, all of which are incorporated herein by reference in their entireties.

[0187] In some embodiments, the iPSCs can be derived from somatic cells. Somatic cells, as that term is used herein, refer to any cells forming the body of an organism, excluding germline cells. Every cell type in the mammalian body—apart from the sperm and ova, the cells from which they are made (gametocytes) and undifferentiated stem cells—is a differentiated somatic cell. For example, internal organs, skin, bones, blood, and connective tissue are all made up of differentiated somatic 32 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT cells. In one embodiment of any method, cells, or composition described herein, the mature cells from which iPS cells are made include any somatic cells such as B lymphocytes (B-cells), T lymphocytes, (T-cells), and fibroblasts and keratinocytes.

[0188] Additional somatic cell types for use with the compositions and methods described herein include: a fibroblast (e.g., a primary fibroblast), a muscle cell (e.g., a myocyte), a cumulus cell, a neural cell, a mammary cell, a hepatocyte and a pancreatic islet cell. In some embodiments, the somatic cell is a primary cell line or is the progeny of a primary or secondary cell line. In some embodiments, the somatic cell is obtained from a human sample, e.g., a hair follicle, a blood sample, a biopsy (e.g., a skin biopsy or an adipose biopsy), a swab sample (e.g., an oral swab sample), and is thus a human somatic cell.

[0189] Some non-limiting examples of differentiated somatic cells include, but are not limited to, epithelial, endothelial, neuronal, adipose, cardiac, skeletal muscle, skin, immune cells, hepatic, splenic, lung, peripheral circulating blood cells, gastrointestinal, renal, bone marrow, and pancreatic cells. In some embodiments, a somatic cell can be a primary cell isolated from any somatic tissue including, but not limited to brain, liver, gut, stomach, intestine, fat, muscle, uterus, skin, spleen, endocrine organ, bone, etc. Further, the somatic cell can be from any mammalian species, with non- limiting examples including a murine, bovine, simian, porcine, equine, ovine, or human cell. In some embodiments, the somatic cell is a human somatic cell.

[0190] When reprogrammed cells are used for generation of progenitor cells to be used in the therapeutic treatment of disease, it is desirable, but not required, to use somatic cells isolated from the patient being treated. For example, somatic cells involved in diseases, and somatic cells participating in therapeutic treatment of diseases and the like can be used. In some embodiments, a method for selecting the reprogrammed cells from a heterogeneous population comprising reprogrammed cells and somatic cells they were derived or generated from can be performed by any known means. For example, a drug resistance gene or the like, such as a selectable marker gene can be used to isolate the reprogrammed cells using the selectable marker as an index.

[0191] Reprogrammed somatic cells as disclosed herein can express any number of pluripotent cell markers, including: alkaline phosphatase (AP); ABCG2; stage specific embryonic antigen-1 (SSEA- 1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49 / 6E; ERas / ECAT5, E-cadherin; beta-III- tubulin; alpha-smooth muscle actin (α-SMA); fibroblast growth factor 4 (Fgf4), Cripto, Dax1; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); (ES cell associated transcript 1 (ECAT1); ESG1 / DPPA5 / ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15- 2; Fthl17; Sal14; undifferentiated embryonic cell transcription factor (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 33 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT lymphoma breakpoint 1 (Tcl1); DPPA3 / Stella; DPPA4; other general markers for pluripotency, etc. Other markers can include Dnmt3L; Sox15; Stat3; Grb2; β-catenin, and Bmi1. Such cells can also be characterized by the down-regulation of markers characteristic of the somatic cell from which the induced pluripotent stem cell is derived. In one embodiment, the iPSCs are derived from mature, differentiated, somatic cells.

[0192] 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 cord blood or bone marrow samples. 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 a preferred embodiment, the population of pluripotent stem cells comprise iPSCs, such as those derived from a somatic cell sample from a patient. See e.g., Tabatabaei-Zavareh et al., J Immunol May 1, 2017, 198 (1 Supplement) 202.9. Hemogenic Endothelium

[0193] In some embodiments, the methods described herein comprise differentiating a population of pluripotent stem cells (e.g., iPSCs) into a population of cells with hematopoietic potential. In some embodiments, the population of cells with hematopoietic potential comprises hemogenic endothelium and / or hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs). The cells with hematopoietic potential (e.g., hemogenic endothelium, HSCs, HPCs) can be produced using any method known in the art.

[0194] One aspect provided herein is a method of differentiating any of the cells or stable cell lines (i.e., expressing any of the synthetic nucleic acids, viruses, or lentiviruses described herein) into a CD34+ hemogenic endothelium having increased potential for T cell differentiation as compared to a hemogenic endothelium differentiated from a cell not expressing any of the synthetic nucleic acids, viruses, or lentiviruses described herein. Methods for differentiating a cell into a CD34+ hemogenic endothelium are described herein and known in the art. It is specifically contemplated herein that any known method for differentiating a cell into a CD34+ hemogenic endothelium can be used with the cells or stable cells described herein (i.e., expressing any of the synthetic nucleic acids, viruses, or lentiviruses described herein).

[0195] In one embodiment, the hemogenic endothelial cells are hemogenic endothelial cells or hematopoietic stem cells or hematopoietic progenitor cells or lymphoid progenitor cells.

[0196] In one aspect herein, any of the cells or stable cell lines described herein are differentiated into a CD34+ hemogenic endothelium having increased potential for T cell differentiation as compared to a hemogenic endothelium differentiated from a cell not expressing any of the synthetic nucleic acids, viruses, or lentiviruses described herein. In one embodiment, the differentiated CD34+ 34 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT hemogenic endothelium described herein have at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, or more, or at least 10x, at least 20x, at least 30x, at least 40x, at least 50x, at least 60x, at least 70x, at least 80x, at least 90x, at least 100x or more increased potential for T cell differentiation as compared to a hemogenic endothelium differentiated from a cell not expressing any of the synthetic nucleic acids, viruses, or lentiviruses described herein. T cell differentiation potential can be assessed by subjecting the CD34+ hemogenic endothelium having increased potential for T cell differentiation to any known T cell differentiation protocol (e.g., as described herein or known in the art).

[0197] One exemplary approach to generate HSCs from hPSCs is to specify HSCs from its ontogenetic precursors. It is now widely accepted that HSCs originate from hemogenic endothelium (HE) in the aorta-gonad-mesonephros (AGM) and arterial endothelium in other anatomical sites. Recent work on the 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.

[0198] 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 the developing mouse, HSCs arise beginning embryonic day 10.5 from a small population of endothelial cells with hemogenic potential (hemogenic endothelium) located within the aorta-gonad-mesonephros region. In a process known as endothelial to hematopoietic transition (EHT), endothelial cells in the floor of the aorta round up and bud into the extravascular space followed by reentry into the circulation via the underlying vein. In some embodiments, a population of cells comprising the properties of hemogenic endothelium is differentiated in vitro from a population of pluripotent stem cells (e.g., iPSCs). Said “cells comprising the properties of hemogenic endothelium” can also be referred to herein as hemogenic endothelium.

[0199] Efforts 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 thus the lymphoid lineage. Definitive hematopoiesis, as measured by T-lymphoid potential, emerges after the establishment of the primitive hematopoietic program and develops from a progenitor population that displays characteristics of hemogenic endothelium.

[0200] In some embodiments, the stroma-free T cell differentiation method comprises differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium. In some embodiments, the resultant CD34+ hemogenic endothelium can undergo definitive hematopoiesis and / or exhibits 35 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT lymphoid potential. In some embodiments, the hemogenic endothelium differentiates or is differentiated into hematopoietic stem cells (HSCs).

[0201] In some embodiments, the 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 produced and cultured in vitro in the presence of serum. The EBs can generate a mixture of primitive and definitive hematopoietic progenitor cell types. Primitive progenitors equate to those that arise in vivo naturally in the earliest stages of embryonic development, whereas at later stages of maturation the embryonic populations give rise to definitive progenitor cells, which behave similarly to the cells typical of adult hematopoiesis.

[0202] In some embodiments, the sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium is at least 8 days (e.g., at least 7, at least 8, at least 9, at least 10 days, or more). In some embodiments, the sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium is at most 8 days, at most 9 days, at most 10 days or more.

[0203] In some embodiments, the aggregation media comprises BMP4, SB-431542, CHIR99021, bFGF, VEGF, IL-6, IL-11, IGF-1, SCF, and EPO or any combination of the same. In some embodiments, the aggregation media comprises 10 ng / ml BMP4, 6 µM SB-431542, 3 µM 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.

[0204] In some embodiments, the components of the aggregation media are varied during the 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. and 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.

[0205] In some embodiments, the aggregation media comprises BMP (e.g., 10 ug / mL BMP) during days 0, 1, and / or 2 of differentiation. In some embodiments, the aggregation media does not comprise BMP during days 3, 4, 5, 6, 7, or 8 of differentiation.

[0206] In some embodiments, the aggregation media comprises SB-431542 (e.g., 6 µM SB-431542) and / or CHIR99021 (e.g., 3 µM CHIR99021) during day 2 of differentiation. SB-431542 is a small- molecule antagonist of activin-nodal signaling. CHIR99021 is a GSK-3 inhibitor and a Wnt agonist. Inhibition of activin-nodal signaling and activation of Wnt signaling has been shown to drive PSC 36 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT differentiation into definitive progenitors (KDR+CD235a−) with lymphoid potential (see e.g., Sturgeon 2014, supra, which is incorporated herein by reference). In some embodiments, the aggregation media does not comprise SB-431542 and / or CHIR99021 during days 0, 1, 3, 4, 5, 6, 7, and / or 8 of differentiation. In some embodiments, the aggregation media comprises SB-431542 and / or CHIR99021 during days 0, 1, 3, 4, 5, 6, 7, and / or 8 of differentiation.

[0207] In some embodiments, the aggregation media comprises bFGF (e.g., 5 ng / ml bFGF) during days 1, 2, 3, 4, 5, 6, 7, and / or 8 of differentiation. In some embodiments, the aggregation media does not comprise bFGF during day 0 of differentiation.

[0208] In some embodiments, the aggregation media comprises VEGF (e.g., 15 ng / ml VEGF) during days 3, 4, 5, 6, 7, and / or 8 of differentiation. In some embodiments, the aggregation media does not comprise VEGF during days 0, 1, or 2 of differentiation.

[0209] In some embodiments, the aggregation media comprises hematopoietic cytokine(s) during days 6, 7, and / or 8 of differentiation. In some embodiments, the aggregation media does not comprise hematopoietic cytokine(s) during days 0, 1, 2, 3, 4, or 5 of differentiation. 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).

[0210] In some embodiments, the differentiation method further comprises selecting or isolating the resultant population of CD34+ hemogenic endothelium using expression of surface markers on the population of CD34+ hemogenic endothelium. Non-limiting examples of methods for selecting or isolating hemogenic endothelium include magnetic-activated cell sorting (MACS) and fluorescence- activated cell sorting (FACS). In some embodiments, the surface marker for hemogenic endothelium is CD34 (e.g., high CD34 surface expression).

[0211] In one embodiment, the negative markers for CD34+ hemogenic endothelium is CD45 and CD73. In one embodiment, the population of CD34+ hemogenic endothelium is CD45 negative / low. In one embodiment, the population of CD34+ hemogenic endothelium is CD73 negative. In one embodiment, the population of CD34+ hemogenic endothelium is CD45 negative / low and CD73 negative.

[0212] In alternative embodiments, additional positive or negative markers for hemogenic endothelium can 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. 37 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0213] In some embodiments, the hemogenic endothelium and / or HSCs are produced using any method known in the art. As a non-limiting example, the method of differentiating PSCs into hemogenic endothelium can comprise the introduction of transcription factors such as ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, and / or SPI1; see e.g., International Application No. WO 2018 / 048828, US Patent Application No.2019 / 0225940, Doulatov et al., Cell Stem Cell.2013 October 3, 13(4); Vo et al., Nature 2018, 553(7689): 506–510; the contents of each of which are incorporated herein by reference in their entireties.

[0214] In some embodiments, the hemogenic endothelium is not derived from PSCs but is rather 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 of with transcription factors (e.g., Fosb, Gfi1, Runx1, and Spi1) and co-culture with an immortalized endothelial cell line; the endothelial cells can be further exposed to cell-extrinsic factors (e.g., serum, SB-431542, and / or endothelial mitogen). 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. Stroma-Free T Cell Differentiation

[0215] In some embodiments, the differentiation method comprises differentiating the resultant population of CD34+ hemogenic endothelium in a CD3+-T-cell differentiation media in the presence of a Notch ligand for a sufficient time to promote differentiation into a population of CD3+ T cells. The method described herein is a stroma-free T cell differentiation method. Compared to differentiation with stromal cells expressing a Notch ligand, stroma-free differentiation unexpectedly results in an increased number of differentiated T cells, with a smaller portion of these T cells being innate-like cells. Unexpectedly, the inventors found that the stroma-free protocol described herein requires starting with hemogenic endothelium (HE), not iPSC or HE-derived progenitors (e.g., lymphoid progenitor).

[0216] In nature, the hematopoietic stem cells (HSCs) in the bone marrow give rise to multipotent progenitors (MPPs) before differentiating into common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs). CLPs migrate from the bone marrow to the thymus, where thymic epithelial cells that express Delta-like ligand 4 (DLL4) trigger canonical Notch 1 signaling in early thymic progenitors (ETPs). This Notch 1 signal is essential for T cell lineage commitment and is further required during early phases of thymocyte differentiation up to the double-negative 3 (DN3) stage. 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 consequence of pre-T cell receptor signaling. Thus subsequent stages of T cell development exhibit very low levels of Notch signaling. Notch was also 38 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT suggested to influence the development of regulatory T (TReg) cells (specifically, thymic TRegcells). Notch signaling is mediated by the Notch 2 receptor. Notch signaling pathway is highly conserved in both vertebrate and invertebrate species and it regulates many different cell fate decisions. It is important for pattern formation during development such as neurogenesis, angiogenesis or myogenesis and regulates T cell development and stem cell maintenance. Notch signaling is also involved in cellular processes throughout adulthood. Signaling via Notch occurs between neighboring cells and both the receptor and its ligands are transmembrane proteins. See, e.g., Schmitt T.M., Zúñiga-Pflücker J.C. (2002) Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17:749-756; Mohtashami M. (2010) Direct Comparison of Dll1- and Dll4-Mediated Notch Activation Levels Shows Differential Lymphomyeloid Lineage Commitment Outcomes. J Immunol.185(2):867-76; Ohishi K et al, which 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. Notch ligands

[0217] Accordingly, to initiate differentiation in the lymphoid lineage and T cell lineage commitment, the hemogenic endothelium is exposed to a Notch ligand to activate the Notch signaling pathway therein. Unexpectedly, the inventors found that the stroma-free protocol described herein, which comprising exposure to a Notch ligand requires starting with hemogenic endothelium (HE), not iPSC or HE-derived progenitors (e.g., lymphoid progenitor). Accordingly, in some embodiments, iPSC or HE-derived progenitors are not the initial population that is differentiated into T cells in the presence of a Notch ligand.

