Genomic engineering of pluripotent cells

Abstract

To provide technology related to the field of genetic editing and genomic engineering of stem cells, more particularly, provide the use of genetic modulation of clonal pluripotent stem cells with molecular strategies that target specific loci and ensure continuous retaining and / or functioning of edited genetic materials.SOLUTION: Provided are methods and compositions for obtaining genome-engineered iPSCs, and derivative cells with stable and functional genome editing at selected sites. Also provided are cell populations or clonal cell lines derived from genome-engineered iPSCs, which comprise targeted integration of one or more exogenous polynucleotides, and / or insertion / deletions in one or more selected endogenous genes.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 251,032, filed November 4, 2015; U.S. Provisional Patent Application No. 62 / 337,258, filed May 16, 2016; and U.S. Provisional Patent Application No. 62 / 366,503, filed July 25, 2016, the disclosures of which are incorporated by reference in their entireties herein.

[0002] The present disclosure generally relates to the field of stem cell gene editing and genome modification. More specifically, the present disclosure relates to the use of gene regulation of clonal pluripotent stem cells using molecular strategies that target specific loci and ensure the continued maintenance and / or function of edited genetic material. [Background technology]

[0003] As the field of human induced pluripotent stem cells (hiPSCs) research continues to advance, and clinical studies of cell therapy with genetically modified hiPSCs begin, there is a need to address and mitigate safety concerns regarding the administration of genetically modified cells. To address these safety issues, several strategies have been explored, including recombinant peptides, monoclonal antibodies, small molecules regulating enzyme activity, and gene-specific modifications to facilitate selective elimination of abnormal cells. In general, previous studies have used viral vectors such as lentiviruses and short promoters to stably introduce suicide genes, including herpes complex virus thymidine kinase or inducible caspase-9 (inducible caspase-9), into human cells. However, the use of viral vectors can result in random integration events that can disrupt or activate disease-related genes and cause adverse effects. Other issues with currently used methods include, but are not limited to, low insertion rates; random insertions; mutations; high insertion copy numbers; and laborious cell sorting to sort heterogeneous cell populations with varying insertion copy numbers due to random insertions. Furthermore, in iPSC genome modification, many artificial promoters and genomic regions are prone to epigenetic gene expression silencing in both pluripotent and differentiated states, which results in unresponsiveness of the promoter or inserted gene to events such as cell proliferation, passaging, reprogramming, differentiation, and / or dedifferentiation. Thus, it is of great importance to identify optimal genome modification strategies, integration sites, promoters, and other elements to maintain the response of the inserted functional modality without compromising safety, especially when developing genetically modified immune cells for therapeutic use. Summary of the Invention

[0004] It is an object of the present invention to provide methods and compositions for generating clonal iPSC lines derived from a single cell, or derived cells thereof, that contain one or several genetic modifications, including polynucleotide insertions, deletions, and substitutions, at selected sites that are retained and maintain functionality in derived cells after differentiation, de-differentiation, reprogramming, expansion, passaging, and / or transplantation.

[0005] The object of the present invention is to generate a single cell-derived clonal iPSC line containing one or several genetic modifications at a selected site through reprogramming of non-pluripotent cells containing the same genetic modifications, including targeted integration and / or targeted insertion (in) / deletion (del). In particular, the object of the present invention is to reprogram the non-pluripotent cells and then genomically modify the reprogrammed cells, so that iPSCs or less differentiated cells for subsequent genome editing are first obtained. The object of the present invention is also to reprogram the non-pluripotent cells and then genomically modify the non-pluripotent cells, so that genomically modified non-pluripotent cells for subsequent cell reprogramming are first obtained. A further object of the present invention is to reprogram the non-pluripotent cells simultaneously with their genomic modification, so that intermediate cells are not isolated from or because of either the reprogramming or the genomic modification, and so that the two processes occur simultaneously in a single cell pool.

[0006] The object of the present invention is to generate non-pluripotent cells derived from iPSCs (or less differentiated cells), including but not limited to CD34 cells, hemogenic endothelium cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell precursors, NK cell precursors, T cells, NKT cells, NK cells, and B cells, which contain one or several genetic modifications at a selected site, through differentiation of iPSCs or less differentiated cells containing the same genetic modification at a selected site. In particular, the object of the present invention is to differentiate less differentiated cells, including precursor cells and pluripotent cells, after genomic modification of said less differentiated cells, such that a genomically modified less differentiated cell for subsequent cell differentiation is first obtained. A further object of the present invention is to differentiate less differentiated cells simultaneously with genomic modification of the less differentiated cells such that intermediate cells are not isolated, either from or because of the differentiation or genomic modification, and such that the two processes occur simultaneously in a single pool of cells.

[0007] One aspect of the present invention provides a construct comprising: (1) one or more exogenous polynucleotides of interest operably linked to one or more exogenous promoters contained in the construct or operably linked to endogenous promoters contained in a selected site after integration; and (2) a pair of homology arms specific to the selected site and flanking the exogenous polynucleotides of interest for targeted integration of the exogenous polynucleotides at the selected site in iPSCs; the one or more exogenous polynucleotides remain integrated and functional in the subsequently expanded iPSCs, differentiated cells derived from the iPSCs, and / or dedifferentiated cells derived therefrom. In some embodiments, the one or more exogenous polynucleotides are linked to each other by a linker sequence. In some embodiments, the linker sequence encodes a self-cleaving peptide. In some embodiments, the linker sequence provides an internal ribosome entry sequence (IRES). In some other embodiments, the above construct further comprises at least one polynucleotide encoding a marker. In other embodiments, the construct comprises a polynucleotide encoding a marker driven by the endogenous promoter at the selected site. In some embodiments, the selected site is a safe harbor locus, a highly expressed locus, a time-expressed locus, or a locus for disruption. In some embodiments, the safe harbor locus can be AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or a locus that meets the criteria for intragenomic safe harbor as defined herein. In some embodiments, the locus for disruption includes TAP1, TAP2, or tapasin, or other genes of interest associated with a cellular function or property whose disruption is desired, such as NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region.

[0008] In some embodiments of the construct, at least one of the exogenous polynucleotides is operably linked to an exogenous promoter, including CMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoter. In one embodiment, the exogenous promoter included in the construct is CAG. In some other embodiments, the one or more polynucleotides encode one or more of a safety switch protein; a targeting modality; a receptor; a signaling molecule; a transcription factor; a pharmacologic active protein or peptide; a potential drug target; and a protein that promotes engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of an iPSC or derived cell incorporating the construct. In some particular embodiments, the one or more polynucleotides encode a safety switch protein, including a caspase, a thymidine kinase, a cytosine deaminase, a modified EGFR, a B-cell CD20, or any combination thereof. In some embodiments, the caspase can be caspase 9, caspase 3, or caspase 7. In some other embodiments, the polynucleotide encoding the safety-switch protein is operably linked to CAG in the construct.