[0218] Notch ligands are single-pass transmembrane proteins with a DSL (Delta, Serrate, LAG-2)- domain and varying numbers of EGF-like repeats. There are two classes of canonical Notch ligands, the Delta / Delta-like and the Serrate / Jagged class. The later has an additional domain of cysteine rich repeats close to the transmembrane domain. There are 5 canonical Notch ligands in mammals: Jagged-1, Jagged-2, DLL1, DLL3 and DLL4. These can bind to the four Notch receptors Notch 1-4. DLL1, also known as Notch Delta ligand, Delta-like 1, is a protein which interacts with a NOTCH2 receptor. See e.g., 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. DLL1 is a protein that in humans is encoded by the DLL1 gene. DLL1 is a human homolog of the Notch Delta ligand.

[0219] In some embodiments, the Notch ligand is selected from the group consisting of Delta-like-1 (DLL1, also referred to as DL1), Delta-like-4 (DLL4, also referred to as DL4), immobilized 39 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT Delta1ext-IgG, and immobilized Delta4ext-IgG. In some embodiments, immobilized Delta1ext-IgG consists of an extracellular domain of human Delta-like-1 fused to the Fc domain of human IgG1. “Immobilized Delta1ext-IgG” refers to recombinant Notch ligand made by fusing the extracellular domain of Delta-like 1 to the Fc domain of human IgG1. This is a synthetic way of providing a titratable dose of NOTCH ligand. See e.g., Varnum-Finney et al., J Cell Sci.2000 Dec;113 Pt 23:4313-8, which is incorporated herein by reference in its entirety. Recombinant Notch ligands and Fc-fusions are commercially available at AdipoGen™. “Immobilized Delta4ext-IgG” refers to recombinant Notch ligand made by fusing the extracellular domain of Delta-like 4 to the Fc domain of human IgG1.

[0220] In some embodiments, the IgG domain of Delta1ext-IgG or Delta4ext-IgG can comprise any known IgG domain in the art. In some embodiments, Delta1ext-IgG or Delta4ext-IgG can be immobilized to a solid substrate (e.g., tissue culture plate) by coating the solid substrate with a composition that binds IgG Fc, including but not limited to anti-human IgG antibody, Protein G, or Protein A.

[0221] Nucleic acid sequences of Notch ligands are known in the art. For example, US Patent Application No.17 / 794,747 describes various Notch ligands sequences useful in the methods described herein; the contents of which are incorporated herein by reference.

[0222] There are several ways to provide a Notch ligand, for example by providing a purified recombinant form of a Notch ligand or a Notch receptor-binding fragment, the receptor-binding fragment being sufficient to elicit cell signaling events in vivo upon contact and binding with the extracellular Notch receptors on these cells. In some embodiments, the Notch ligand is attached to a solid substrate, for example using a covalent or non-covalent bond or linkage. In some embodiments, the Notch ligand is attached to a cell culture dish.

[0223] In some embodiments, the Notch ligand further comprises a domain to immobilize the Notch ligand to a solid substrate. As a non-limiting example, the Notch ligand comprises a first member of an affinity pair, and the solid substrate comprises a second member of an affinity pair. In some embodiments, the first and second members of the affinity pair are selected from the group consisting of: a haptenic or antigenic compound in combination with a corresponding antibody or binding portion or fragment thereof (e.g., FLAG and anti-FLAG monoclonal antibody, the sequence of which are known in the art); digoxigenin and anti-digoxigenin; mouse immunoglobulin and goat anti-mouse immunoglobulin; a non-immunological binding pair; biotin and avidin; biotin and streptavidin; a hormone and a hormone-binding protein; thyroxine and cortisol-hormone binding protein; a receptor and a receptor agonist; a receptor and a receptor antagonist; acetylcholine receptor and acetylcholine or an analog thereof; IgG and protein A; lectin and carbohydrate; an enzyme and an enzyme cofactor; an enzyme and an enzyme inhibitor; complementary oligonucleotide pairs capable of forming nucleic 40 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT acid duplexes; and a first molecule that is negatively charged and a second molecule that is positively charged.

[0224] In some embodiments, the population of hemogenic endothelium is differentiated into a population of CD3+ T cells by culturing in a non-tissue culture treated culture vessel; said another way, the culture vessel is not exposed to a plasma gas in order to modify the hydrophobic plastic surface to make it more hydrophilic. As used herein, the term “culture vessel” includes dishes, flasks, plates, multi-well plates, and the like. In some embodiments, the culture vessel is coated with recombinant human DL1-Fc protein (e.g., commercially available via R&D SYSTEMS, item number 10184-DL), recombinant human DL4-Fc protein (e.g., commercially available via ACRO BIOSYSTEMS, item number DL4-H5259), or a mixture of both Notch ligands, or any Notch ligand as described herein. In some embodiments, the culture vessel is coated with Notch ligand for at least 0.5 hour, at least 1.0 hour, at least 1.5 hours, at least 2.0 hours, at least 2.5 hours, at least 3.0 hours, at least 3.5 hours, at least 4.0 hours, at least 4.5 hours, or at least 5.0 hours. In some embodiments, the culture vessel is coated with Notch ligand at room temperature.

[0225] In some embodiments, the non-stromal-derived Notch ligand (e.g., the Notch ligand immobilized on a tissue culture plate) is provided at a concentration of 1 µg / mL to 100 µg / mL or a concentration of 5 µg / mL to 15 µg / mL. In some embodiments, the non-stromal-derived Notch ligand is provided at a concentration of at least 1 µg / mL, at least 2 µg / mL, at least 3 µg / mL, at least 4 µg / mL, at least 5 µg / mL, at least 6 µg / mL, at least 7 µg / mL, at least 8 µg / mL, at least 9 µg / mL, at least 10 µg / mL, at least 11 µg / mL, at least 12 µg / mL, at least 13 µg / mL, at least 14 µg / mL, at least 15 µg / mL, at least 16 µg / mL, at least 17 µg / mL, at least 18 µg / mL, at least 19 µg / mL, at least 20 µg / mL, at least 25 µg / mL, at least 30 µg / mL, at least 35 µg / mL, at least 40 µg / mL, at least 45 µg / mL, at least 50 µg / mL, at least 55 µg / mL, at least 60 µg / mL, at least 65 µg / mL, at least 70 µg / mL, at least 75 µg / mL, at least 80 µg / mL, at least 85 µg / mL, at least 90 µg / mL, at least 95 µg / mL, or at least 100 µg / mL. In a preferred embodiment, the non-stromal-derived Notch ligand is provided at a concentration of 10 µg / mL.

[0226] In some embodiments, the cells are cultured exposed to a non-stromal-derived Notch ligand (e.g., a Notch ligand immobilized on a tissue culture plate) for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, at least 31 days, at least 32 days, at least 33 days, at least 34 days, at least 35 days, at least 36 days, at least 37 days, at least 38 days, at least 39 days, at least 40 days, at least 41 days, at least 42 days, at least 43 days, at least 44 days, at 41 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 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. Stroma-free differentiation

[0227] The method described herein is a stroma-free T cell differentiation method, i.e., a method that does not comprise co-culturing with stromal cells or any other type of supporting cell. Co-culture with stromal cells such as mouse stromal cells limits the translational potential of iPSC-derived T cells; for example, there can be fears of transplantation rejection due to the presence of stromal cells. Furthermore, T cells differentiated using stromal cells exhibit an innate-like phenotype (e.g., as measured by TCRgd expression, which is a marker for gamma delta T cells). It is preferred that T cells exhibit an adaptive phenotype, for example characterized by expression of TCR α and β. Additionally, as described herein, stroma-free T cell differentiation methods result in increased numbers of CD3+ T cells (e.g., CD4+CD8+ cells) compared to differentiation methods comprising stromal co-culture.

[0228] Accordingly, T cells differentiated using stromal-free methods exhibit at least the following unexpected benefits compared to stromal co-culture methods: (1) increased potential for transplantation in humans; (2) decreased number of innate-like T cells; (3) increased number and / or percentage of resultant T cells (e.g., CD5+CD7+ Pro-T cells; CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells; alpha-beta T cells); (4) gene expression profiles most similar to alpha beta T cells; (5) a more diverse TCR repertoire; and / or (6) increased TCR CDR length.

[0229] As used herein, the term “supporting cell or stromal cell” when used in the context of cell differentiation refers to any cells that are capable of creating, promoting, or supporting a microenvironment for the growth, proliferation, differentiation, or expansion of multipotent hematopoietic progenitor cells or T cells or B cells. Non-limiting examples of supporting cells that are not comprised by the differentiation methods described herein include, but are not limited to, stromal cells and fibroblast cells.

[0230] Supporting cells used previously in co-cultures for cell differentiation purposes are typically stromal cells. However, the methods described herein do not comprise co-cultures comprising stromal cells. Examples of stromal cell lines that are not comprised by the differentiation methods described herein include, but are not limited to, murine MS5 stromal cell line; murine bone marrow-derived stromal cell lines, such as S10, S17, OP9 (e.g., OP9-DL1 cells or OP9-DL4 cells) and BMS2 cell lines; human marrow stromal cell lines such as those described in U.S. Patent No.5,879,940, which is incorporated herein by reference in its entirety; or any other similar cells that express and display extracellular or secretes a Notch ligand. OP9-DL1 cells are a bone-marrow-derived stromal cell line that ectopically expresses the Notch ligand, Delta-like 1 (DLL1). Method of differentiating pluripotent stem cells to T-cells using OP9-Notch ligand expressing cells are known in the art. See, 42 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT e.g., US Patent Nos: 7575925, 8772028, 8871510, and 9206394 and US Patent Publication Nos: 20090217403, 20110123502, 2011005255420110027881, 20110236363, 20120149100, 20130281304, 20140322808, 20140248248, and 20140037599. These references are incorporated herein by reference in their entirety.

[0231] Described herein are methods of differentiating T cells from pluripotent stem cells, wherein the methods do not comprise a step of co-culturing the cells with supporting cells or stromal cells. In some embodiments, the Notch ligand used herein is not derived from a stromal cell. In some embodiments, differentiating the hemogenic endothelium in the presence of a Notch ligand does not comprise co-culturing with a stromal cell expressing a Notch ligand. In some embodiments, differentiating the hemogenic endothelium in the presence of a Notch ligand does not comprise co- culturing with OP9-DL1 cells or OP9-DL4 cells. T cell differentiation medias

[0232] In some embodiments, the differentiation method comprises differentiating the resultant population of CD34+ hemogenic endothelium in a CD3+-T-cell differentiation media for a sufficient time to promote differentiation into a population of CD3+ T cells. In some embodiments, the sufficient time to promote differentiation into a population of CD3+ T cells is at least 3 weeks, at least 3.5 weeks, at least 4 weeks, at least 4.5 weeks, at least 5 weeks, at least 5.5 weeks, at least 6 weeks, or more. In some embodiments, the sufficient time to promote differentiation into a population of CD3+ T cells is at most 6 weeks. Any known method for differentiating a population of a CD34+ hemogenic endothelium into a T cell, e.g., a CD3+ T cell, can be used. Exemplary methods for differentiating a CD34+ hemogenic endothelium into a T cell are described, for example, as described on the world wide web at https: / / pubmed.ncbi.nlm.nih.gov / 35931029 / ; https: / / pubmed.ncbi.nlm.nih.gov / 35941192 / ; or https: / / pubmed.ncbi.nlm.nih.gov / 33462228 / ; and US Patent Application No.17 / 794,747; the contents of which are incorporated herein by reference in their entirety.

[0233] In some embodiments, a polypeptide (e.g., growth or differentiation factors) that can be expressed by the supporting cell or stromal cell can be provided in the cell culture medium. Non limiting examples of polypeptides that support the differentiation of T cells 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 it is involved in B and T cell development. 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 referred to as 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 chiefly responsible for megakaryocyte production but also has a role in maintaining hematopoietic stem cells (HSCs). See, 43 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 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.

[0234] In some embodiments, the CD3+-T-cell-differentiation media is serum-free. In some embodiments, the CD3+-T-cell-differentiation media comprises at least one of SCF, FLT3, and / or IL7. In some embodiments, the CD3+-T-cell-differentiation media comprises SCF, FLT3, and IL7. In some embodiments, the CD3+-T-cell-differentiation media comprises 30 ng / ml SCF, 15 ng / ml FLT3, and 25 ng / ml IL7. In some embodiments, the CD3+-T-cell-differentiation media comprises 100 ng / ml SCF, 100 ng / ml FLT3, and 50 ng / ml IL7. In some embodiments, the CD3+-T-cell-differentiation media comprises FLT3 and IL7. In some embodiments, the CD3+-T-cell-differentiation media comprises 15 ng / ml FLT3 and 25 ng / ml IL7. In some embodiments, the CD3+-T-cell-differentiation media comprises 100 ng / ml FLT3 and 50 ng / ml IL7.

[0235] The concentrations of SCF, FLT3, and / or IL7 should be used such that they promote the differentiation of hemogenic endothelium into a population of CD3+ T cells. The concentration of SCF can range from 1 ng / mL to 200 ng / mL. In some embodiments, the concertation of SCF (e.g., in the CD3+-T-cell-differentiation media) is 30 ng / mL. In some embodiments, the concertation of SCF (e.g., in the CD3+-T-cell-differentiation media) is 100 ng / ml. The concentration of FLT3 can range from 1 ng / mL to 200 ng / mL. In some embodiments, the concertation of FLT3 (e.g., in the CD3+-T- cell-differentiation media) is 15 ng / ml. In some embodiments, the concertation of FLT3 (e.g., in the CD3+-T-cell-differentiation media) is 100 ng / ml. The concentration of IL7 can range from 1 ng / mL to 200 ng / mL. In some embodiments, the concertation of IL7 (e.g., in the CD3+-T-cell-differentiation media) is 25 ng / ml. In some embodiments, the concertation of IL7 (e.g., in the CD3+-T-cell- differentiation media) is 50 ng / ml.

[0236] Tables 1-3 described media, growth factor formulations and other recipes useful in methods of T cell differentiation described herein.44 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT45 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0237] In one embodiment, the cells are contacted with a lymphoid induction media. In one embodiment, the lymphoid induction media comprises the T cell differentiation medium further comprising SCF, FLT3, TPO, IL-3, and IL-7. In one embodiment the cells are contacted with the lymphoid induction media for at least 7 days, i.e., from day 0 to day 7 of method. 46 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0238] In one embodiment, the cells are contacted with a T-cell specification media. In one embodiment, the lymphoid induction media comprises the T cell differentiation medium further comprising SCF, FLT3, TPO, and IL-7. In one embodiment, the cells are contacted with the T-cell specification media at least 7 days, i.e., from day 7 to day 14 of method.

[0239] In one embodiment, the cells are contacted with a T-cell maturation media. In one embodiment, the lymphoid induction media comprises the T cell differentiation medium further comprising FLT3 and IL-7. In one embodiment, the cells are contacted with the T-cell maturation media at least 20 days, i.e., from day 14 to day 35 of method.