[0009] One embodiment of the invention provides a method of generating genomically modified iPSCs comprising at least one targeted genome edit at one or more selected sites within the genome, comprising: (I), (II) or (III)

[0010] (I): Genetically modifying iPSCs by one or both of (i) and (ii) in any order: (i) introducing one or more constructs of claim 1 into the iPSCs for targeted integration at a selected site; (ii)(a) introducing one or more double strand breaks into the iPSCs at the selected site using one or more endonucleases capable of recognizing the selected site; and (b) culturing the iPSCs of step (I)(ii)(a) to allow endogenous DNA repair to occur and generate a targeted insertion / deletion at the selected site; thereby obtaining genomically modified iPSCs that contain at least one functional targeted genome edit and are capable of being differentiated into partially or fully differentiated cells.

[0011] (II): Genetically modifying the non-pluripotent cells undergoing reprogramming to obtain genomically modified iPSCs by: (i) contacting the non-pluripotent cells with one or more reprogramming factors and, optionally, a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and / or a ROCK inhibitor to trigger reprogramming of said non-pluripotent cells; and (ii) contacting the non-pluripotent cells undergoing reprogramming of step (II)(i) with (a) and (b): (a) a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and / or a ROCK inhibitor to trigger reprogramming of said non-pluripotent cells. (b) introducing, in any order, one or both of the constructs described in claim 1; and (b) one or more double-stranded breaks at the selected sites using at least one endonuclease capable of recognizing the selected sites, and culturing the cells of step (II)(ii)(b) to allow endogenous DNA repair to occur and generate targeted insertions / deletions at the selected sites; thereby obtaining genomically modified iPSCs that contain at least one functional targeted genome edit and are capable of being differentiated into partially or fully differentiated cells.

[0012] (III): Genetically modifying non-pluripotent cells for reprogramming to obtain genomically modified iPSCs, comprising the following (i) and (ii): (i) introducing into the non-pluripotent cells one or both of (a) and (b): (a) one or more constructs according to claim 1 for enabling targeted integration at the selected site; (b) one or more double-strand breaks at the selected site using at least one endonuclease capable of recognizing the selected site, in any order, and culturing the cells of step (III)(i)(b), thereby obtaining endogenous D allowing NA repair to occur, resulting in a targeted insertion / deletion at the selected site; and (ii) contacting the cells of step (III)(i) with one or more reprogramming factors, and optionally a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and / or a ROCK inhibitor, to obtain a genomically modified iPSC comprising a targeted edit at the selected site; thereby obtaining a genomically modified iPSC that contains at least one functional targeted genomic edit and is capable of being differentiated into a partially or fully differentiated cell.

[0013] In one embodiment of the above method, at least one targeted genome edit at the one or more selected sites comprises insertion of one or more exogenous polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of the genomically modified iPSCs or derived cells. In some embodiments, the exogenous polynucleotides for insertion are operably linked to (1) one or more exogenous promoters, including CMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters; or (2) one or more endogenous promoters contained at the selected sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria for a genomic safe harbor. In some embodiments, the genomically modified iPSCs comprise one or more different exogenous polynucleotides encoding a protein including a caspase, a thymidine kinase, a cytosine deaminase, a modified EGFR, or B cell CD20, and when the genomically modified iPSCs comprise more than one suicide gene, the suicide genes are integrated into different safe harbor loci including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1.

[0014] In some other embodiments, the genomically modified iPSCs generated using the methods provided herein comprise insertions / deletions in one or more endogenous genes associated with targeting modalities, receptors, signaling molecules, transcription factors, potential drug targets, immune response control and regulation, or proteins that inhibit the engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or derived cells. In some embodiments, the endogenous genes for disruption comprise at least one of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region.

[0015] In still other embodiments, the genomically modified iPSCs generated using the methods provided herein comprise an exogenous polynucleotide encoding a caspase at the AAVS1 locus and an exogenous polynucleotide encoding a thymidine kinase at the H11 locus.

[0016] In yet some other embodiments, approaches (I), (II) and / or (III) further comprise maintaining the pluripotency of the genomically modified iPSCs by contacting the genomically modified iPSCs with a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor and a ROCK inhibitor. In one embodiment, the resulting genomically modified iPSCs containing at least one targeted genome edit are functional, potent and capable of differentiation into non-pluripotent cells containing the same functional genome edit.

[0017] Another aspect of the present invention provides genomically modified iPSCs generated from any one of the above methods. In one embodiment, the generated genomically modified iPSCs comprise one or more exogenous polynucleotides introduced using the constructs provided above, the exogenous polynucleotides being integrated into one or more selected sites of the iPSCs. In one embodiment, the generated genomically modified iPSCs comprise one or more insertions / deletions in endogenous genes associated with targeting modalities; receptors; signaling molecules; transcription factors; potential drug targets; immune response control and regulation; and proteins that inhibit the engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or derived cells. In one embodiment, the endogenous genes comprise one or more of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region. In some other embodiments, the genomically modified iPSCs comprise a deletion or reduced expression of at least one of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region. In some other embodiments, the genomically modified iPSCs comprise a deletion or reduced expression of at least one of HLA-E, HLA-G, CD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2AThe method includes the introduction or upregulation of at least one of R, CAR, TCR, Fc receptor, engager, and surface triggering receptor for binding to a bispecific, multispecific, or universal engager. In some embodiments, the introduction or upregulation is via integrative or non-integrative methods for introducing a polynucleotide encoding an exogenous protein. In some embodiments, the genomically modified iPSCs are deficient in HLA class I and / or class II. In some embodiments, the genomically modified iPSCs include null or low expression of B2M, null or low expression of TAP1, and / or null or low expression of TAP2. In some embodiments, the genomically modified iPSCs include integrated or non-integrated exogenous polynucleotides encoding one or more of HLA-E, HLA-G, CD16, 41BBL, and PDL1 proteins; or the genomically modified iPSCs include the introduction of one or more of HLA-E, HLA-G, CD16, 41BBL, and PDL1 proteins. In some embodiments, the transduction is an increase in expression from a non-expressed or under-expressed gene contained in the cell, hi some embodiments, the non-integrated exogenous polynucleotide is introduced using a Sendai virus, an episome, or a plasmid.

[0018] In some other embodiments, the genomically modified iPSCs comprise at least one exogenous polynucleotide encoding a high affinity CD16 receptor; at least one exogenous polynucleotide encoding a non-cleavable CD16 receptor; at least one exogenous polynucleotide encoding a high affinity and non-cleavable CD16 receptor (hnCD16); at least one exogenous polynucleotide encoding a non-cleavable HLA-E; or at least one exogenous polynucleotide encoding a non-cleavable HLA-G. In some embodiments, the iPSCs comprise one or more exogenous polynucleotides encoding a protein, including a safety switch protein; a targeting modality; a receptor; a signaling molecule; a transcription factor; a pharmacologic active protein or peptide; a potential drug target; or a protein that promotes engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of the iPSC or a derived cell thereof.

[0019] In some further embodiments, the exogenous polynucleotides in the one or more constructs are operably linked after integration to one or more exogenous promoters, including CMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters; or, (2) one or more endogenous promoters in selected sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1. In some further embodiments, the iPSCs include at least one exogenous polynucleotide encoding a safety switch protein, including a caspase, a thymidine kinase, a cytosine deaminase, a modified EGFR, or a B-cell CD20. In some specific embodiments, the iPSCs comprise exogenous polynucleotides encoding at least two of the same or different safety switch proteins integrated into the same safe harbor locus, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other locus that meets the criteria for a genomic safe harbor. In one embodiment, the iPSCs comprise exogenous polynucleotides encoding two of the same or different safety switch proteins integrated into different safe harbor loci, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other locus that meets the criteria for a genomic safe harbor. In a particular embodiment, the iPSCs comprise exogenous polynucleotides encoding two of the same or different safety switch proteins integrated into different safe harbor loci, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR or RUNX1, or any other locus that meets the criteria for a genomic safe harbor.