[0240] In one embodiment, the cells are contacted with a SP T-cell induction media. In one embodiment, the lymphoid induction media comprises the T cell differentiation medium further comprising FLT3, IL-7, CD3 / CD28 T cell activator, and IL-15. In one embodiment, the cells are contacted with the SP T-cell induction media at least 7 days, i.e., from day 35 to day 42 of method.

[0241] In some embodiments, the CD3+-T-cell-differentiation media further comprises thrombopoietin (TPO) for at least the first 2 weeks of differentiating in the CD3+-T-cell- differentiation media. As a non-limiting example, the CD3+-T-cell-differentiation media further comprises thrombopoietin (TPO) for at least the first 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days of differentiating in the CD3+-T-cell-differentiation media. In some embodiments, CD3+-T-cell-differentiation media comprising TPO promotes differentiation into a population of CD5+ CD7+ ProT cells. Such CD5+ CD7+ ProT cells can be detected after at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days of differentiating in the CD3+-T-cell-differentiation media. In some embodiments, CD5+ CD7+ ProT cells can be detected after at least 2 weeks of differentiating in the CD3+-T-cell- differentiation media.

[0242] In some embodiments, the concentration of TPO should be used such that it promotes the 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 concertation of TPO (e.g., in the CD3+-T-cell-differentiation media) is 5 ng / mL. In some embodiments, the concertation of TPO (e.g., in the CD3+-T-cell-differentiation media) is 50 ng / ml.

[0243] In some embodiments, the CD3+-T-cell-differentiation media (e.g., comprising IL-7 and / or FLT3) further comprises SCF for at least the first 2 weeks of differentiating in the CD3+-T-cell- differentiation media. As a non-limiting example, the CD3+-T-cell-differentiation media further comprises SCF for at least the first 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days of differentiating in the CD3+-T-cell-differentiation media. In some embodiments, 47 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT CD3+-T-cell-differentiation media comprising SCF promotes differentiation into a population of CD5+ CD7+ ProT cells.

[0244] In some embodiments, SCF, FLT3, IL7, and / or TPO are provided in the CD3+-T-cell- differentiation media at a concentration of at least 1 ng / mL, at least 2 ng / mL, at least 3 ng / mL, at least 4 ng / mL, at least 5 ng / mL, at least 6 ng / mL, at least 7 ng / mL, at least 8 ng / mL, at least 9 ng / mL, at least 10 ng / mL, at least 11 ng / mL, at least 12 ng / mL, at least 13 ng / mL, at least 14 ng / mL, at least 15 ng / mL, at least 16 ng / mL, at least 17 ng / mL, at least 18 ng / mL, at least 19 ng / mL, at least 20 ng / mL, at least 25 ng / mL, at least 30 ng / mL, at least 35 ng / mL, at least 40 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 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 concentration of SCF, FLT3, IL7, and / or TPO can be the same or different.

[0245] In some embodiments, CD3+ T cells can be detected after at least 5.0 weeks of differentiating in the CD3+-T-cell-differentiation media. In some embodiments, CD3+ T cells can be detected after at least 1.5 weeks, 2 weeks, 2.5 weeks, 3.0 weeks, 3.5 weeks, 4.0 weeks, 4.5 weeks, or 5.0 weeks of differentiating in the CD3+-T-cell-differentiation media. In some embodiments, the population of CD3+ T cells comprises a population of CD4+CD8+ T cells, also referred to herein as double-positive or DP T cells. Such CD4+CD8+ CD3+ T cells can be detected after at least 1.5 weeks, 2 weeks, 2.5 weeks, 3.0 weeks, 3.5 weeks, 4.0 weeks, 4.5 weeks, or 5.0 weeks of differentiating in the CD3+-T- cell-differentiation media.

[0246] In some embodiments, the method further comprises differentiating the population of CD4+CD8+ T cells in a single-positive-T-cell-differentiation media for a sufficient time to promote differentiation into a population of CD4+ cells and a population of CD8+ cells. In some embodiments, the sufficient time to promote differentiation from the population of CD4+CD8+ T cells into a population of CD4+ T cells and a population of CD8+ cells is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days. In some embodiments, the sufficient time to promote differentiation from the population of CD34+ hemogenic endothelium into a population of CD4+ T cells and a population of CD8+ cells is at least 4.0 weeks, 4.5 weeks, 5.0 weeks, 5.5. weeks, or 6.0 weeks.

[0247] In some embodiments, the single-positive-T-cell-differentiation media comprises 10 ng / ml 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, a variety of concentrations of IL-15 can be used as long as it still promotes the 48 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT differentiation of CD4+CD8+ T cells into single positive CD4+ cells and CD8+ cells. In some embodiments, the concentration of IL15 can range from 1 ng / mL to 200 ng / mL, with a preferred concentration of 10 ng / ml.

[0248] In some embodiments, the T cell activator comprises components (e.g., soluble tetrameric antibody complexes) that bind CD3 and CD28 (and optionally CD2) cell surface ligands. Binding of the T cell activator results in the cross-linking of CD3 and CD28 (and optionally CD2) cell surface ligands, thereby providing the required primary and co-stimulatory signals for T cell activation.

[0249] In some embodiments, the T cell activator comprises a CD3 / CD28 T cell activator (e.g., at a concentration of 10ul / ml). Such a CD3 / CD28 T cell activator is available commercially (e.g., via StemCell Technology™, item #10970). In some embodiments, the concentration of the CD3 / CD28 T cell activator should be used such that it promotes the differentiation of CD4+CD8+ T cells into single positive CD4+ cells and CD8+ cells. In some embodiments, the concentration can range from 1 ul / mL to 200 ul / mL, with a preferred concentration of 10 ul / ml.

[0250] 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 available commercially (e.g., via ThermoFisher™ #11132D). In some embodiments, the concentrations of CD3 / CD28 T cell activator Dynabeads should be used such that it promotes the differentiation of CD4+CD8+ T cells into single positive CD4+ cells and CD8+ cells. In some embodiments, the concentration can range from 1 bead / cell to 20 beads / cell, with a preferred concentration of 1 bead / cell.

[0251] In some embodiments, the method further comprises, after at least 1 week (e.g., in the single- positive-T-cell-differentiation media), a step of CD4+ cell enrichment and / or CD8+ cell enrichment. In some embodiments, a step of CD4+ cell enrichment and / or CD8+ cell enrichment can occur at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days of culturing in the single-positive-T-cell-differentiation media.

[0252] Methods of enriching for CD4+ or CD8+ cells are known in the art. As non-limiting examples, the CD4+ or CD8+ cells can be enriched using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS) with anti-CD4 or anti-CD8 antibodies accordingly.

[0253] In some embodiments, the entire T cell differentiation protocol described herein occurs in a stromal-free environment, e.g., the cells are cultured exposed to a non-stromal-derived Notch ligand (e.g., Notch ligand immobilized on a tissue culture plate). In some embodiments, at least a portion of the T cell differentiation protocol (e.g., comprising culturing in the CD3+-T-cell-differentiation media and in the single-positive-T-cell-differentiation media) occurs in a stromal-free environment, e.g., the cells are cultured exposed to a non-stromal-derived Notch ligand (e.g., Notch ligand immobilized on a tissue culture plate). 49 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT Derived T Cell Population

[0254] As described herein, the population of T cells derived, e.g., using stromal-free methods as described herein, exhibits at least the following unexpected benefits compared to stromal co-culture methods: (1) increased potential for transplantation in humans; (2) decreased number of innate-like T cells; (3) increased number and / or percentage of resultant T cells (e.g., CD5+CD7+ Pro-T cells; CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells; alpha-beta T cells); (4) gene expression profiles most similar to alpha beta T cells; (5) a more diverse TCR repertoire; and / or (6) increased TCR CDR length.

[0255] Use of the cells or stable cell lines described herein, e.g., expressing any of the synthetic nucleic acids, viruses or lentiviruses thereof, eliminates the need for lentiviral transduction at the CD34+ stage in the methods described in US Patent Application No.17 / 794,747, the contents of which are incorporated herein by reference in its entirety.

[0256] In some embodiments, the population of T cells derived from methods described herein are produced more efficiently as compared to other methods, e.g., using the method as described in US Patent Application No.17 / 794,747. In one embodiment, the population of T cells derived from methods described herein are produced at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% or more, or at least 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 500x, 1,000x, or more efficiently as compared to other methods, e.g., using the method as described in US Patent Application No.17 / 794,747

[0257] In some embodiments, the population of T cells derived from methods described herein are produced a higher percentage of CD3+TCRa+b+ cells as compared to other methods, e.g., using the method as described in US Patent Application No.17 / 794,747. In one embodiment, the population of T cells derived from methods described herein are produce at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% or more, or at least 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 500x, 1,000x, or more CD3+TCRa+b+ cells as compared to other methods, e.g., using the method as described in US Patent Application No.17 / 794,747.

[0258] In some embodiments, the population of T cells derived from methods described herein are produced a T cells having higher viability as compared to other methods, e.g., using the method as 50 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT described in US Patent Application No.17 / 794,747. In some embodiments, the population of T cells derived from methods described herein are produced a T cells having at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% or more, or at least 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 500x, 1,000x, or more greater viability as compared to other methods, e.g., using the method as described in US Patent Application No.17 / 794,747.

[0259] In some embodiments, the population of T cells (e.g., CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells) derived using stromal-free methods as described herein exhibits at least a 10% higher transplantation or engraftment rate than a population of T cells derived using a stromal method. In some embodiments, the population of T cells derived using stromal-free methods as described herein exhibits at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% or more, or at least 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 500x, 1,000x, or more higher transplantation or engraftment rate than a population of T cells derived using a stromal method.

[0260] In some embodiments, a minority of the population of T cells (e.g., CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells) derived using stromal-free methods as described herein are TCRgd+(i.e., innate-like gamma delta T cells). Gamma delta T cells (γδ T cells) are T cells that have a distinctive 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 that is made up of one γ (gamma) chain and one δ (delta) chain. Like other 'unconventional' T cell subsets bearing invariant TCRs, such as CD1d- restricted Natural Killer T cells, gamma delta T cells exhibit several characteristics that place them at the border between the more evolutionarily primitive innate immune system that permits a rapid beneficial response to a variety of foreign agents and the adaptive immune system, where B and T cells coordinate a slower but highly antigen-specific immune response leading to long-lasting memory against subsequent challenges by the same antigen. Gamma delta T cells may be considered a component of adaptive immunity in that they rearrange TCR genes to produce junctional diversity and can develop a memory phenotype. However, the various subsets may also be considered part of the innate immunity in which a specific TCR can function as a pattern recognition receptor. See, e.g., 51 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT Born WK, Reardon CL, O'Brien RL (February 2006). "The function of gammadelta T cells in innate immunity". Current Opinion in Immunology.18 (1): 31–8.

[0261] 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) derived using stromal-free as 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 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% of the population of T cells (e.g., CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells) are TCRgd+.

[0262] In some embodiments, the population of T cells (e.g., CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells) derived using stromal-free methods as described herein comprises at least 10% more T cells than a population of T cells derived using a stromal method. In some embodiments, the population of T cells derived using stromal-free methods as described herein comprises at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% or more, or at least 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 500x, 1,000x, or more T cells than a population of T cells derived using a stromal method.

[0263] In some embodiments, the population of T cells (e.g., CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells) derived using stromal-free as described herein exhibits a gene expression profile that is more similar to αβ T cells, than to other cells (e.g., γδ T cells; NK cells; iPSCs derived T cells using a OP9-DL4 co-culture system; T cells differentiated from cord blood CD34+ HSPCs), e.g., the gene profile of the derived T cells is at least 0.5% more similar to a αβ T cells as compared to another cell type. In one embodiment, the population of T cells derived using stromal-free methods as described herein exhibits a gene expression profile of T cell signature genes and / or αβ T cell signature genes that is at most 10% divergent from the gene expression profile of αβ T cells. In one embodiment, the population of T cells derived using stromal-free methods as described herein exhibits a gene expression profile of T cell signature genes and / or αβ T signature cell genes that is at most 20% (e.g., at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most 9%, at most 10%, at most 11%, at most 12%, at most 13%, at most 52 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 14%, at most 15%, at most 16%, at most 17%, at most 18%, at most 19%, or more) divergent from the gene expression profile of αβ T cells. In one embodiment, the population of T cells derived using stromal-free methods as described herein exhibits a gene expression profile of T cell signature genes and / or αβ T cell signature genes that is 1%-5%, 2%-6%, 3%-7%, 4%-8%, 5%-9%, 5%-10%, 5%-15%, 10%-15%, or 15%-20% divergent from the gene expression profile of αβ T cells.

[0264] In one embodiment, the population of T cells derived using stromal-free methods as 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% or more similar to the gene expression profile of αβ T cells compared to a population of T cells derived using a stromal method. In one embodiment, the derived T cell has a greater percentage of similarity to the gene expression profile of an αβ T cell than the gene profile of another cell type. One skilled in the art can determine the similarity of gene expression in a T cell derived from stromal-free methods described herein and an αβ T cell using standard methods, e.g., transcriptome sequencing of specific cell types (FACS-sorted cells).

[0265] In one embodiment, the population of T cells derived using stromal-free methods as described herein exhibits a gene expression profile with a Pearson’s correlation coefficient compared to peripheral blood alpha beta T cells that is at least 0.75, 0.755, 0.76, 0.765, 0.77, 0.775, 0.78, 0.785, 0.79, 0.795, 0.8, 0.805, 0.81, 0.815, 0.82, 0.825, 0.83, 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.

[0266] 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 someembodiments, the population of CD3+T cells exhibits a gene expression profile that is at least 10%,20%, 30%, 40% or more similar to alpha beta T cells. In some embodiments, the population of CD3+ T cells exhibits a gene expression profile with a Pearson’s correlation coefficient compared to peripheral blood alpha beta T cells that is at least 0.85.

[0267] In some embodiments, the immune cell, e.g., derived using stromal-free as described herein, exhibits a gene expression profile that is most similar to alpha beta T cells. In some embodiments, the immune cell exhibits a gene expression profile that is similar or substantially similar to alpha beta T cells. In some embodiments, the immune cell exhibits a gene expression profile that is at least 10%, 20%, 30%, 40% or more similar to alpha beta T cells. In some embodiments, the immune cell exhibits 53 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT a gene expression profile with a Pearson’s correlation coefficient compared to peripheral blood alpha beta T cells that is at least 0.85.

[0268] In some embodiments, the population of T cells derived, e.g., using stromal-free methods as described herein expresses at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 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, at least 150 or more signature genes from an αβ T cell. In one embodiment, the derived T cell expresses a greater number of signature genes from an αβ T cell than signature genes from another cell type. As used herein, the term “signature gene” refers to a gene that exhibits a characteristic expression pattern in a specific cell type (e.g., T cell, αβ T cell); a signature gene can be required for the function of a specific cell type. Non-limiting examples of T cell signature genes and αβ T cell signature genes are described further herein. A specific cell type (e.g., T cell, αβ T cell) exhibits a gene signature or gene expression signature, which comprises a single or combined group of genes in a cell with a uniquely characteristic pattern of gene expression (i.e., signature genes).

[0269] In some embodiments, the population of T cells derived expresses at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 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, at least 54 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 150 or more genes from an αβ T cell. In one embodiment, the derived T cell expresses a greater number of genes from a αβ T cells than signature genes from another cell type.

[0270] In some embodiments, the population of T cells derived using stromal-free methods as described herein expresses at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 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, at least 150 or more genes from an αβ T cell. In one embodiment, the derived T cell expresses a greater number of genes from a αβ T cells than signature genes from another cell type.

[0271] 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 (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 viral 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 55 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT (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 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).

[0272] Non-limiting examples of αβ T cell signature genes include ATP11B (ATPase Phospholipid Transporting 11B); PPP4R3A (Protein Phosphatase 4 Regulatory Subunit 3A); CAB39 (Calcium Binding Protein 39); GLS (Glutaminase); UBE2Z (Ubiquitin Conjugating Enzyme E2 Z); INPP4A (Inositol Polyphosphate-4-Phosphatase Type I A); RAB22A (Ras-Related Protein Rab-22A, Member Ras Oncogene Family); SMARCD2 (SWI / SNF (SWItch / Sucrose Non-Fermentable) Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily D, Member 2); VPS26B (VPS26, Retromer Complex Component B, Vacuolar Protein Sorting-Associated Protein 26B); CERK (Ceramide Kinase); ESYT2 (Extended Synaptotagmin 2); RAC1 (Rac 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 determination protein 1) Family Inhibitor Domain Containing); YWHAH (Tyrosine 3-Monooxygenase / Tryptophan 5-Monooxygenase Activation Protein Eta); MCMBP (Minichromosome Maintenance Complex Binding Protein); GOLPH3 (Golgi Phosphoprotein 3); PTGER4 (Prostaglandin E Receptor 4); B3GNT2 (UDP-GlcNAc:BetaGal Beta- 1,3-N-Acetylglucosaminyltransferase 2, Galactosyltransferase 7); PITPNC1 (Phosphatidylinositol Transfer Protein Cytoplasmic 1); ARAP2 (ArfGAP With RhoGAP Domain, Ankyrin Repeat And PH Domain 2; Arf And Rho GAP Adapter Protein 2); ZFP36L2 (Zinc Finger Protein 36, C3H1 Type- Like 2); EFHD2 (EF-Hand Domain Family Member D2, Swiprosin-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 Transferase Subunits Alpha And Beta); GRSF1 (G-Rich RNA Sequence Binding Factor 1); TARP (TCR Gamma Alternate Reading Frame Protein, T-Cell Receptor Gamma-Chain); ZBTB16 (Zinc Finger And BTB (for BR-C, ttk and 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); CD4 (T-Cell Surface Glycoprotein CD4); LRRN3 (Leucine Rich Repeat Neuronal 3); SLC40A1 56 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT (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); CCR7 (C-C 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 Of Lysosomal Organelles Complex 1 Subunit 4); SIAH2 (Siah E3 Ubiquitin Protein Ligase 2); MXD4 (Max 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 (Family With Sequence Similarity 50 Member A); PIM3 (Pim-3 Proto-Oncogene, Serine / Threonine Kinase); SIPA1 (Signal-Induced Proliferation- Associated 1); FAM89B (Family With Sequence Similarity 89 Member B); ZBTB7A (Zinc Finger And BTB (for BR-C, ttk and bab) Domain Containing 7A, Factor That Binds To Inducer Of Short Transcripts Protein 1); NIN (Ninein); 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 Lin-12- Like Protein 3); IL12RB2 (Interleukin 12 Receptor Subunit Beta 2); COTL1 (Coactosin Like F-Actin Binding Protein 1); PIK3AP1 (Phosphoinositide-3-Kinase Adaptor Protein 1); TBX21 (T-Box Transcription Factor 21); FAM43A (Family With Sequence Similarity 43 Member A); KLRD1 (Killer Cell Lectin Like Receptor D1); SLAMF7 (signaling lymphocytic activation molecule (SLAM) family member 7); S1PR5 (Sphingosine-1-Phosphate Receptor 5); LAG3 (Lymphocyte Activating 3); ABCG1 (ATP Binding Cassette Subfamily G Member 1); S100B (S100 Calcium-Binding Protein, Beta); CCL22 (C-C Motif Chemokine Ligand 22); CEBPD (CCAAT box Enhancer Binding Protein Delta); IL17F (Interleukin 17F); and CEACAM1 (CEA Cell Adhesion Molecule 1).

[0273] In some embodiments, the population of T cells derived using stromal-free methods as described herein exhibits a more diverse TCR repertoire compared to T cells not derived using such stromal-free methods. In some embodiments, the population of T cells derived using stromal-free methods as described herein exhibits a Productive Simpson Clonality value of about 0.000-0.025. A value closer to 0 represents a higher level of diversity compared to clonality. A value closer to 1 represents a higher level of clonality compared to diversity. In some embodiments, the population of 57 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT T cells derived using stromal-free methods as described herein exhibits a Productive Simpson Clonality value of at most 0.01, at most 0.015, at most 0.02, at most 0.025, at most 0.03, at most 0.035, at most 0.04, at most 0.045, at most 0.05, at most 0.055, at most 0.06, at most 0.065, at most 0.07, at most 0.075, at most 0.08, at most 0.085, at most 0.09, at most 0.095, or at most 0.1. In some embodiments, the population of T cells derived using stromal-free methods as described herein exhibits a Productive Simpson Clonality value of about 0.025.

[0274] The variable domain of both the T-cell receptor (TCR) α-chain and β-chain each have three hypervariable or complementarity-determining regions (CDRs; e.g., CDR1, CDR2, CDR3). In some embodiments, the population of T cells derived using stromal-free methods as described herein exhibits an increased CDR (e.g., CDR1, CDR2, CDR3) length compared to T cells derived using stromal methods. In some embodiments, the population of T cells derived using stromal-free methods as described herein exhibits CDR (e.g., CDR1, CDR2, CDR3) length that is, on average, about 3 nucleotides (nt), 6 nt, 9 nt, or 12 nt or more longer than the CDRs of T cells derived using stromal methods. In some embodiments, the population of T cells derived using stromal-free methods as described herein exhibits CDR (e.g., CDR1, CDR2, CDR3) length that is, on average, about 27 nt, 30 nt, 33 nt, 36 nt, 39 nt, 42 nt, 45 nt, 48 nt, 51 nt, 54 nt, 57 nt, or 60 nt or longer. In some embodiments, the population of T cells derived using stromal-free methods as described herein exhibits a CDR3 length that is, on average, about 42 nt long, compared to 39 nt on average for control iPSC-derived T cells, or 45 on average for peripheral blood mononuclear cell (PBMC)-derived T cells.

[0275] One aspect provided herein is a immune cell produced by any of the methods described herein, or an methods known in the art.

[0276] Another aspect herein is a composition comprising any of the immune cells described herein or population thereof. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are described herein below.

[0277] Another aspect herein is a pharmaceutical composition comprising any of the immune cell described herein or population thereof, and a pharmaceutically acceptable carrier.

[0278] In one embodiment, the immune cell or population, or any composition thereof is for use in cellular therapy in a subject. Genetic Modifications of T Cells

[0279] In some embodiments, the resultant population of CD34+ hemogenic endothelium or another population as described herein (e.g., ESCs; iPSCs; HSCs; CD5+CD7+ ProT cells; CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells) are genetically modified. In some embodiments, the native T cell receptor locus can be removed and / or replaced to enhance targeted specificity. In some embodiments, an endogenous HLA (e.g., class I and / or class II major histocompatibility complexes) can be edited or removed. In some embodiments, the genetic modification can comprise 58 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT introduction and expression of non-canonical HLA-G and HLA-E to prevent NK cell-mediated lysis (see e.g., Riolobos L et al.2013), which can provide a source of universal T cells for immunotherapy, e.g., cancer immune therapy.

[0280] In some embodiments, the genetic modification comprises expressing a chimeric antigen receptor (CAR). Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors) are receptor proteins that have been engineered to give T cells the new ability to target a specific protein. The receptors are chimeric because they combine both antigen-binding and T-cell activating functions into a single receptor. Methods of engineering chimeric antigen receptor T cells (also known as CAR T cells) are known in the art. See e.g., US Patents US7446190, US8399645, US8822647, US9212229, US9273283, US9447194, US9587020, US9932405, US10125193, US10221245, US10273300, US10287354; US patent publication US20160152723; PCT publication WO2009091826, WO2012079000, WO2014165707, WO2015164740, WO2016168595A1, WO2017040945, WO2017100428, WO2017117112, WO2017149515, WO2018067992, WO2018102787, WO2018102786, WO2018165228, WO2019084288; the contents of each of which are incorporated herein by reference in their entireties.

[0281] In some embodiments, methods of genetically modifying a cell to express a CAR can comprise but are not limited to: transfection or electroporation of a cell with a vector encoding a CAR; transduction with a viral vector (e.g., retrovirus, lentivirus) encoding a CAR; gene editing using zin finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganuclease- TALENs, or CRISPR-Cas; or any other methods known in the art of genetically modifying a cell to express a CAR.

[0282] Preferably, a population of cells at an early stage of differentiation (e.g., ESCs; PSCs; iPSCs; hemogenic endothelium; HSCs) is genetically modified with the CAR.

[0283] 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, autoimmune disease, or heart disease (e.g., cardiac fibrosis). As used herein, the term “cancer” relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.

[0284] 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 59 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., intrusion on and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via lymph or blood). As used herein, the term “metastasize” refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.” The metastatic tumor contains cells that are like those in the original (primary) tumor. As used herein, the term “benign” or “non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.

[0285] A “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue. A tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancer cells form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.

[0286] 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 the normal tissues. Thus, a neoplasm can be a benign neoplasm, premalignant neoplasm, or a malignant neoplasm.

[0287] A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastases. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.

[0288] Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelial neoplasm.; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin’s and non-Hodgkin’s lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade / follicular non-Hodgkin’s 60 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 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; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs’ syndrome. Preferably, in the case of CAR T therapy, the cancer is a blood cancer such as a leukemia or lymphoma.

[0289] Immunotherapy with chimeric antigen receptor (CAR) T cells offers a promising method to improve cure rates and decrease morbidities for patients with cancer. In this regard, CD19-specific CAR T cell therapies have achieved dramatic objective responses for a high percent of patients with CD19-positive leukemia or lymphoma. Accordingly, in some embodiments, the antigen-binding region of the CAR is directed against CD19; see e.g., US patents US10221245, US10357514; US patent publication US20160152723; PCT publication WO2016033570; the contents of each of which are incorporated herein by reference in their entireties.

[0290] Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses. The selection of the antigen binding domain of the invention will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-11Ra, IL-13Ra, EGFR, B7H3, Kit, CA-IX, CS-1, MUC1, BCMA, bcr-abl, HER2, β- human chorionic gonadotropin, alphafetoprotein (AFP), ALK, CD19, CD123, cyclin B 1, lectin- reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RU1, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAX5, SART3, CLL-1, fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, plysialic acid, PLAC1, RU1, RU2 (AS), intestinal carboxyl esterase, lewisY, sLe, LY6K, mut 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-carcinoma tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAD-CT-1, MAD-CT-2, MelanA / MART1, XAGE1, ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1, ephrinB2, 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, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. In a preferred embodiment, the tumor antigen is selected from the group consisting of folate receptor 61 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT (FRa), mesothelin, EGFRvIII, IL-13Ra, CD123, CD19, CD33, BCMA, GD2, CLL-1, CA-IX, MUC1, HER2, and any combination thereof; see e.g., US Patent publications 20170209492 and 20180022795, the contents of each of which are incorporated herein by reference in their entireties. Cellular therapy

[0291] In one embodiment, provided herein a population of engineered immune cells produced by a method described herein, where in the T cell population is produced using a stroma-free differentiation method as described herein. In some embodiments, the population of engineered immune cells comprises an immune cell differentiated using methods described herein, including but not limited to: PSCs; iPSCs; hemogenic endothelium; HSCs; CD5+CD7+ ProT cells; CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells. In some embodiments, the immune cell exhibits a gene expression profile that is most similar to alpha beta T cells.

[0292] In one embodiment, the population of cells further comprises a pharmaceutically acceptable carrier. These engineered immune cells can be culture expanded to increase the number of cells for use.

[0293] The engineered immune cells described herein are useful in the laboratory for biological studies. For examples, these cells can be derived from an individual having a genetic disease or defect, and used in the laboratory to study the biological aspects of the disease or defect, and to screen and test for potential remedy for that disease or defect.

[0294] Alternatively, the engineered immune cells described herein are useful in cellular therapy and other medical treatment in subjects having the need. For example, patients who have undergone chemotherapy or irradiation or both, and manifest deficiencies in immune function and / or lymphocyte reconstitution, or in cancer immune therapy.

[0295] In various embodiments, the engineered immune cells described herein are administered (i.e., implanted or transplanted) to a subject in need of cellular therapy.

[0296] In one embodiment, provided herein is a method of cellular therapy, or for the treatment of cancer, autoimmune disorders, hematological diseases, or other genetic diseases and disorders in a subject, comprising (a) providing a somatic cell from a donor subject, (b) generating multilineage hematopoietic progenitor cells (e.g., hemogenic endothelium, HSPCs) from pluripotent stem cells derived from the somatic cell as described in any of the preceding paragraphs; (c) optionally inhibiting a histone methyltransferase in the resultant population of multilineage hematopoietic progenitor cells as described in any of the preceding paragraphs; (d) differentiating the resultant population of multilineage hematopoietic progenitor cells in the presence of a notch ligand to promote differentiation into the lymphoid lineage (e.g., T cells) as described in any of the preceding paragraphs, and (e) implanting or administering the resultant differentiated lymphoid cells into a recipient subject. 62 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0297] In one embodiment, the host subject and the recipient subject are the same individual. Alternatively, the host subject and the recipient subject are not the same individual, but are at least HLA compatible.

[0298] Hematological diseases are disorders which primarily affect the blood. Non-limiting such diseases or disorders include myeloid derived disorders such as hemoglobinopathies (congenital abnormality of the hemoglobin molecule or of the rate of hemoglobin synthesis), examples, sickle-cell disease, thalassemia, and methemoglobinemia; Anemias (lack of red blood cells or hemoglobin), Pernicious anemia; disorders resulting in decreased numbers of cells, such as myelodysplastic syndrome, neutropenia (decrease in the number of neutrophils), and thrombotic thrombocytopenic purpura (TTP), thrombocytosis, hematological malignancies such as lymphomas, myelomas, and leukemia. 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, Waldenström macroglobulinemia, Plasmacytoma; leukemias that increases defect WBC such as Acute lymphocytic leukemia (ALL), Chronic lymphocytic leukemia (CLL), Acute myelogenous leukemia (AML), Chronic Idiopathic Myelofibrosis (MF), Chronic myelogenous leukemia (CML), T-cell prolymphocytic leukemia (T-PLL), B-cell prolymphocytic leukemia (B-PLL), Chronic neutrophilic leukemia (CNL), Hairy cell leukemia (HCL), T-cell large granular lymphocyte leukemia (T-LGL), and Aggressive NK-cell leukemia.