[0020] In some embodiments, the genomically modified iPSCs generated using the provided methods have improved persistence, increased resistance to immune cells, increased immune tolerance, or increased resistance to T cells and / or NK cells compared to iPSCs not having the same genomic modification; and maintain pluripotency; and maintain the ability to differentiate into non-pluripotent cells having the same functional genomic modification. In one embodiment, the generated genomically modified iPSCs are capable of differentiating into partially differentiated cells or fully differentiated cells, the partially differentiated cells or fully differentiated cells comprising at least one targeted genome edit contained in the iPSC. In one embodiment, the generated genomically modified iPSCs are capable of differentiating into hematopoietic lineage cells, including mesodermal cells, CD34 cells, hemogenic endothelial cells, hematopoietic stem and progenitor cells, hematopoietic pluripotent progenitor cells, T cell progenitors, NK cell progenitors; T cells, NKT cells, NK cells, or B cells.

[0021] Another aspect of the invention provides genomically modified iPSCs comprising: (i) null or low expression of B2M; (ii) null or low expression of TAP1; (iii) null or low expression of TAP2; (iv) null or low expression of tapasin; (v) expression of HLA-E or uncleavable HLA-E; (vi) expression of HLA-G or uncleavable HLA-G; (vii) expression of PDL1; (viii) high affinity uncleavable CD16 (hnCD16); (ix) Fc receptor; (x) T cell receptor (TCR); (xi) chimeric antigen receptor (CAR); (xii) one or more suicide genes expressing a safety switch protein; (xiii) a bispecific, multispecific or universal engager, or any combination thereof. In some embodiments, the genomically modified iPSCs are B2M null, TAP1 null, TAP2 null, or tapasin null. In some embodiments, the genomically modified iPSCs are B2M null and have one or more of HLA-E, HLA-G, PDL1, and hnCD16 introduced. In some embodiments, the genomically modified iPSCs are B2M null and have one or more of HLA-E, HLA-G, PDL1, hnCD16, and CAR introduced. In some embodiments, the genomically modified iPSCs are B2M null and have one or more of HLA-E, HLA-G, PDL1, hnCD16, CAR, and at least one safety switch protein introduced. In some embodiments, the introduced expression is an upregulation of a non-expressed or low-expressed gene contained in the cell. In some other embodiments, the introduced expression is exogenous expression. In some embodiments, the TCR, CAR, engager, Fc receptor, or suicide gene is inducible. In some embodiments, the genomically modified iPSCs have improved persistence, increased resistance to immune cells, increased resistance to T cells and / or NK cells, or increased immune tolerance compared to iPSCs that do not have the same genomic modification; and maintain pluripotency; and maintain the ability to differentiate into non-pluripotent cells with the same functional genomic modification.

[0022] Further provided are modified HLA-deficient iPSCs that are deficient in HLA class I and / or II and further comprise: (1) one or more of exogenous HLA-E, HLA-G, CD16, 41BBL, CD47, CD113, and PDL1; or (2) one or more of HLA-E, HLA-G, CD16, 41BBL, CD47, CD113, and PDL1 proteins; and, optionally, (3) one or more of TCR, CAR, engager, Fc receptor, and single or dual safety-switch proteins. In some embodiments, the expression is increased expression from a non-expressed or low-expressed gene contained in the cells. In one embodiment, the TCR, CAR, engager, Fc receptor, or suicide gene contained in the modified HLA-deficient iPSCs is inducible. In some embodiments, the modified HLA-deficient iPSCs have improved persistence, increased resistance to immune cells, increased resistance to T cells and / or NK cells, or increased immune tolerance compared to iPSCs that do not have the same genomic modification; and maintain pluripotency; and maintain the ability to differentiate into non-pluripotent cells that are HLA-deficient and have the same functional genomic modification.

[0023] Yet another aspect of the invention provides a method of making genomically modified non-pluripotent cells derived from genomically modified iPSCs, comprising: (i) obtaining genomically modified iPSCs or modified HLA-deficient iPSCs as provided above, comprising at least one functional genome edit; and (ii) differentiating the genomically modified iPSCs or modified HLA-deficient iPSCs to obtain derived non-pluripotent cells comprising the same functional targeted genome edit comprised in the genomically modified iPSCs. In some embodiments, the differentiation step does not require the formation of embryoid bodies. In some embodiments, the differentiation is feeder-free, stromal-free, or serum-free. In some embodiments, the derived non-pluripotent cells comprise hematopoietic cells. In some embodiments, the derived non-pluripotent cells comprise mesodermal cells, CD34 cells, hemogenic endothelial cells, hematopoietic stem cells and progenitors, hematopoietic pluripotent progenitors, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, or B cells.

[0024] A further aspect of the invention provides a method of generating genomically modified non-pluripotent cells from iPSCs, the method comprising: (i) exposing iPSCs to conditions sufficient to initiate lineage-specific differentiation; and (ii) genetically modifying the differentiating cells of step (i) to obtain genomically modified non-pluripotent cells by one or both of (a) and (b) in any order: (a) introducing into the differentiating cells of step (i) one or more constructs of claim 1 for targeted integration at selected sites; (b)(1) introducing into the differentiating cells of step (i) one or more double strand breaks at the selected sites using one or more endonucleases capable of recognizing the selected sites; and (2) culturing the cells resulting from step (ii)(b)(1) to allow endogenous DNA repair to occur and generate targeted insertions / deletions at the selected sites; thereby obtaining genomically modified non-pluripotent cells comprising one or more functional targeted edits at one or more selected sites. In some embodiments, the genomically modified non-pluripotent cells comprise hematopoietic cells, hi some other embodiments, the genomically modified non-pluripotent cells comprise mesodermal cells, CD34 cells, hemogenic endothelial cells, hematopoietic stem or progenitor cells, hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, or B cells.

[0025] The invention further provides iPSC-derived genomically modified non-pluripotent cells generated using the above-described conventional methods, wherein the generated non-pluripotent cells comprise one or more functional targeted genome edits. In one embodiment, the iPSC-derived genomically modified non-pluripotent cells comprise mesodermal cells, hemogenic endothelial cells, CD34 cells, hematopoietic stem or progenitor cells, hematopoietic pluripotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, or B cells. In some embodiments, the iPSC-derived genomically modified non-pluripotent cells comprise one or more exogenous polynucleotides encoding proteins, including safety-switch proteins; targeting modalities; receptors; signaling molecules; transcription factors; pharmacologic active proteins or peptides; drug target candidates; or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of the non-pluripotent cells. In yet some other embodiments, the iPSC-derived genomically modified non-pluripotent cells comprise one or more insertions / deletions in endogenous genes associated with targeting modalities; receptors; signaling molecules; transcription factors; potential drug targets; immune response control and regulation; and proteins that inhibit the engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of the non-pluripotent cells. In some particular embodiments, the iPSC-derived genomically modified non-pluripotent cells comprise endogenous genes with one or more insertions / deletions, the endogenous genes comprising B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, or any gene in the chromosome 6p21 region. In some embodiments, the derived genomically modified non-pluripotent cells comprise a deletion or reduced expression of at least one of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region. In some other embodiments, the derived genomically modified non-pluripotent cells comprise a deletion or reduced expression of at least one of HLA-E, HLA-G, CD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2AThe introduction or increased expression of at least one of the following surface triggering receptors for binding to an R, a CAR, a TCR, an Fc receptor, an engager, and a bispecific or multispecific or universal engager.

[0026] In still other embodiments, the iPSC-derived genomically modified non-pluripotent cell of any one of claims 54-60 comprises: (i) at least two exogenous polynucleotides, each integrated into the same safe harbor locus, encoding the same or different safety switch proteins; or (ii) at least two exogenous polynucleotides, each integrated into different safe harbor loci, encoding the same or different safety switch proteins. In some embodiments of the iPSC-derived genomically modified non-pluripotent cell, the exogenous polynucleotides encoding safety switch proteins are selected from caspase, thymidine kinase, cytosine deaminase, modified EGFR, B cell CD20, and any combination thereof. In some embodiments of the iPSC-derived genomically modified non-pluripotent cell, the safe harbor loci comprise AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria for intragenomic safe harbor. In some embodiments, the iPSC-derived genomically modified non-pluripotent cells can be reprogrammed into iPSCs that contain the same functional exogenous polynucleotides contained in the non-pluripotent cells. In some embodiments of the iPSC-derived genomically modified non-pluripotent cells, the iPSCs reprogrammed from the iPSC-derived genomically modified non-pluripotent cells can be differentiated into partially differentiated or fully differentiated cells. In other embodiments, the iPSC-derived genomically modified non-pluripotent cells can be transdifferentiated into non-pluripotent cells of different pre-determined fates.

[0027] Further aspects of the invention provide iPSC-derived genomically modified hematopoietic cells comprising: (i) null or low expression of B2M; (ii) null or low expression of TAP1; (iii) null or low expression of TAP2; (iv) null or low expression of tapasin; (v) expression of HLA-E or uncleavable HLA-E; (vi) expression of HLA-G or uncleavable HLA-G; (vii) expression of PDL1; (viii) high affinity uncleavable CD16 (hnCD16); (ix) Fc receptor; (x) T cell receptor (TCR); (xi) chimeric antigen receptor (CAR); (xii) one or more suicide genes expressing a safety switch protein; (xiii) a bispecific, multispecific or universal engager, or any combination thereof. In some embodiments, the iPSC-derived genomically modified hematopoietic cells are B2M null, TAP1 null, TAP2 null, or tapasin null. In some embodiments, the iPSC-derived genomically modified hematopoietic cells are B2M null and have one or more of HLA-E, HLA-G, PDL1, and hnCD16 introduced. In some embodiments, the iPSC-derived genomically modified hematopoietic cells are B2M null and have one or more of HLA-E, HLA-G, PDL1, hnCD16, and CAR introduced. In some embodiments, the iPSC-derived genomically modified hematopoietic cells are B2M null and have one or more of HLA-E, HLA-G, PDL1, hnCD16, CAR, and at least one safety switch protein introduced. In some embodiments, the introduced expression is an upregulation of a non-expressed or low-expressed gene contained in the cell. In some other embodiments, the introduced expression is an exogenous expression. In some embodiments, the TCR, CAR, engager, Fc receptor, or suicide gene contained in the iPSC-derived genomically modified hematopoietic cells is inducible.In some embodiments, the iPSC-derived genomically modified hematopoietic cells have improved persistence, increased resistance to immune cells, increased resistance to T cells and / or NK cells, or increased immune tolerance; and the less differentiated genomically modified hematopoietic cells maintain the ability to differentiate into more differentiated hematopoietic cells with the same functional genomic modifications.

[0028] The present invention further provides compositions comprising iPSC-derived hematopoietic cells, including (a) iPSC-derived genomically modified CD34 cells; (b) iPSC-derived genomically modified hematopoietic stem or progenitor cells; (c) iPSC-derived genomically modified hematopoietic pluripotent progenitor cells; (d) iPSC-derived genomically modified T cell progenitor cells; (e) iPSC-derived genomically modified NK cell progenitor cells; (f) iPSC-derived genomically modified T cells; (g) iPSC-derived genomically modified NK cells, and any combination thereof, as described above.

[0029] Further provided are genomically modified hematopoietic cells comprising: (i) null or low expression of B2M; (ii) null or low expression of TAP1; (iii) null or low expression of TAP2; (iv) null or low expression of tapasin; (v) expression of HLA-E or uncleavable HLA-E; (vi) expression of HLA-G or uncleavable HLA-G; (vii) expression of PDL1; (viii) high affinity uncleavable CD16 (hnCD16); (ix) Fc receptor; (x) T cell receptor (TCR); (xi) chimeric antigen receptor (CAR); (xii) one or more suicide genes expressing a safety switch protein; (xiii) a bispecific, multispecific or universal engager, or any combination thereof. In some embodiments, the genomically modified hematopoietic cells are B2M null, TAP1 null, TAP2 null, or tapasin null. In some embodiments, the genomically modified hematopoietic cells are B2M null and have one or more of HLA-E, HLA-G, PDL1, and hnCD16 introduced. In some embodiments, the genomically modified hematopoietic cells are B2M null and have one or more of HLA-E, HLA-G, PDL1, hnCD16, and CAR introduced. In some embodiments, the genomically modified hematopoietic cells are B2M null and have one or more of HLA-E, HLA-G, PDL1, hnCD16, CAR, and at least one safety switch protein introduced. In some embodiments, the introduced expression is an upregulation of a non-expressed or low-expressed gene contained in the cell. In some other embodiments, the introduced expression is an exogenous expression. In some embodiments, the TCR, CAR, or suicide gene contained in the genomically modified hematopoietic cells is inducible. In some embodiments, the genomically modified hematopoietic cells have improved persistence, increased resistance to immune cells, increased resistance to T cells and / or NK cells, or increased immune tolerance; and the less differentiated genomically modified hematopoietic cells maintain the ability to differentiate into more differentiated hematopoietic cells with the same functional genomic modifications.

[0030] Further provided are modified HLA-deficient hematopoietic cells that are HLA class I and / or II deficient and further comprise: (1) one or more of exogenous HLA-E, HLA-G, CD16, 41BBL, CD47, CD113, and PDL1; or (2) one or more of HLA-E, HLA-G, CD16, 41BBL, CD47, CD113, and PDL1 proteins; and, optionally, (3) one or more of TCR, CAR, engager, Fc receptor, and single or dual safety-switch proteins. In some embodiments, the expression is increased expression from a non-expressed or low-expressed gene contained in the cell. In some embodiments, the TCR, CAR, engager, Fc receptor, or suicide gene contained in the modified HLA-deficient hematopoietic cell is inducible. In some embodiments, the modified HLA-deficient hematopoietic cells provided herein have improved persistence, increased resistance to immune cells, increased resistance to T cells and / or NK cells, or increased immune tolerance; the less differentiated modified HLA-deficient hematopoietic cells maintain the ability to differentiate into more differentiated hematopoietic cells with the same functional genomic modifications.