[0299] Provided herein is a method of treating an autoimmune disease, which comprises administering an effective amount of an immune cell or population thereof, or a composition, or a pharmaceutical composition as 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 are autoreactive to endogenous self-peptides and cause destruction of tissue. Thus, an immune response is mounted against a subject's own antigens, referred to as self-antigens. A “self-antigen” as used herein refers to an antigen of a normal host tissue. Normal host tissue does not include neoplastic cells.

[0300] Non-limiting examples of autoimmune diseases that can be treated include pemphigus (pemphigus vulgaris, pemphigus foliaceus or paraneoplastic pemphigus), Crohn's disease, idiopathic thrombocytopenic purpura (ITP), heparin induced thrombocytopenia (HIT), thrombotic thrombocytopenic purpura (TTP), Myasthenia Gravis (MG), and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP). Additional non-limiting autoimmune diseases include autoimmune thrombocytopenia, immune neutropenia, antihemophilic FVIII inhibitor, antiphospholipid syndrome, Kawasaki Syndrome, ANCA-associated disease, polymyositis, bullous pemphigoid, multiple sclerosis (MS), Guillain-Barre Syndrome, chronic polyneuropathy, ulcerative colitis, diabetes mellitus, autoimmune thyroiditis, Graves' opthalmopathy, rheumatoid arthritis, 63 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT ulcerative colitis, primary sclerosing cholangitis, systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, Hashimoto's thyroiditis, Goodpasture's syndrome, autoimmune hemolytic anemia, scleroderma with anticollagen antibodies, mixed connective tissue disease, pernicious anemia, idiopathic Addison's disease, autoimmune-associated infertility, glomerulonephritis (e.g., crescentic glomerulonephritis, proliferative glomerulonephritis), insulin resistance, and autoimmune diabetes mellitus (type 1 diabetes mellitus; insulin dependent diabetes mellitus). Autoimmune disease has been recognized also to encompass atherosclerosis and Alzheimer's disease. In another embodiment, the autoimmune diseases include hepatitis, autoimmune hemophilia, autoimmune lymphoproliferative syndrome (ALPS), autoimmune uveoretinitis, glomerulonephritis, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, autoimmune urticarial neuropathy, autoimmune axonal neuropathy, Balo disease, Behçet's disease, Castleman disease, celiac disease, Chagas disease, chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid, benign mucosal 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-Schonlein purpura, Herpes gestationis, Idiopathic hypocomplementemic tubulointestitial nephritis, multiple myeloma, multifocal motor neuropathy, NMDA receptor antibody encephalitis, IgG4-related disease, IgG4-related sclerosing disease, inflammatory aortic aneurysm, inflammatory pseudotumour, inclusion body myositis, interstitial cystitis, juvenile arthritis, Kuttner's tumour, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lyme disease, chronic, mediastinal fibrosis, Meniere's disease, Microscopic polyangiitis, Mikulicz's syndrome, Mooren's ulcer, Mucha-Habermann disease, multifocal fibrosclerosis, narcolepsy, optic neuritis, Ormond's disease (retroperitoneal fibrosis), palindromic rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with Streptococcus), paraneoplastic cerebellar degeneration, paraproteinemic polyneuropathies, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, periaortitis, periarteritis, peripheral neuropathy, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, Type I, II, & III autoimmune polyglandular syndromes, polymyalgia rheumatic, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, 64 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT restless legs syndrome, rheumatic fever, Riede's thyroiditis, sarcoidosis, Schmidt syndrome, scleritis, Sjogren's syndrome, sperm and testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, Tolosa-Hunt syndrome, transverse myelitis, undifferentiated connective tissue disease (UCTD), vesiculobullous dermatosis, vitiligo, Rasmussen's encephalitis, Waldenstrom's macroglobulinaemia.

[0301] As used herein, the terms "administering," "introducing" and "transplanting" are used interchangeably in the context of the placement of described cells, e.g., hematopoietic progenitor cells, into a subject, by a method or route which results in at least partial localization of the introduced cells at a desired site, such as a site of injury or repair, such that a desired effect(s) is produced. The cells e.g., hematopoietic progenitor cells, or their differentiated progeny (e.g., T cells) can be administered by any appropriate route which results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable.

[0302] In various embodiments, the engineered immune cells described herein are optionally expanded ex vivo prior to administration to a subject. In other embodiments, the engineered immune cells are optionally cryopreserved for a period, then thawed prior to administration to a subject.

[0303] The engineered immune cells used for cellular therapy can be autologous / autogenic ("self") or non-autologous ("non-self," e.g., allogeneic, syngeneic or xenogeneic) in relation to the recipient of the cells. "Autologous," as used herein, refers to cells from the same subject. "Allogeneic," as used herein, refers to cells of the same species that differ genetically to the cell in comparison. "Syngeneic," as used herein, refers to cells of a different subject that are genetically identical to the cell in comparison. "Xenogeneic," as used herein, refers to cells of a different species to the cell in comparison. In preferred embodiments, the cells of the invention are allogeneic.

[0304] In various embodiments, the engineered immune cell described herein that is to be implanted into a subject in need thereof is autologous or allogeneic to the subject.

[0305] In various embodiments, the engineered immune cell described herein can be derived from one or more donors, or can be obtained from an autologous source. In some embodiments, the engineered immune cells are expanded in culture prior to administration to a subject in need thereof.

[0306] In various embodiments, the engineered immune cell described herein can be derived from one or more donors, or can be obtained from an autologous source.

[0307] In various embodiments, prior to implantation, the recipient subject is treated with chemotherapy and / or radiation.

[0308] In various embodiments, the recipient subject is a human.

[0309] In various embodiments, the subject has been previously diagnosed with HIV or other viral disease, a hematological disease, or undergoing a cancer treatment. 65 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0310] In one embodiment, a subject is selected to donate a somatic cell which would be used to produce iPSCs and an engineered immune cell described herein. In one embodiment, the selected subject has a genetic disease or defect.

[0311] In various embodiments, the donor subject is a human, non-human animal, rodent or non- rodent. For example, the subject can be any mammal, e.g., a human, other primate, pig, rodent such as mouse or rat, rabbit, guinea pig, hamster, cow, horse, cat, dog, sheep or goat, or a non-mammal such as a bird.

[0312] In various embodiments, the donor has been previously diagnosed with HIV, a hematological disease or cancer.

[0313] In one embodiment, a biological sample, a population of embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells is obtained from the donor subject.

[0314] In various embodiments, the biological sample, a population of embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells described herein can be derived from one or more donors, or can be obtained from an autologous source.

[0315] In one embodiment, the embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, hematopoietic progenitor cells are isolated from the donor subject, transfected, cultured (optional), and transplanted back into the same subject, i.e., an autologous cell transplant. Here, the donor and the recipient subject is the same individual. In another embodiment, the embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells are isolated from a donor who is an HLA- type match with a subject (recipient). Donor-recipient antigen type-matching is well known in the art. The HLA-types include HLA-A, HLA-B, HLA-C, and HLA-D. These represent the minimum number of cell surface antigen matching required for transplantation. That is the transfected cells are transplanted into a different subject, i.e., allogeneic to the recipient host subject. The donor’s or subject’s embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells can be transfected with a vector or nucleic acid comprising the nucleic acid molecule(s) described herein, the transfected cells are cultured, inhibited, and differentiated as disclosed, optionally expanded, and then transplanted into the recipient subject. In one embodiment, the transplanted engineered immune cells engraft in the recipient subject. In one embodiment, the transplanted engineered immune cells reconstitute the immune system in the recipient subject. The transfected cells can also be cryopreserved after transfected and stored, or cryopreserved after cell expansion and stored.

[0316] The engineered immune cells or the histone methyltransferase inhibited, multilineage hematopoietic progenitor cells or T cells differentiated using a stroma-free method as described herein 66 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT may be administered as part of a bone marrow or cord blood transplant in an individual that has or has not undergone bone marrow ablative therapy. In one embodiment, genetically modified cells contemplated herein are administered in a bone marrow transplant to an individual that has undergone chemoablative or radioablative bone marrow therapy.

[0317] In one embodiment, a dose of cells is delivered to a subject intravenously. In one embodiment, the cells are intravenously administered to a subject.

[0318] In particular embodiments, patients receive a dose of the modified cells described herein, e.g., engineered immune cells or the histone methyltransferase inhibited, multilineage hematopoietic progenitor cells or T cells differentiated using a stroma-free method as described herein, of about 1 x 105cells / kg, about 5 x 105cells / kg, about 1 x 106cells / kg, about 2 x 106cells / kg, about 3 x 106cells / kg, about 4 x 106cells / kg, about 5 x 106cells / kg, about 6 x 106cells / kg, about 7 x 106cells / kg, about 8 x 106cells / kg, about 9 x 106cells / kg, about 1 x 107cells / kg, about 5 x 107cells / kg, about 1 x 108cells / kg, or more in one single intravenous dose.

[0319] In certain embodiments, patients receive a dose of the modified cells described herein, e.g., engineered immune cells or the histone methyltransferase inhibited, multilineage hematopoietic progenitor cells or T cells differentiated using a stroma-free method as described herein, of at least 1 x 105cells / kg, at least 5 x 105cells / kg, at least 1 x 106cells / kg, at least 2 x 106cells / kg, at least 3 x 106cells / kg, at least 4 x 106cells / kg, at least 5 x 106cells / kg, at least 6 x 106cells / kg, at least 7 x 106cells / kg, at least 8 x 106cells / kg, at least 9 x 106cells / kg, at least 1 x 107cells / kg, at least 5 x 107cells / kg, at least 1 x 108cells / kg, or more in one single intravenous dose.

[0320] In an additional embodiment, patients receive a dose of the modified cells described herein, e.g., engineered immune cells, multilineage hematopoietic progenitor cells or T cells differentiated using a stroma-free method as described herein, of about 1 x 105cells / kg to about 1 x 108cells / kg, about 1 x 106cells / kg to about 1 x 108cells / kg, about 1 x 106cells / kg to about 9 x 106cells / kg, about 2 x 106cells / kg to about 8 x 106cells / kg, about 2 x 106cells / kg to about 8 x 106cells / kg, about 2 x 106cells / kg to about 5 x 106cells / kg, about 3 x 106cells / kg to about 5 x 106cells / kg, about 3 x 106cells / kg to about 4 x 108cells / kg, or any intervening dose of cells / kg.

[0321] In general, the engineered immune cells or the histone methyltransferase inhibited, multilineage hematopoietic progenitor cell described herein or T cells differentiated using a stroma- free method as described herein are administered as a suspension with a pharmaceutically acceptable carrier. For example, as therapeutic compositions. Therapeutic compositions contain a physiologically tolerable carrier together with the cell composition and optionally at least one additional bioactive agent as described herein, dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the therapeutic composition is not substantially immunogenic when administered to a mammal or human patient for therapeutic purposes, unless so desired. One of skill in the art will recognize that a pharmaceutically acceptable carrier to be used in a cell composition will not include 67 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT buffers, compounds, cryopreservation agents, preservatives, or other agents in amounts that substantially interfere with the viability of the cells to be delivered to the subject. A formulation comprising cells can include, e.g., osmotic buffers that permit cell membrane integrity to be maintained, and optionally, nutrients to maintain cell viability or enhance engraftment upon administration. Such formulations and suspensions are known to those of skill in the art and / or can be adapted for use with the cells as described herein using routine experimentation.

[0322] As used herein, the terms “pharmaceutically acceptable”, “physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like. A pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired. The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectable either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified or presented as a liposome composition. The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient. The therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and 68 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT water-oil emulsions. The amount of an active agent used in the methods described herein that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field of art. For example, a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of active ingredient in 0.9% sodium chloride solution.

[0323] In one embodiment, the “pharmaceutically acceptable” carrier does not include in vitro cell culture media.

[0324] In some embodiments, the composition of engineered immune cells described further comprises a pharmaceutically acceptable carrier.

[0325] In various embodiments, at least a second or subsequent dose of cells is administered to the recipient subject. For example, a second administration can be given between about one day to 30 weeks from the previous administration. Two, three, four or more total subsequent administrations can be delivered to the individual, as needed, e.g., determined by a skilled clinician.

[0326] A cell composition can be administered by any appropriate route which results in effective cellular replacement treatment in the subject, i.e. administration results in delivery to a desired location in the subject where at least a portion of the composition delivered, i.e. at least 1 x 104cells are delivered to the desired site for a period of time. Modes of administration include injection, infusion, or instillation, “Injection” includes, without limitation, intravenous, intra-arterial, intraventricular, intracardiac injection and infusion. For the delivery of cells, administration by injection or infusion is generally preferred.

[0327] Efficacy testing can be performed during the course of treatment using the methods described herein. Measurements of the degree of severity of a number of symptoms associated with a particular ailment are noted prior to the start of a treatment and then at later specific time period after the start of the treatment. In some embodiments, a pharmaceutical composition comprising an immune as described herein or a population thereof can be used for cellular therapy in a subject.

[0328] Accordingly, it is also the objective of this the present disclosure to provide compositions of modified (also referred to as engineered) cells for use in in vivo cellular therapy, medical therapy such as cancer immune therapy, and for the in vitro studies of disease modeling, drug screening, and hematological diseases.

[0329] The advantage of the disclosure protocols is the methods permit semi-permanent bulk production of desired immune cells or other types of hematopoietic cells (i.e. cells differentiated from multipotent HSCs,) from a variety of types of cell source, from stem cells, hematopoietic progenitor cells, and mature and differentiated somatic cells, all of which can be readily collected from the patient's body. 69 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0330] The produced engineered immune cells or engineered histone methyltransferase-inhibited, CD34+ / CD 38lo / -hematopoietic progenitor cells (e.g., hemogenic endothelium) or T cells differentiated using a stroma-free method as described herein can be transplanted into a patient for various medical treatments such as immune system reconstruction therapy (e.g., after bone marrow ablation) or immunotherapy (e.g., in cancer therapy or autoimmune diseases). One added advantage is that if the donor of the source cells and recipient of the engineered immune cells are the same person, the produced engineered immune cells have HLA that are identical to the recipient and this avoids host-graft immune rejection after the transplantation. For recipient patients that are HLA allogeneic to the donor person of the source cells, host-graft immune rejection is greatly reduced.

[0331] The produced engineered immune cells or engineered histone methyltransferase-inhibited, CD34+ / CD 38- hematopoietic progenitor cells or T cells differentiated using a stroma-free method as described herein can also be cryopreserved till needed in the future.

[0332] Currently, bone marrow transplantation is the most established cellular therapy for a variety of hematological disorders. The functional unit of a bone marrow transplant 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 severely limits the ability to carry out transplantation, disease modeling and drug screening. As such, many studies have aimed to generate HSCs from alternative sources. Advances in reprogramming to induced pluripotent stem cells (iPSCs) has provided access to a wide array of patient-specific pluripotent cells, a promising source for disease modeling, drug screens and cellular therapies. However, the inability to derive engraftable hematopoietic stem and progenitor cells from human pluripotent stem cells (hPSCs) has limited the characterization of hematological diseases to in vitro assays. Generation of HSCs by directed differentiation has remained elusive, and there is a need for novel approaches to this problem.