[0031] Another aspect of the invention provides a composition for obtaining genomically modified iPSCs, the composition comprising: (i) a genomically modified non-pluripotent cell as provided herein comprising at least one targeted genome edit at one or more selected sites within its genome; (ii) one or more reprogramming factors; and, optionally, (iii) a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor, the composition being useful for obtaining iPSCs comprising the same targeted genome edit contained in the genomically modified non-pluripotent cell. In some embodiments, the at least one targeted genome edit of the cell at one or more selected sites comprises insertion of one or more exogenous polynucleotides encoding a safety switch protein, a targeting modality, a receptor, a signaling molecule, a transcription factor, a pharmacologic active protein or peptide, a drug target candidate, or a protein that promotes engraftment, trafficking, homing, viability, self-renewal, self-renewal, persistence, and / or survival of the non-pluripotent cell and iPSCs reprogrammed therefrom.

[0032] In some embodiments of the composition, the one or more exogenous polynucleotides are operably linked to one or more exogenous promoters, including: (1) CMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters; or (2) one or more endogenous promoters contained within a selected site, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1. In some embodiments of the composition, the one or more exogenous polynucleotides are operably linked to one or more endogenous promoters contained within a selected insertion site, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1. In yet other embodiments of the composition, the genomically modified non-pluripotent cell comprises one or more exogenous polynucleotides encoding a safety switch protein, including a caspase, a thymidine kinase, a cytosine deaminase, a modified EGFR, a B-cell CD20, or any combination thereof. In some embodiments of the composition, the genomically modified non-pluripotent cell comprises exogenous polynucleotides encoding two or more of the same or different safety switch proteins integrated into different safe harbor loci, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria for a genomic safe harbor. In one particular embodiment of the composition, the genomically modified non-pluripotent cell comprises a polynucleotide encoding a caspase at the AAVS1 locus and a polynucleotide encoding a thymidine kinase at the H11 locus.In some other embodiments of the compositions, the genomically modified non-pluripotent cells comprise one or more insertions / deletions in endogenous genes associated with targeting modalities; receptors; signaling molecules; transcription factors; potential drug targets; immune response control and regulation; and proteins that inhibit the engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of the non-pluripotent cells and iPSCs derived therefrom.

[0033] In some embodiments of the compositions, the one or more endogenous genes for disruption by insertion / deletion are selected from B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region. In some embodiments of the compositions, the genomically modified non-pluripotent cells comprise a deletion or reduced expression of at least one of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region. In some other embodiments of the compositions, the genomically modified non-pluripotent cells comprise a deletion or reduced expression of at least one of HLA-E, HLA-G, CD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2AThe composition comprises at least one of the following: R, CAR, TCR, Fc receptor, engager, and surface triggering receptor for binding with a bispecific or multispecific or universal engager. In some embodiments of the composition, the introduction or upregulation is via integrative or non-integrative methods for introducing a polynucleotide encoding an exogenous protein. In some embodiments, the non-integrative exogenous polynucleotide is introduced using a Sendai virus, or an episome, or a plasmid. In one embodiment of the composition, the genomically modified non-pluripotent cell is HLA class I and / or class II deficient. In one embodiment, the genomically modified non-pluripotent cell comprises null or low expression of B2M, null or low expression of TAP1, null or low expression of TAP2, and / or null or low expression of tapasin. In some other embodiments of the composition, the genomically modified non-pluripotent cell comprises an integrated or non-integrated exogenous polynucleotide encoding one or more of an HLA-E protein, an HLA-G protein, a CD16 protein, a 41BBL protein, and a PDL1 protein. In some embodiments, the non-integrated exogenous polynucleotide is introduced using a Sendai virus, or an episome, or a plasmid. In some embodiments, the genomically modified non-pluripotent cell comprises an expression of one or more of an HLA-E protein, an HLA-G protein, a CD16 protein, a 41BBL protein, and a PDL1 protein, wherein the expression is increased expression from a non-expressed or under-expressed gene contained in the cell. In some embodiments of the composition, the genomically modified non-pluripotent cells comprise at least one exogenous polynucleotide encoding a high affinity CD16 receptor; at least one exogenous polynucleotide encoding a non-cleavable CD16 receptor; at least one exogenous polynucleotide encoding a high affinity and non-cleavable CD16 receptor (hnCD16); at least one exogenous polynucleotide encoding a non-cleavable HLA-E; or at least one exogenous polynucleotide encoding a non-cleavable HLA-G.In still other embodiments of the compositions, the genomically modified non-pluripotent cells comprise mesodermal cells, CD34 cells, hemogenic endothelial cells, hematopoietic stem or progenitor cells, hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, or B cells. In some embodiments of the compositions, the genomically modified non-pluripotent cells have improved persistence, increased resistance to immune cells, or increased immune tolerance; or the genomically modified non-pluripotent cells have increased resistance to T cells and / or NK cells.

[0034] Yet another aspect of the invention provides a composition for obtaining genomically modified iPSCs, the composition comprising: (i) non-pluripotent cells; (ii) one or more constructs for targeted editing at one or more selected loci; (iii) one or more reprogramming factors; and optionally, (iv) a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor, the composition being useful for obtaining iPSCs comprising a targeted genome edit, whereby the genomically modified iPSCs have improved persistence, increased resistance to immune cells, or increased immune resistance; or the genomically modified iPSCs have increased resistance to T cells and / or NK cells.

[0035] In some embodiments, the composition further comprises one or more endonucleases capable of recognizing a selected site to introduce a double-stranded break at the selected site; and / or a construct comprising one or more exogenous polynucleotides encoding safety-switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of non-pluripotent cell reprogrammed iPSCs or derived cells. In some embodiments, the one or more exogenous polynucleotides are operably linked to (1) one or more exogenous promoters, including CMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters; or (2) one or more endogenous promoters contained at the selected site, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1. In some embodiments of the compositions, the resulting genomically modified iPSCs comprise one or more exogenous polynucleotides encoding a safety switch protein, including a caspase, a thymidine kinase, a cytosine deaminase, a modified EGFR, a B cell CD20, or any combination thereof. In some other embodiments, the resulting genomically modified iPSCs comprise exogenous polynucleotides encoding two or more of the same or different safety switch proteins integrated into different safe harbor loci, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or any other locus that meets the criteria for a genomic safe harbor.

[0036] In some other embodiments, the resulting genomically modified iPSCs comprise an exogenous polynucleotide encoding a caspase at the AAVS1 locus and an exogenous polynucleotide encoding a thymidine kinase at the H11 locus. In still other embodiments, the resulting genomically modified iPSCs comprise one or more insertions / deletions in endogenous genes associated with targeting modalities; receptors; signaling molecules; transcription factors; potential drug targets; immune response control and regulation; and proteins that inhibit engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or derived cells. In some embodiments, the resulting genomically modified iPSCs comprising one or more insertions / deletions comprise one or more of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region.