[0333] Accordingly, in one aspect described herein is a method of cellular therapy, the method comprising administering an immune cell as described herein or population thereof, or a composition comprising said immune cell or population thereof, or a pharmaceutical composition comprising said immune cell or population thereof to a recipient subject in need thereof.

[0334] In some embodiments, the recipient subject has undergone chemotherapy and / or irradiation. In some embodiments, the recipient subject has deficiencies in immune function and / or lymphocyte reconstitution. Definitions

[0335] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the 70 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 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 within the specification shall prevail.

[0336] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[0337] As used herein, the term “cell” refers to a single cell as well as to a population of (i.e., more than one) cells. The population may be a pure population comprising one cell type, such as a population of pluripotent stem cells or a population of differentiated T cells. As used herein, the term “population” refers to a pure population or to a population comprising a majority (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%) of one cell type. Alternatively, the population may comprise more than one cell type, for example a mixed cell population. It is not meant to limit the number of cells in a population; for example, a mixed population of cells may comprise at least one differentiated cell. In the present invention, there is no limit on the number of cell types that a mixed cell population may comprise.

[0338] As used herein, in one embodiment, the term “hematopoietic stem cell” or “HSC” refers to a stem cell that has self-renewal capacity and also give rise to all the blood cell types of the three hematopoietic lineages, erythroid, lymphoid, and myeloid. These cell types include the myeloid lineages (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and the lymphoid lineages (T-cells, B-cells, NK-cells). Human HSCs are determined as CD34+, CD59+, CD90 / Thy1+, CD38low / -, c-kit / CD117- / low, and Lin-. Mouse HSC- are considered CD34low / -, SCA-1+, CD90 / Thy1+ / low, CD38+, c-Kit / CD117+, and Lin-. Detecting the expression of these marker panels allows separation of specific cell populations via techniques like fluorescence-activated cell sorting (FACS). In one embodiment, the term “hematopoietic stem cell” or “HSC” refers to a stem cell that has self-renewal capacity and that have the following cell surface markers: CD34+, CD59+, Thy1 / CD90+, CD38lo / -, 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 cellthat has self-renewal capacity and that is at least CD34+and c-kit / CD117lo / -. In one embodiment, theterm “hematopoietic stem cell” or “HSC” refers to a stem cell that has self-renewal capacity and that is at least CD38low / -, c-kit / CD117- / low. The term HSC can be used interchangeably with the term “hematopoietic stem and progenitor cell” (HSPC).

[0339] As used herein, the terms “iPS cell”, “iPSC”, and “induced pluripotent stem cell” are used interchangeably and refers to a pluripotent cell artificially derived by the transfection of the following reprogramming factors OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28, from a 71 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT differentiated cell, e.g., a somatic cell. Alternative combinations of reprogramming factors include OCT4, SOX2, NANOG, and LIN28. The term hPSC refers to a human pluripotent stem cell.

[0340] 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 development pathway, which the cell can take to becoming a fully differentiated cell. For example, a HSC has three hematopoietic lineages, erythroid, lymphoid, and myeloid; the HSC has the potential, i.e., the ability, to differentiate and develop into those terminally differentiated cell types known for all these three lineages. When the term “multilineage” used, it means the cell is able to, in the future, differentiate and develop into those terminally differentiated cell types known for more than one lineage. For example, the HSC has multilineage potential. When the term “limited lineage” used, it means the cell can differentiate and develop into those terminally differentiated cell types known for one lineage. For example, a common myeloid progenitor cell (CMP) or a megakaryocyte-erythroid progenitor (MEP) has a limited lineage because the cell can only differentiate and develop into those terminally differentiated cell types of the myeloid lineage and not that 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); and terminally differentiated cells of the lymphoid lineage include T lymphocytes / T cells, B lymphocytes / B cells, dendritic cells, and natural killer cells.

[0341] As used herein, the term “a progenitor cell” refers to an immature or undifferentiated cell that has the potential later on to mature (differentiate) into a specific cell type (a fully differentiated or terminally differentiated cell), for example, a blood cell, a skin cell, a bone cell, or hair cells. Progenitor cells have a cellular phenotype that is more primitive (e.g., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell) relative to a cell, which it can give rise to by differentiation. Often, progenitor cells also have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate. A progenitor cell also can proliferate to make more progenitor cells that are similarly immature or undifferentiated.

[0342] The term "differentiated cell" is meant any primary cell that is not, in its native form, pluripotent as that term is defined herein. The term a "differentiated cell" also encompasses cells that are partially differentiated, such as multipotent cells (e.g., adult somatic stem cells). In some embodiments, the term "differentiated cell" also refers to a cell of a more specialized cell type derived from a cell of a less specialized cell type (e.g., from an undifferentiated cell or a reprogrammed cell) where the cell has undergone a cellular differentiation process.

[0343] In the context of cell ontogeny, the term "differentiate", or "differentiating" is a relative term meaning a "differentiated cell" is a cell that has progressed further down the developmental pathway 72 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT than its precursor cell. Thus in some embodiments, a reprogrammed cell as this term is defined herein, can differentiate to lineage-restricted precursor cells (such as a mesodermal stem cell or a endodermal stem cell), which in turn can differentiate into other types of precursor cells further down the pathway (such as an tissue specific precursor, for example, a cardiomyocyte precursor, or a pancreatic precursor), and then to an end-stage differentiated cell, which plays a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further.

[0344] The term "multipotent" when used in reference to a "multipotent cell" refers to a cell that is able to differentiate into some but not all of the cells derived from all three germ layers. Thus, a multipotent cell is a partially differentiated cell. Multipotent cells are well known in the art, and examples of multipotent cells include adult somatic stem cells, such as for example, hematopoietic stem cells and neural stem cells, hair follicle stem cells, liver stem cells etc. Multipotent means a stem cell may form many types of cells in a given lineage, but not cells of other lineages. For example, a multipotent blood stem cell can form the many different types of blood cells (red, white, platelets, etc.), but it cannot form neurons; cardiovascular progenitor cell (MICP) differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types; pancreas-derived multipotent progenitor (PMP) colonies produce cell types of pancreatic lineage (cells that produces insulin, glucagon, amylase or somatostatin) and neural lineage (cells that are morphologically neuron-like, astrocytes-like or oligodendrocyte-like).

[0345] The term a "reprogramming gene", as used herein, refers to a gene whose expression, contributes to the reprogramming of a differentiated cell, e.g., a somatic cell to an undifferentiated cell (e.g., a cell of a pluripotent state or partially pluripotent state, multipotent state). A reprogramming gene can be, for example, genes encoding master transcription factors Sox2, Oct3 / 4, Klf4, Nanog, Lin-28, c-myc and the like. The term "reprogramming factor" refers to the protein encoded by the reprogramming gene.

[0346] The term "exogenous" refers to a substance present in a cell other than its native source. The terms "exogenous" when used herein refers to a nucleic acid (e.g., a nucleic acid encoding a reprogramming transcription factor, e.g., Sox2, Oct3 / 4, Klf4, Nanog, Lin-28, c-myc and the like) or a protein (e.g., a transcription factor polypeptide) that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found or in which it is found in lower amounts. A substance (e.g., a nucleic acid encoding a sox2 transcription factor, or a protein, e.g., a SOX2 polypeptide) will be considered exogenous if it is introduced into a cell or an ancestor of the cell that inherits the substance.

[0347] The term "isolated" as used herein signifies that the cells are placed into conditions other than their natural environment. The term "isolated" does not preclude the later use of these cells thereafter in combinations or mixtures with other cells. 73 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0348] As used herein, the term “expanding” refers to increasing the number of like cells through cell division (mitosis). The term “proliferating” and “expanding” are used interchangeably.

[0349] As used herein, a “cell-surface marker” refers to any molecule that is expressed on the surface of a cell. Cell-surface expression usually requires that a molecule possesses a transmembrane domain. Some molecules that are normally not found on the cell-surface can be engineered by recombinant techniques to be expressed on the surface of a cell. Many naturally occurring cell-surface markers are termed “CD” or “cluster of differentiation” molecules. Cell-surface markers often provide antigenic determinants to which antibodies can bind to. A cell-surface marker of particular relevance to the methods described herein is CD34. The useful hematopoietic progenitor cells (e.g., hemogenic endothelium) according to the present disclosure preferably express CD34 or in other words, they are CD34 positive.

[0350] A cell can be designated “positive” or “negative” for any cell-surface marker, and both such designations are useful for the practice of the methods described herein. A cell is considered “positive” for a cell-surface marker if it expresses the marker on its cell-surface in amounts sufficient to be detected using methods known to those of skill in the art, such as contacting a cell with an antibody that binds specifically to that marker, and subsequently performing flow cytometric analysis of such a contacted cell to determine whether the antibody is bound the cell. It is to be understood that while a cell may express messenger RNA for a cell-surface marker, in order to be considered positive for the methods described herein, the cell must express it on its surface. Similarly, a cell is considered “negative” or “negative / low” (abbreviated as “- / lo” or “lo / -”) for a cell-surface marker if the cell does not express the marker on its cell surface in amounts sufficient to be detected using methods known to those of skill in the art, such as contacting a cell with an antibody that binds specifically to that marker and subsequently performing flow cytometric analysis of such a contacted cell to determine whether the antibody is bound the cell. In some embodiments, where agents specific for cell-surface lineage markers used, the agents can all comprise the same label or tag, such as fluorescent tag, and thus all cells positive for that label or tag can be excluded or removed, to leave uncontacted hematopoietic stem or progenitor cells for use in the methods described herein.

[0351] The term "inhibitory RNA" is meant to include a nucleic acid molecule that contains a sequence that is complementary to a target nucleic acid (e.g., a target microRNA) that mediates a decrease in the level or activity of the target nucleic acid. Non-limiting examples of inhibitory RNAs include interfering RNA, shRNA, siRNA, ribozymes, antagomirs, and antisense oligonucleotides. Methods of making inhibitory RNAs are described herein. Additional methods of making inhibitory RNAs are known in the art. In one embodiment, the EZH1, TET2, or TP53 microRNA described herein is an inhibitory RNA that causes a decrease in the activity of EZH1, TET2, or TP53 mRNA, respectfully. 74 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0352] As used herein, "an interfering RNA" refers to any double stranded or single stranded RNA sequence, capable - either directly or indirectly (i.e., upon conversion) of inhibiting or down- regulating gene expression by mediating RNA interference. Interfering RNA includes, but is not limited to, small interfering RNA ("siRNA") and small hairpin RNA ("shRNA"). "RNA interference" refers to the selective degradation of a sequence-compatible messenger RNA transcript.

[0353] As used herein "an shRNA" (small hairpin RNA) refers to an RNA molecule comprising an antisense region, a loop portion and a sense region, wherein the sense region has complementary nucleotides that base pair with the antisense region to form a duplex stem. Following post- transcriptional processing, the small hairpin RNA is converted into a small interfering RNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family. As used herein, the phrase "post-transcriptional processing" refers to mRNA processing that occurs after transcription and is mediated, for example, by the enzymes Dicer and / or Drosha.

[0354] A "small interfering RNA" or "siRNA" as used herein refers to any small RNA molecule capable of inhibiting or down regulating gene expression by mediating RNA interference in a sequence specific manner. The small RNA can be, for example, about 18 to 21 nucleotides long. Each siRNA duplex is formed by a guide strand and a passenger strand. The endonuclease Argonaute 2 (Ago 2) catalyzes the unwinding of the siRNA duplex. Once unwound, the guide strand is incorporated into the RNA Interference Specificity Complex (RISC), while the passenger strand is released. RISC uses the guide strand to find the mRNA that has a complementary sequence leading to the endonucleolytic cleavage of the target mRNA.

[0355] Retroviruses are RNA viruses that utilize reverse transcriptase during their replication cycle. The term "retrovirus" refers to any known retrovirus (e.g., type c retroviruses, such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus.

[0356] The retroviral genomic RNA is converted into double-stranded DNA by reverse transcriptase. This double-stranded DNA form of the virus is capable of being integrated into the chromosome of the infected cell; once integrated, it is referred to as a “provirus.” The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules, which encode the structural proteins and enzymes needed to produce new viral particles.

[0357] At each end of the provirus are structures called “long terminal repeats” or “LTRs.” The term “long terminal repeat (LTR)” refers to domains of base pairs located at the ends of retroviral DNAs which, in their natural sequence context, are direct repeats and contain U3, R, and U5 regions. LTRs generally provide functions fundamental to the expression of retroviral genes (e.g., promotion, initiation and polyadenylation of gene transcripts) and to viral replication. The LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals and sequences needed for replication and integration of the viral genome. The viral LTR is divided into 75 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT three regions called U3, R and U5. The U3 region contains the enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. The LTR composed of U3, R, and U5 regions, appears at both the both the 5' and 3' ends of the viral genome. In one embodiment of the invention, the promoter within the LTR, including the 5' LTR, is replaced with a heterologous promoter. Examples of heterologous promoters that can be used include, for example, a spleen focus-forming virus (SFFV) promoter, a tetracycline-inducible (TET) promoter, a β-globin locus control region and a β-globin promoter (LCR), and a cytomegalovirus (CMV) promoter.

[0358] The term “lentivirus” refers to a group (or genus) of retroviruses that give rise to slowly developing disease. Viruses included within this group include HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2), the etiologic agent of the human acquired immunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, the caprine arthritis-encephalitis virus, which causes immune deficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia, and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immune deficiency in cats; bovine immune deficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possibly central nervous system infection in cattle; and simian immunodeficiency virus (SIV), which cause immune deficiency and encephalopathy in sub-human primates. Diseases caused by these viruses are characterized by a long incubation period and protracted course. Usually, the viruses latently infect monocytes and macrophages, from which they spread to other cells. HIV, FIV, and SIV also readily infect T lymphocytes, i.e., T-cells.

[0359] The term “R region” refers to the region within retroviral LTRs beginning at the start of the capping group (i.e., the start of transcription) and ending immediately prior to the start of the poly A tract. The R region is also defined as being flanked by the U3 and U5 regions. The R region plays an important role during reverse transcription in permitting the transfer of nascent DNA from one end of the genome to the other.

[0360] The term “promoter / enhancer” refers to a segment of DNA which contains sequences capable of providing both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. The enhancer / promoter may be “endogenous,” “exogenous,” or “heterologous.” An “endogenous” enhancer / promoter is one which is naturally linked with a given gene in the genome. An “exogenous” or “heterologous” enhancer / promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. 76 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0361] As used herein, the term “synthetic nucleic acid” or “synthetic nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The synthetic nucleic acid can be either single-stranded or double-stranded. A single-stranded synthetic nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded synthetic nucleic acid not derived from any double-stranded DNA. In one aspect, the synthetic nucleic acid can be DNA. In another aspect, the synthetic nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA, iRNA, miRNA, siRNA, etc.

[0362] The synthetic inhibitory nucleic acid can include, for example, from a group including: nucleic acid encoding a protein of interest, oligonucleotides, nucleic acid analogues, for example peptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), and locked nucleic acid (LNA). Such nucleic acid sequences include, for example, but are not limited to, nucleic acid sequence encoding proteins, for example that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi, siRNA, microRNAi (miRNA), and antisense oligonucleotides.

[0363] As used herein, the term “engraftment” in reference to a recipient host is when the new blood-forming cells start to grow and which are derived from the implanted cells and make healthy blood stem cells that show up in recipient’s blood after a minimum period of 10 days after implantation. Engraftment can occur as early as 10 days after transplant but is more common around 14-20 days.

[0364] As used herein, the term “reconstitution” with respect to the immune system or the blood system in a recipient host refers to the rebuilding the innate reservoir or working system, or part thereof within the body of recipient host to a natural or a functionally state. For example, such as bone marrow after chemotherapy had obliterated the bone marrow stem cells.

[0365] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder. 77 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0366] 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% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.

[0367] As used herein, a "subject" means a human or animal. Usually, the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. 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.

[0368] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of cellular therapy. A subject can be male or female.

[0369] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., hematologic disease, cancer, etc.) or one or more complications related to such a condition, and optionally, have already undergone treatment for a hematologic disease or the one or more complications related to a hematologic disease. Alternatively, a subject can also be one who has not been previously diagnosed as having a hematologic disease or one or more complications related to a hematologic disease. For example, a subject can be one who exhibits one or more risk factors for a hematologic disease, or one or more complications related to a hematologic disease or a subject who does not exhibit risk factors.

[0370] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0371] A variant amino acid or DNA sequence can be at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by 78 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g., BLASTp or BLASTn with default settings).

[0372] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are very well established and include, for example, those disclosed by Walder et al. (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 herein incorporated by reference in their entireties. Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

[0373] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and / or to the translation of mRNA into a polypeptide.

[0374] In some embodiments, the expression of a biomarker(s), target(s), or gene / polypeptide described herein is / are tissue-specific. In some embodiments, the expression of a biomarker(s), target(s), or gene / polypeptide described herein is / are global. In some embodiments, the expression of a biomarker(s), target(s), or gene / polypeptide described herein is systemic.

[0375] "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g., 5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0376] In some embodiments, a polypeptide, nucleic acid, or cell as described herein can be engineered. As used herein, “engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when at least one aspect of the 79 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell are typically still referred to as “engineered" even though the actual manipulation was performed on a prior entity.

[0377] In some embodiments, the differentiated and / or engineered T cell described herein is exogenous. In some embodiments, the differentiated and / or engineered T cell described herein is ectopic. In some embodiments, the differentiated and / or engineered T cell described herein is not endogenous.

[0378] The term "exogenous" refers to a substance present in a cell other than its native source. The term "exogenous" when used herein can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, “exogenous” can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to the biological system or cell. As used herein, “ectopic” refers to a substance that is found in an unusual location and / or amount. An ectopic substance can be one that is normally found in a given cell, but at a much lower amount and / or at a different time. Ectopic also includes substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given cell in its natural environment.

[0379] Synthetic nucleic acids encoding a polypeptide as described herein can be comprised by a vector. The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.

[0380] The vector can be recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments, the vector comprises sequences originating from at least two different species. In some embodiments, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like). 80 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0381] In some embodiments, the vector or nucleic acid described herein is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence, but will be transcribed and / or translated at an improved efficiency in a desired expression system. In some embodiments, the expression system is an organism other than the source of the native / wild-type sequence (or a cell obtained from such organism). In some embodiments, the vector and / or nucleic acid sequence described herein is codon- optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments, the vector and / or nucleic acid sequence described herein is codon- optimized for expression in a human cell. In some embodiments, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell. In some embodiments, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell.

[0382] As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.

[0383] As used herein, the term “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non- essential viral genes. The vector and / or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. Non-limiting examples of a viral vector of this invention include an AAV vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, a herpesvirus vector, an alphavirus vector, a poxvirus vector a baculovirus vector, and a chimeric virus vector.

[0384] It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. For example, the use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.

[0385] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g., a hematological disease or cancer. The term “treating" includes reducing or alleviating at least one 81 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT adverse effect or symptom of a condition, disease or disorder associated with a hematological disease or cancer. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0386] As used herein, the term "administering," refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and / or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and / or the subject being treated.

[0387] As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and / or decanting; and / or manipulation of a delivery device or machine.

[0388] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0389] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[0390] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0391] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment. 82 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT

[0392] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0393] As used herein, the term “corresponding to” refers to an amino acid or nucleotide at the enumerated position in a first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to an enumerated amino acid or nucleotide in a second polypeptide or nucleic acid. Equivalent enumerated amino acids or nucleotides can be determined by alignment of candidate sequences using degree of homology programs known in the art, e.g., BLAST.

[0394] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0395] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0396] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 83 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0397] In some embodiments, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.

[0398] Other terms are defined herein within the description of the various aspects of the invention.

[0399] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0400] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of 84 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT the disclosure. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0401] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0402] The invention disclosed herein can further be described in the following numbered paragraphs: 1. A synthetic nucleic acid encoding at least one promoter, a transgene, and at least one inhibitory nucleic acid sequence. 2. The synthetic nucleic acid of paragraph 1, wherein at least one promoter is selected from the group consisting of an EF1a promoter, a U6 promoter, an H1 promoter, a PGK1 promoter, or Ubiquitin-C (UBC) promoter. 3. The synthetic nucleic acid of any of the preceding paragraphs, wherein the transgene encodes a function BCL-XL protein. 4. The synthetic nucleic acid of any of the preceding paragraphs, wherein the inhibitory nucleic acid sequence is a shRNA sequence, miRNA sequence, CRISPRi sgRNA sequence, antisense sequence, or sequence encoding an inhibitory polypeptide or protein. 5. The synthetic nucleic acid of any of the preceding paragraphs, wherein the inhibitory nucleic acid sequence inhibits a target gene selected from the group consisting of EZH1, TP53, or TET2. 6. The synthetic nucleic acid of any of the preceding paragraphs, wherein each of the transgene and at least one inhibitory nucleic acid sequence are operatively linked to the at least one promoter. 7. The synthetic nucleic acid of any of the preceding paragraphs, wherein the transgene is operatively linked to a EF1a promoter. 8. The synthetic nucleic acid of any of the preceding paragraphs, wherein the at least one inhibitory nucleic acid sequence is operatively linked to a U6 promoter. 9. The synthetic nucleic acid of any of the preceding paragraphs, further comprising a sequence encoding terminal repeats flanking the at least one promoter, the transgene, and the least one shRNA. 85 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 10. The synthetic nucleic acid of any of the preceding paragraphs, comprising from 5’ to 3’, a. a 5’ terminal repeat, b. at least one inhibitory nucleic acid sequence operatively linked to a promoter, c. the transgene operatively linked to a promoter, and d. a 3’ terminal repeat. 11. The synthetic nucleic acid of any of the preceding paragraphs, comprising from 5’ to 3’, a. a 5’ terminal repeat, b. at least one inhibitory nucleic acid sequence operatively linked to a promoter, c. the BCL-XL operatively linked to the EF1a promoter, and d. a 3’ terminal repeat. 12. The synthetic nucleic acid of any of the preceding paragraphs, comprising from 5’ to 3’, a. a 5’ terminal repeat, b. at least one of EZH1 shRNA operatively linked to a U6 promoter, TP53 shRNA operatively linked to a U6 promoter, and TET2 shRNA operatively linked to a promoter, c. the BCL-XL operatively linked to the EF1a promoter, and d. a 3’ terminal repeat. 13. The synthetic nucleic acid of any of the preceding paragraphs, further comprising a sequence encoding a marker gene. 14. The synthetic nucleic acid of any of the preceding paragraphs, wherein the terminal repeat is a long terminal repeat (LTR). 15. The synthetic nucleic acid of any of the preceding paragraphs, wherein the synthetic nucleic acid is a plasmid. 16. A synthetic nucleic acid comprising from 5’ to 3’ a sequence encoding, a. a 5’ terminal repeat, b. at least one of EZH1 shRNA operatively linked to a U6 promoter, TP53 shRNA operatively linked to a U6 promoter, and TET2 shRNA operatively linked to a promoter, c. the BCL-XL operatively linked to the EF1a promoter, and d. a 3’ terminal repeat. 17. A virus expressing any of the synthetic nucleic acids of any of the preceding paragraphs. 86 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 18. The virus of any of any of the preceding paragraphs, wherein the virus is selected from the group consisting of Retroviruses (alpha-retrovirus, gamma-retrovirus, or lentivirus), adeno- associated virus, paramyxovirus, alphanodavirus . 19. A lentivirus expressing any of the synthetic nucleic acids of any of the preceding paragraphs. 20. A cell comprising any of the synthetic nucleic acids of any of the preceding paragraphs, any of the viruses of any of the preceding paragraphs, or lentivirus of any of the preceding paragraphs. 21. The cell of any of any of the preceding paragraphs, wherein the cell is a human pluripotent stem cell. 22. The cell of any of any of the preceding paragraphs, wherein the cell is an iPSC. 23. A stable cell line comprising any of the synthetic nucleic acids of any of the preceding paragraphs, any of the viruses of any of the preceding paragraphs, or lentivirus of any of the preceding paragraphs. 24. The stable cell line of any of the preceding paragraphs, wherein the cell is a human pluripotent stem cell. 25. The stable cell of any of the preceding paragraphs, wherein the cell is an iPSC. 26. A method comprising differentiating a population of any of the cells of any of the preceding paragraphs or stable cell line of any of the preceding paragraphs for a sufficient time to promote differentiation into a population of T cells. 27. A method comprising: a) differentiating a population of any of the cells of any of the preceding paragraphs or stable cell line of any of the preceding paragraphs for a sufficient time to promote differentiation into a population of CD34+hemogenic endothelium; and b) differentiating the resultant population of CD34+hemogenic endothelium in a CD3+- T-cell-differentiation media in the presence of a Notch ligand for a sufficient time to promote differentiation into a population of CD3+T cells. 28. The method of any of the preceding paragraphs, wherein the Notch ligand is attached to a solid substrate. 29. The method of any of the preceding paragraphs, wherein the Notch ligand is attached to a cell culture dish. 30. The method of any of the preceding paragraphs, wherein the Notch ligand is not derived from a stromal cell. 87 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 31. The method of any of the preceding paragraphs, wherein differentiating the hemogenic endothelium in the presence of a Notch ligand does not comprise co-culturing with a stromal cell expressing a Notch ligand. 32. The method of any of the preceding paragraphs, wherein differentiating the hemogenic endothelium in the presence of a Notch ligand does not comprise co-culturing with OP9-DL1 cells or OP9-DL4 cells. 33. The method of any of the preceding paragraphs, wherein the Notch ligand is selected from the group consisting of Delta-like-1 (DLL1), Delta-like-4 (DLL4), immobilized Delta1ext-IgG, and immobilized Delta4ext-IgG. 34. The method of any of the preceding paragraphs, wherein immobilized Delta1ext-IgGconsists of an extracellular domain of human Delta-like-1 fused to the Fc domain of human IgG1. 35. The method of any of the preceding paragraphs, wherein the sufficient time to promote differentiation into a population of CD3+T cells is at least 4 weeks. 36. The method of any of the preceding paragraphs, wherein the CD3+-T-cell-differentiation media is serum-free. 37. The method of any of the preceding paragraphs, wherein the cells are contacted with a lymphoid induction media comprising SCF, FLT3, TPO, IL-3, and IL-7. 38. The method of any of the preceding paragraphs, wherein the cells are contacted with a T-cell specification media comprising SCF, FLT3, TPO, and IL-7. 39. The method of any of the preceding paragraphs, wherein the cells are contacted with a T-cell maturation media comprising FLT3 and IL-7. 40. The method of any of the preceding paragraphs, wherein the cells are contacted with a SP T- cell induction media comprising FLT3, IL-7, CD3 / CD28 T cell activator, and IL-15. 41. The method of any of the preceding paragraphs, wherein the cells are contacted with the lymphoid induction media for at least 7 days, i.e., from day 0 to day 7 of method. 42. The method of any of the preceding paragraphs, wherein the cells are contacted with the T- cell specification media at least 7 days, i.e., from day 7 to day 14 of method. 43. The method of any of the preceding paragraphs, wherein the cells are contacted with the T- cell maturation media at least 20 days, i.e., from day 14 to day 35 of method. 44. The method of any of the preceding paragraphs, wherein the cells are contacted with the SP T-cell induction media at least 7 days, i.e., from day 35 to day 42 of method. 45. The method of any of the preceding paragraphs, wherein the population of CD34+hemogenic endothelium is CD45 negative / low. 46. The method of any of the preceding paragraphs, wherein the population of CD34+hemogenic endothelium is CD73 negative. 88 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 47. The method of any of the preceding paragraphs, wherein the population of CD34+hemogenic endothelium has improved potential for T cell differentiation. 48. The method of any of the preceding paragraphs, wherein the resultant population of T cells are CD3+TCRa+b+ cells. 49. The method of any of the preceding paragraphs, further comprising the step of genetically modifying the resultant population of CD34+hemogenic endothelium or the resultant population of CD3+TCRa+b+ cells T cells. 50. The method of any of the preceding paragraphs, wherein the genetic modification is editing an endogenous HLA, removing an endogenous TCR, and / or expressing a chimeric antigen receptor (CAR). 51. The method of any of the preceding paragraphs, wherein the sufficient time to promote differentiation from a cell into a population of CD34+hemogenic endothelium is about 8 days. 52. The method of any of the preceding paragraphs, wherein the sufficient time to promote differentiation from the population of CD34+ cells into a population of resultant T-cells is about 14 days. 53. The method of any of the preceding paragraphs, wherein the resultant T cells comprise a high percentage of CD3+TCRa+b+ cells. 54. An immune cell produced by the method of any of the preceding paragraphs. 55. A composition comprising an immune cell of any of the preceding paragraphs or population thereof. 56. The composition of any of the preceding paragraphs, further comprising a pharmaceutically acceptable carrier. 57. A pharmaceutical composition comprising an immune cell of any of the preceding paragraphs or population thereof, and a pharmaceutically acceptable carrier. 58. The pharmaceutical composition of any of the preceding paragraphs for use in cellular therapy in a subject. 59. A method of cellular therapy, the method comprising administering an immune cell of any of the preceding paragraphs or population thereof, or a composition of any of the preceding paragraphs, or a pharmaceutical composition of any of the preceding paragraphs to a recipient subject in need thereof. 60. The method of cellular therapy of any of the preceding paragraphs, wherein the recipient subject has undergone chemotherapy and / or irradiation. 61. The method of cellular therapy of any of the preceding paragraphs, wherein the recipient subject has deficiencies in immune function and / or lymphocyte reconstitution. 89 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 62. The method of cellular therapy of any of the preceding paragraphs, wherein the recipient subject has cancer. 63. The method of cellular therapy of any of the preceding paragraphs, wherein the recipient subject has an auto-immune disease. 64. The method of cellular therapy of any of the preceding paragraphs, wherein the immune cell or population thereof is autologous to the recipient subject. 65. The method of cellular therapy of any of the preceding paragraphs, wherein the immune cell or population thereof is HLA type matched with the recipient subject. 66. The method of cellular therapy of any of the preceding paragraphs, wherein the immune cell or population thereof is not HLA type matched with the recipient subject. 67. Use of any of the cells of any of the preceding paragraphs or stable cell line of any of the preceding paragraphs for differentiation into a population of CD34+hemogenic endothelium. 68. Use of any of the cells of any of the preceding paragraphs or stable cell line of any of the preceding paragraphs for differentiation into a population of resultant T cells. EXAMPLES EXAMPLE 1

[0403] Described herein is a approach to address the challenges of conventional autologous CAR T cells. The inventors developed a transgenic approach to drastically increase the yield, expandability, and robustness of functional, mature hiPSC-derived CD3+TCRa+b+CD4-CD8+ cytotoxic T cells, thereby enabling cost-efficient production of therapeutically relevant cell doses. Through confocal live imaging we were able to observe efficient killing of CD19+ leukemic cells by allogeneic CD19- directed CAR T cells derived with this system. Data presented herein presents a multifaceted approach promises to significantly lower production costs and to enhance the therapeutic potential of “off the shelf” CAR T cells while also addressing critical safety concerns. Overall, this strategy represents a significant step forward in overcoming limitations and moving towards more effective and safer cell-based therapies for a broader spectrum of cancers, autoimmune syndromes, and other degenerative diseases.