[0037] In one embodiment, the non-pluripotent cells of the composition comprise mesodermal cells, CD34 cells, hemogenic endothelial cells, hematopoietic stem or progenitor cells, hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, or B cells. In another embodiment, the composition further comprises a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor to maintain pluripotency of the resulting genomically modified iPSCs.

[0038] Further aspects of the invention include compositions for maintaining pluripotency of genomically modified iPSCs, said compositions comprising (i) genomically modified iPSCs and (ii) a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor and a ROCK inhibitor. In some embodiments, said genomically modified iPSCs are obtained from reprogramming of genomically modified non-pluripotent cells, said obtained iPSCs comprising the same targeted integration and / or targeted insertion / deletion at selected sites of said genomically modified non-pluripotent cells. In some embodiments, said genomically modified iPSCs are obtained from genetic modification of clonal iPSCs or pools of iPSCs by introducing one or more targeted integrations and / or targeted insertion / deletions at one or more selected sites. In some other embodiments, said genomically modified iPSCs are obtained from genomic modifications by introducing one or more targeted integrations and / or targeted insertions / deletions at one or more selected sites into a pool of non-pluripotent cells undergoing reprogramming that have been contacted with one or more reprogramming factors and optionally a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and / or a ROCK inhibitor.

[0039] The genomically modified iPSCs of the composition comprise one or more exogenous polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of the non-pluripotent cell reprogrammed iPSCs or their derived cells; and / or insertions / deletions in one or more endogenous genes associated with targeting modalities, receptors, signaling molecules, transcription factors, drug target candidates, immune response control and regulation, or proteins that inhibit engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of the non-pluripotent cell reprogrammed iPSCs or their derived cells. In some embodiments, the one or more exogenous polynucleotides are operably linked to: (1) one or more exogenous promoters, including CMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters; or (2) one or more endogenous promoters contained within a selected site, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1. In some embodiments, the composition further comprises one or more endonucleases capable of recognizing a selected site to introduce a double-stranded break at the selected site.

[0040] In some embodiments, the genomically modified iPSCs of the composition comprise one or more exogenous polynucleotides encoding a safety switch protein selected from a caspase, a thymidine kinase, a cytosine deaminase, a modified EGFR, a B cell CD20, and any combination thereof. In some embodiments, the genomically modified iPSCs comprise exogenous polynucleotides encoding two or more of the same or different safety switch proteins integrated into different safe harbor loci, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria for a genomic safe harbor. In some embodiments, the genomically modified iPSCs comprise a gene encoding a caspase at the AAVS1 locus and a gene encoding a thymidine kinase at the H11 locus. In some embodiments, the genomically modified iPSCs comprise one or more insertions / deletions, including B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region. In some embodiments, the genomically modified iPSCs comprise a deletion or reduced expression of at least one of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region. In some embodiments, the genomically modified iPSCs of the composition comprise one or more insertions / deletions, including ... 2A In some embodiments, the genomically modified iPSCs include the introduction or upregulation of at least one of an R, a CAR, a TCR, an Fc receptor, an engager, and a surface triggering receptor for binding to a bispecific, multispecific, or universal engager. In some embodiments, the genomically modified iPSCs include the introduction or upregulation of a polynucleotide encoding an exogenous protein via an integrative or non-integrative method.

[0041] In some embodiments, the genomically modified iPSCs of the composition are deficient in HLA class I and / or class II. In some embodiments, the genomically modified iPSCs of the composition comprise null or under-expressed B2M, null or under-expressed TAP1 and / or null or under-expressed TAP2. In some embodiments, the genomically modified iPSCs of the composition comprise an integrated or non-integrated exogenous polynucleotide encoding one or more of an HLA-E protein, an HLA-G protein, a CD16 protein, a 41BBL protein, and a PDL1 protein; or the genomically modified iPSCs comprise an inducible expression of one or more of an HLA-E protein, an HLA-G protein, a CD16 protein, a 41BBL protein, and a PDL1 protein. In some embodiments, the genomically modified iPSCs of the composition comprise at least one exogenous polynucleotide encoding a high affinity CD16 receptor; at least one exogenous polynucleotide encoding a non-cleavable CD16 receptor; at least one exogenous polynucleotide encoding a high affinity, non-cleavable CD16 receptor (hnCD16); at least one exogenous polynucleotide encoding a non-cleavable HLA-E; or at least one exogenous polynucleotide encoding a non-cleavable HLA-G.

[0042] In some embodiments, the genomically modified iPSCs of the composition have improved persistence, increased resistance to immune cells, or increased immune tolerance; or the genomically modified iPSCs have increased resistance to T cells and / or NK cells. In some embodiments, the genomically modified iPSCs of the composition have the ability to differentiate into non-pluripotent cells, including hematopoietic cells, with the same functional targeted genome edit. In some embodiments, the genomically modified iPSCs of the composition have the ability to differentiate into mesodermal cells, CD34 cells, hemogenic endothelial cells, hematopoietic stem / progenitor cells, hematopoietic pluripotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, or B cells.

[0043] The invention further provides pharmaceutical compositions comprising (i) one or more of a genomically modified iPSC population, a modified HLA-deficient iPSC population, a genomically modified non-pluripotent cell population, a genomically modified hematopoietic cell population, a modified HLA-deficient hematopoietic cell population, or any combination thereof, provided by the methods and compositions disclosed herein; and (ii) a pharma- ceutically acceptable medium. In some embodiments, the hematopoietic cells comprise mesodermal cells, CD34 cells, hemogenic endothelial cells, hematopoietic stem or progenitor cells, hematopoietic pluripotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, or B cells.

[0044] Further provided are therapeutic uses of the above pharmaceutical compositions by introducing the composition into a subject suitable for adoptive cell therapy having an autoimmune disorder; a hematopoietic tumor; a solid tumor; a cancer, or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0045] Yet another aspect of the present invention provides methods of producing non-pluripotent cells derived from genomically modified iPSCs using the methods and compositions provided herein.

[0046] Various objects and advantages of this use will become apparent from the following description taken in conjunction with the accompanying drawings, in which: FIG. [Brief description of the drawings]

[0047] [Figure 1]Figure 1 illustrates the AAVS1 safe harbor target insertion of an inducible caspase-9 suicide gene driven by a CMV promoter and the cellular response when the suicide gene is activated by AP1903. A. Diagram of the donor construct AAVS1-G1.0 designed to target the AAVS1 locus. B. Flow cytometry for GFP expression in puromycin-selected 293T cells transfected with AAVS1 locus-specific ZFNs and the donor construct. C. Puromycin-selected 293T cells were exposed to AP1903 (or DMSO control) treatment for 24 hours. Treated cells were harvested, stained with 7AAD (which stains dying cells with compromised membranes), and analyzed by flow cytometry. The percentage of 7AAD-negative cells (presumably live cells) was plotted for each treatment (n=2). Significant cell death was detected after AP1903 treatment. D. Junction PCR of genomic DNA obtained from puromycin-selected 293T cells showed targeted insertion of the donor vector into the AAVS1 locus. E. hiPSCs were transfected with a donor construct (AAVS1-G1.0) containing ZFNs specific for the AAVS1 locus and CMV-driven inducible caspase 9. Puromycin-resistant cells were analyzed by flow cytometry for GFP expression. Three populations were sorted based on GFP intensity and expanded for further analysis. F. Sorted and expanded GFP (negative), GFP (low) and GFP (high) populations were exposed to AP1903 (or DMSO control) treatment for 24 hours. Treated cells were harvested, stained with 7AAD and analyzed by flow cytometry. The percentage of 7AAD-negative cells (viable cells) was plotted for each treatment. G. Junction PCR of genomic DNA obtained from puromycin-selected and sorted hiPSCs showed targeted insertion of the donor vector into the AAVS1 locus in the GFP(low) population but not in the GFP(negative) and GFP(high) populations.

[0048] [Diagram 2] Figure 2 shows safe harbor gene coordinate insertion of inducible caspase 9 under various endogenous and exogenous promoters in hiPSCs. A. Diagram of constructs designed to target the AAVs1 locus with the AAVS1 promoter driving gene expression. B. hiPSCs were transfected with a donor construct containing ZFNs specific for the AAVS1 locus and inducible caspase 9 under the control of the AAVS1 endogenous promoter; puromycin-resistant cells were analyzed by flow cytometry for GFP expression. C. Expanded puromycin-resistant iPSCs were exposed to AP1903 (or DMSO control) treatment for 24 hours. Treated cells were harvested and stained with 7AAD. The percentage of 7AAD-negative cells was plotted for each treatment. D. Junction PCR of genomic DNA obtained from the puromycin-resistant pool demonstrated targeted insertion of the donor vector into the AAVS1 locus in this population. E. Diagram of constructs designed to target the AAVs1 locus with various promoters. F. hiPSCs were transfected with ZFNs specific for the AAVS1 locus and donor constructs containing inducible caspase 9 under the control of various endogenous and exogenous promoters, and puromycin-resistant cells were analyzed by flow cytometry for GFP expression.

[0049] [Diagram 3]Figure 3 shows targeted insertion of EF1α promoter-driven inducible caspase-9 into the AAVS1 safe harbor in iPSCs. A. hiPSCs were transfected with a donor construct (see Figure 2) containing ZFNs specific for the AAVS1 locus and EF1α-driven inducible caspase-9, selected with puromycin, and treated with AP1903. The gated region highlights GFP-positive cells responsive to AP1903 cell death induction. B. TRA181-positive and GFP-positive hiPSCs were bulk sorted or individually sorted in 96-well plates. C. GFP-positive bulk-sorted hiPSCs were maintained for an additional time in puromycin-free medium to confirm long-term population characteristics. D. Bright field image of clonal hiPSCs derived from single-cell sorted TRA181- and GFP-cells. E. Clonal hiPSCs were expanded and GFP expression was lost to various degrees following expansion.

[0050] [Figure 4]Figure 4. Targeted insertion of CAG promoter-driven inducible caspase-9 into the AAVS1 safe harbor in hiPSCs. A. hiPSCs were transfected with a donor construct (see Figure 2) containing ZFNs specific for the AAVS1 locus and CAG-driven inducible caspase-9. Puromycin-resistant cells were bulk-sorted or individually sorted in 96-well plates. B. GFP+ bulk-sorted iPSCs were maintained in puromycin-free medium for 21 days and then analyzed by flow cytometry for GFP expression. C. When treated with AP1903, GFP-positive cells stained positive for 7AAD. D. Bright-field image of a typical colony after individual sorting. E. Colonies arising from single cells were expanded and flow cytometric assessment showed high expression of GFP and TRA181. F. Mean fluorescence intensity of selected clones. G. Junction PCR of genomic DNA to detect homologous recombination of the donor construct and the AAVS1 locus in both the parental and CAG hiPSC population pools.

[0051] [Diagram 5] Figure 5 shows the pluripotency evaluation of CAG-hiPSC clones. A. Representative images of CAG clone C38 maintained in feeder-free culture. B. Fluorescent immunostaining of pluripotency markers NANOG and OCT4. C. Quantitative RT-PCR for the expression of various endogenous pluripotency markers. D. Selected CAG clones were directed toward germ layer-specific differentiation and evaluated for germ layer markers: nestin, ectoderm; αSMA, mesoderm, SOX17, endoderm. E. CAG-C38 was evaluated for its ability to generate teratomas composed of various lineages. Teratomas were harvested 6 weeks after injection.

[0052] [Figure 6]Figure 6 shows the inducible suicide gene-mediated killing of hiPSC clones in both pluripotent and differentiated states with CAG-driven inducible caspase 9 targeted to the AAVS1 locus. A. Twenty CAG-inducible caspase 9 targeted hiPSC clones were treated with AP1903 (or DMSO control) for 24 hours and then analyzed for GFP and 7AAD staining by flow cytometry. Flow cytometry plots from clones CAG-C5 and CAG-C38 are also shown in this figure. B. One out of 15 CAG-inducible caspase 9 targeted iPSC clones survived AP1903 (10 nM) treatment. C. Kinetics of coverage of CAG-C5 live cells on 10 cm dishes after AP1903 (10 nM) treatment. D. CAG-inducible caspase 9-targeted hiPSC clones were induced to differentiate into endoderm, mesoderm or ectoderm cells, each treated with AP1903, followed by recovery and differentiation. Replicate wells before and after treatment were stained with crystal violet and imaged. Representative images from the CAG-C5 clone are shown. E. CAG-inducible caspase 9-targeted hiPSC clones were induced to differentiate into CD34+ cells, treated with AP1903 for 48 hours, and analyzed for cell death by flow cytometry.

[0053] [Figure 7]FIG. 7 shows AP1903-induced regression of teratomas derived from inducible caspase 9-iPSC clones in vivo. A. Diagram of subcutaneous injection location in NSG mouse study. B. Images of teratomas harvested 34 days after treatment of parental hiPSC lines and CAG-C38 clones with AP1903 (intraperitoneal, 200 μg total) on days 7-11. C. Volumetric measurements of harvested teratomas. D. Harvested teratomas derived from parental hiPSC or CAG-C38 lines stained with hematoxylin and eosin. While parental hiPSC lines displayed numerous three germ layer differentiated cell types, CAG-C38 appears to be composed mostly of highly organized adipoid cells. E. Parental hiPSC lines and CAG clones were injected with 4E6 cells and treated with AP1903 (intraperitoneal, 200 μg total) on days 40-46. All tumors were volumetrically measured at regular intervals.

[0054] [Figure 8] FIG. 8 shows the characterization of escaping clones. A. Illustrated method for selecting clones surviving AP1903 treatment. B. PCR amplification of the inducible caspase 9 transgene followed by purification and genomic sequencing to confirm whether sequence mutations were present. C. Flow cytometric evaluation of escaping clones showed high expression of GFP and TRA181. D. Identification of a single point mutation, K189E, in all sequenced clones. E. To confirm whether K189E was the direct reason for refractory clones, an inducible caspase 9 mutant was generated and tested. F. The inducible caspase 9 mutant transgene was confirmed to be the reason for survival of AP1903 treatment.