[0404] Following the same IT cell differentiation protocol as described in US Patent Application No. 17 / 794,747, the new method provided herein avoids the need for lentiviral transduction at the CD34+ stage, produces CD3+ T cells more efficiently, and produces a higher percentage of CD3+TCRa+b+ cells, with a much higher viability, as compared to the method described in 17 / 794,747 (See, e.g., Fig. 3). With these improvements, a therapeutic dose of about 50M cytotoxic T cells can be produced in a single culture dish (Larson et al.2023). The modifications described here are expected to increase 90 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT persistence of the cells in vivo and functionality against tumor cells (Figs 5-9). The contents of US Patent Application No.17 / 794,747 are incorporated herein by reference in its entirety. EXAMPLE 2

[0405] Chimeric antigen receptor (CAR) T cell therapy has transformed cancer treatment, yet sustained remission, which correlates with long-term persistence of CAR T cells, is only seen in less than half of treated patients. In addition, the process of manufacturing CAR T cells is complex, costly, and time-consuming and wider use of this approach is limited by insufficient availability of suitable cGMP facilities. While human induced pluripotent stem (hiPSC) cells hold promise as a potentially unlimited source for mass-produced allogeneic T cells, existing in vitro differentiation methods suffer from low efficiency and significant variability depending on the specific hiPSC source used. Furthermore, such “off the shelf CAR T” approaches require additional genetic engineering steps to block graft-versus-host disease and host-versus-graft immune reactions. This involves measures such as inhibiting TCR expression and concealing the CAR T cells from the patient’s immune system. While conventional autologous CAR T cells are subject to normal immunosurveillance and rarely give rise to product derived lymphoma, the clinical consequences of malignant transformation or infection may be considerably higher with genetically engineered, allogeneic CAR T cells. Described herein is an approach to address these challenges.

[0406] The inventors developed a transgenic approach to drastically increase the yield, expandability, and robustness of functional, mature hiPSC-derived CD3+TCRa+b+CD4-CD8+ cytotoxic T cells, thereby enabling cost-efficient production of therapeutically relevant cell doses. Through confocal live imaging, inventors observed efficient killing of CD19+ leukemic cells by allogeneic CD19-directed CAR T cells derived with this system. The invention disclosed herein significantly lowers production costs and enhances the therapeutic potential of “off the shelf” CAR T cells while also addressing critical safety concerns. Overall, this strategy represents a significant step forward in overcoming limitations and moving towards more effective and safer cell-based therapies for a broader spectrum of cancers, autoimmune syndromes, and other degenerative diseases.

[0407] Data provided herein identified a combination of gene overexpression (BCL-XL) and gene silencing (shEZH1, shTET2), which has a strong anti-apoptotic and growth promoting effect during iT cell differentiation, thereby drastically improving yield and purity of the target cell population (Fig.10-14B). This protocol further improves upon the EZ-T cell platform developed by Jing et al., 2022 (see., e.g., Fig.10-14B) and allows for large scale manufacturing of CAR T cells at a clinically relevant scale. It is specifically contemplated herein that the a combination of gene overexpression (BCL-XL) and gene silencing (shEZH1, shTET2) would have markedly reduce the 91 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT presence of tumor cells in vivo, for example, in a murine lymphoma model. Similarly, it is specifically contemplated herein that the a combination of gene overexpression (BCL-XL) and gene silencing (shEZH1, shTET2) would markedly reduce the symptoms and / or presence of an auto- immune disease or disorder in a mouse models of autoimmune disease or disorders. 92 4899-6336-4634.2 701039-000143WOPT

Claims

Attorney Docket No: 701039-000143WOPT CLAIMS What is claimed herein is:

1. A synthetic nucleic acid encoding at least one promoter, a transgene, and at least one inhibitory nucleic acid sequence.

2. The synthetic nucleic acid of claim 1, wherein at least one promoter is selected from the group consisting of an EF1a promoter, a U6 promoter, an H1 promoter, a PGK1 promoter, or Ubiquitin-C (UBC) promoter.

3. The synthetic nucleic acid of claim 1, wherein the transgene encodes a function BCL-XL protein.

4. The synthetic nucleic acid of claim 1, wherein the inhibitory nucleic acid sequence is a shRNA sequence, miRNA sequence, CRISPRi sgRNA sequence, antisense sequence, or sequence encoding an inhibitory polypeptide or protein.

5. The synthetic nucleic acid of claim 1, wherein the inhibitory nucleic acid sequence inhibits a target gene selected from the group consisting of EZH1, TP53, or TET2.

6. The synthetic nucleic acid of any of claims 1-4, wherein each of the transgene and at least one inhibitory nucleic acid sequence are operatively linked to the at least one promoter.

7. The synthetic nucleic acid of claim 6, wherein the transgene is operatively linked to a EF1a promoter.

8. The synthetic nucleic acid of claim 6, wherein the at least one inhibitory nucleic acid sequence is operatively linked to a U6 promoter.

9. The synthetic nucleic acid of any of claims 1-8, further comprising a sequence encoding terminal repeats flanking the at least one promoter, the transgene, and the least one shRNA.

10. The synthetic nucleic acid of any of claims 1-9, comprising from 5’ to 3’, a. at least one inhibitory nucleic acid sequence operatively linked to a promoter, and b. the transgene operatively linked to a promoter.

11. The synthetic nucleic acid of any of claims 1-9, comprising from 5’ to 3’, a. at least one inhibitory nucleic acid sequence operatively linked to a promoter, and b. the BCL-XL operatively linked to the EF1a promoter.

12. The synthetic nucleic acid of any of claims 1-9, comprising from 5’ to 3’, a. at least one of EZH1 shRNA operatively linked to a U6 promoter, TP53 shRNA operatively linked to a U6 promoter, and TET2 shRNA operatively linked to a promoter, and 93 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT b. the BCL-XL operatively linked to the EF1a promoter.

13. The synthetic nucleic acid of any of claims 1-12, further comprising a sequence encoding a marker gene.

14. The synthetic nucleic acid of claim 12, wherein the terminal repeat is a long terminal repeat (LTR).

15. The synthetic nucleic acid of any of claims 1-14, wherein the synthetic nucleic acid is a plasmid.

16. A synthetic nucleic acid comprising from 5’ to 3’ a sequence encoding, a. a 5’ terminal repeat, b. at least one of EZH1 shRNA operatively linked to a U6 promoter, TP53 shRNA operatively linked to a U6 promoter, and TET2 shRNA operatively linked to a promoter, c. the BCL-XL operatively linked to the EF1a promoter, and d. a 3’ terminal repeat.

17. A virus expressing any of the synthetic nucleic acids of claims 1-16.

18. The virus of any of claims 17, wherein the virus is selected from the group consisting of Retroviruses (alpha-retrovirus, gamma-retrovirus, or lentivirus), adeno-associated virus, paramyxovirus, alphanodavirus.

19. A lentivirus expressing any of the synthetic nucleic acids of claims 1-16.

20. A cell comprising any of the synthetic nucleic acids of claims 1-16, any of the viruses of claims 17-18, or lentivirus of claim 19.

21. The cell of any of claims 20, wherein the cell is a human pluripotent stem cell.

22. The cell of any of claims 20, wherein the cell is an iPSC.

23. A stable cell line comprising any of the synthetic nucleic acids of claims 1-16, any of the viruses of claims 17-18, or lentivirus of claim 19.

24. The stable cell line of any of claims 23, wherein the cell is a human pluripotent stem cell.

25. The stable cell of any of claims 23, wherein the cell is an iPSC.

26. A method comprising differentiating a population of any of the cells of claims 20-22 or stable cell line of claims 23-25 for a sufficient time to promote differentiation into a population of T cells.

27. A method comprising: 94 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT a) differentiating a population of any of the cells of claims 20-22 or stable cell line of claims 23-25 for a sufficient time to promote differentiation into a population of CD34+hemogenic endothelium; and b) differentiating the resultant population of CD34+hemogenic endothelium in a CD3+- T-cell-differentiation media in the presence of a Notch ligand for a sufficient time to promote differentiation into a population of CD3+T cells.

28. The method of claim 27, wherein the Notch ligand is attached to a solid substrate.

29. The method of any one of claims 27 or 28, wherein the Notch ligand is attached to a cell culture dish.

30. The method of any one of claims 27-29, wherein the Notch ligand is not derived from a stromal cell.

31. The method of any one of claims 27-30, wherein differentiating the hemogenic endothelium in the presence of a Notch ligand does not comprise co-culturing with a stromal cell expressing a Notch ligand.

32. The method of any one of claims 27-31, wherein differentiating the hemogenic endothelium in the presence of a Notch ligand does not comprise co-culturing with OP9-DL1 cells or OP9- DL4 cells.

33. The method of any one of claims 27-32, wherein the Notch ligand is selected from the group consisting of Delta-like-1 (DLL1), Delta-like-4 (DLL4), immobilized Delta1ext-IgG, and immobilized Delta4ext-IgG.

34. The method of claim 33, wherein immobilized Delta1ext-IgGconsists of an extracellular domain of human Delta-like-1 fused to the Fc domain of human IgG1.

35. The method of any one of claims 27-34, wherein the sufficient time to promote differentiation into a population of CD3+T cells is at least 4 weeks.

36. The method of any one of claims 27-35, wherein the CD3+-T-cell-differentiation media is serum-free.

37. The method of any one of claims 27-36, wherein the cells are contacted with a lymphoid induction media comprising SCF, FLT3, TPO, IL-3, and IL-7.

38. The method of any one of claims 27-37, wherein the cells are contacted with a T-cell specification media comprising SCF, FLT3, TPO, and IL-7.

39. The method of any one of claims 27-38, wherein the cells are contacted with a T-cell maturation media comprising FLT3 and IL-7.

40. The method of any one of claims 27-38, wherein the cells are contacted with a SP T-cell induction media comprising FLT3, IL-7, CD3 / CD28 T cell activator, and IL-15.

41. The method of any one of claims 27-40, wherein the cells are contacted with the lymphoid induction media for at least 7 days, i.e., from day 0 to day 7 of method. 95 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 42. The method of any one of claims 27-40, wherein the cells are contacted with the T-cell specification media at least 7 days, i.e., from day 7 to day 14 of method.

43. The method of any one of claims 27-40, wherein the cells are contacted with the T-cell maturation media at least 20 days, i.e., from day 14 to day 35 of method.

44. The method of any one of claims 27-40, wherein the cells are contacted with the SP T-cell induction media at least 7 days, i.e., from day 35 to day 42 of method.

45. The method of any one of claims 27-44, wherein the population of CD34+hemogenic endothelium is CD45 negative / low.

46. The method of any one of claims 27-44, wherein the population of CD34+hemogenic endothelium is CD73 negative.

47. The method of any one of claims 27-46, wherein the population of CD34+hemogenic endothelium has improved potential for T cell differentiation.

48. The method of any one of claims 27-46, wherein the resultant population of T cells are CD3+TCRa+b+ cells.

49. The method of claim 48, further comprising the step of genetically modifying the resultant population of CD34+hemogenic endothelium or the resultant population of CD3+TCRa+b+ cells T cells.

50. The method of claim 49, wherein the genetic modification is editing an endogenous HLA, removing an endogenous TCR, and / or expressing a chimeric antigen receptor (CAR).

51. The method of any one of claims 27-50, wherein the sufficient time to promote differentiation from a cell into a population of CD34+hemogenic endothelium is about 8 days.

52. The method of any one of claims 27-50, wherein the sufficient time to promote differentiation from the population of CD34+ cells into a population of resultant T-cells is about 14 days.

53. The method of claim 52, wherein the resultant T cells comprise a high percentage of CD3+TCRa+b+ cells.

54. An immune cell produced by the method of any one of claims 27-53.

55. A composition comprising an immune cell of any one of claims 54 or population thereof.

56. The composition of claim 55, further comprising a pharmaceutically acceptable carrier.

57. A pharmaceutical composition comprising an immune cell of any one of claims 54 or population thereof, and a pharmaceutically acceptable carrier.

58. The pharmaceutical composition of claim 57 for use in cellular therapy in a subject.

59. A method of cellular therapy, the method comprising administering an immune cell of claim 54 or population thereof, or a composition of claims 55-56, or a pharmaceutical composition of claims 57-58 to a recipient subject in need thereof.

60. The method of cellular therapy of claim 59, wherein the recipient subject has undergone chemotherapy and / or irradiation. 96 4899-6336-4634.2 701039-000143WOPTAttorney Docket No: 701039-000143WOPT 61. The method of cellular therapy of claim 59, wherein the recipient subject has deficiencies in immune function and / or lymphocyte reconstitution.

62. The method of cellular therapy of claim 59, wherein the recipient subject has cancer.

63. The method of cellular therapy of claim 59, wherein the recipient subject has an auto-immune disease.

64. The method of cellular therapy of any one of claims 59-62, wherein the immune cell or population thereof is autologous to the recipient subject.

65. The method of cellular therapy of any one of claims 59-63, wherein the immune cell or population thereof is HLA type matched with the recipient subject.

66. The method of cellular therapy of any one of claims 59-63, wherein the immune cell or population thereof is not HLA type matched with the recipient subject.

67. Use of any of the cells of claims 20-22 or stable cell line of claims 23-25 for differentiation into a population of CD34+hemogenic endothelium.

68. Use of any of the cells of claims 20-22 or stable cell line of claims 23-25 for differentiation into a population of resultant T cells. 97 4899-6336-4634.2 701039-000143WOPT

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