[0055] [Figure 9]FIG. 9 shows genome editing of the human ROSA26 locus using CRISPR / caspase 9 for targeted or nuclease-independent insertion of inducible caspase 9. A. Diagram of constructs designed to target the ROSA26 locus with CRISPR / caspase 9. B. hiPSCs were transfected with a gRNA / caspase 9-RFP plasmid specific for the ROSA26 locus and a donor construct containing CAG-driven inducible caspase 9 as shown in A. Two days after transfection, cell populations were analyzed by flow cytometry for GFP expression to determine transfection efficiency (approximately 89%). Twenty days after transfection, puromycin-resistant cells were analyzed by flow cytometry for GFP expression. C. AP1903-induced cell death analysis in 293T cells transfected with the construct. D. Targeted insertion of the construct in 293T cells. E. hiPSCs transfected with the construct and enriched for target cells by puromycin selection.

[0056] [Figure 10]FIG. 10 shows the generation of iPSC clones with monoallelic and biallelic inducible caspase 9 targeted integration. A. Quantitative PCR-based assessment of transgene (inducible caspase 9-GFP) copy number in the indicated CAG promoter-driven inducible caspase 9 clones. B. Mean fluorescence intensity (MFI) of inducible caspase 9-GFP in sorted pooled cultures (black) or in clones with monoallelic (light grey) or biallelic (dark grey) inducible caspase 9 targeted insertions as determined by flow cytometry. C. PCR analysis of transgene integration in targeted pooled cells and clones using primers overlapping the AAVS1-transgene junction. D. Assessment of the number of targeted integrations into the AAVS1 allele using a PCR-based method and primers specific for the AAVS1 integration site. E. Assessment of plate surface coverage by unsorted targeted pool cells, targeted pool cells sorted for inducible caspase 9-GFP, and clonal CAG-C38 and CAG-C5 cell lines immediately following addition of 10 nM AP1903 using live cell imaging.

[0057] [Figure 11]FIG. 11: Exogenous CAG promoter enabled inducible caspase 9-mediated elimination of established iPSC-derived teratomas. A. Schematic description of study design. B. Bioluminescence imaging of the three indicated groups of NSG mice (n=2) before AP1903 administration (top panel) and 3 days after completion of AP1903 administration (125 μg, i.p., from days 19-25, bottom panel). C. Luminescence (photons / sec) of teratomas arising from the indicated cell types shown as mean and SD. D. Ratio of post-treatment luminescence to pre-treatment luminescence (mean- / +SD) for the indicated cell types. E. Results of mice injected with both clonal CAG-C38 cells and CAG pool cells. F. End-stage volume measurements (day 28) of teratomas derived from the indicated cell types. G. Images of teratomas at day 28. No teratomas were detected in the sides injected with CAG-C38 clones or CAG-induced caspase 9 pooled cells. *p value less than 0.05, as determined by unpaired t-test. ns; not significant difference.

[0058] [Figure 12] FIG. 12 shows more effective inducible killing of transplanted biallelic inducible caspase 9 clones compared to that of monoallelic inducible caspase 9 clones. A. Schematic description of study design. B. Bioluminescence imaging of NSG mice transplanted with the indicated cell types (biallelic CAG-C5 clones, monoallelic CAG-C38 clones and parental iPSCs) at days 7, 14 and 42 after iPSC transplantation. Imaging on day 7 was performed immediately prior to AP1903 administration (125 μg i.p.; days 7-13). C. Bottom panel shows images of teratomas taken on day 42, if present. D. Luminescence yield (photons / sec) of teratomas resulting from the indicated groups shown as mean and SD. *p value less than 0.05; **p value less than 0.01; *** **p value less than 0.001, as determined by unpaired t-test. ns; not significant difference.

[0059] [Figure 13]Figure 13. Creation of a safety monitoring system with dual suicide genes targeted to separate safe harbor loci. A. PCR analysis of specific genomic integration of sr39TK into the H11 safe harbor locus using primers specific for sr39TK, the sr39TK-blasticidin (Bsd) junction or the endogenous H11 sequence-transgene junction. GAPDH is used as a loading control. B. CAG-C38 inducible caspase-9 iPSC clones with targeted insertion of sr39TK into the H11 locus treated as follows: (i) growing without either GCV or AP1903 treatment; (ii) treated with AP1903 alone for 2 days; (iii) treated with GCV alone for 9 days; and (iv) combined treatment with both AP1903 and GCV for 2 days, after which AP1903 was removed by washing and treatment with GCF continued for an additional 7 days.

[0060] [Figure 14] Figure 14: Generation of B2M- / -, HLA-I deficient iPSCs. A. Flow cytometric analysis of GFP expression before (left panel) and after (middle panel) transfection, as well as B2M and HLA-I expression. Cells negative for both B2M and HLA-I were bulk sorted (right panel). B. Flow cytometric analysis of clonally sorted B2M-negative and HLA-I-negative cells into 96-well plates.

[0061] [Figure 15A] Figure 15 shows the enhanced persistence of B2M- / -, HLA-I-deficient iPSCs. A. B2M- / - hiPSCs were not killed by prestimulated T cells. B. B2M- / - hiPSCs were not recognized by NK cells in co-culture in a single well. C. B2M- / - hiPSCs were not recognized by NK cells in co-culture in separate wells. D. B2M- / - hiPSCs have increased persistence in immune-competent mice. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above.

[0062] [Figure 16A] Figure 16 shows that modulation of HLA class I on iPSCs increases persistence of iPSCs in immunocompetent recipients. A. Genetically modified HLA-modified iPSCs express HLA-E on the cell surface and maintain a pluripotent phenotype. B. Luciferin imaging of teratomas 72 hours after injection of B2M- / -HLAE iPSCs showing increased persistence compared to wild-type iPSCs in vivo. [Figure 16B] Same as above.

[0063] [Figure 17] Figure 17 shows that iPSCs genetically modified to express the high affinity uncleavable CD16 receptor and the 41BBL costimulatory molecule retain expression throughout differentiation into iCD34 cells. A. Undifferentiated cells at day 0. B. Differentiated cells at day 10.

[0064] [Figure 18] FIG. 18 shows the reprogramming of CAR-T cells into iPSCs (TiPSCs) that retain the same genetic imprint of the source T cells by PCR analysis of CAR (FTV106) integration in iPSCs derived from FTV106 genetically modified T cells.

[0065] [Figure 19A] Figure 19 shows that iPSCs genetically modified to express CD19 chimeric antigen receptor (CAR) and truncated LNGFR cell surface marker as a common identifier for CAR retain expression through differentiation into iCD34 cells. A. Undifferentiated cells at day 0. B. Differentiated cells at day 10. [Figure 19B] Same as above.

[0066] [Figure 20]Figure 20 shows cell type-specific expression of the CAR driven by the endogenous TCR promoter and knockout of TCR expression and function by locus-specific insertion of the CAR.

[0067] [Figure 21] Figure 21 shows that hiPSCs modified to express immunosuppressive proteins can give rise to CD34 cells. A. Expression of HLAE, HLAG and PDL1 on the cell surface of modified HLA-I-deficient iPSCs; B. All modified HLA-I-deficient iPSCs differentiate into CD34+HE after 10 days of differentiation culture.

[0068] [Figure 22] FIG. 22 shows that hiPSCs engineered to express immunosuppressive proteins are capable of giving rise to hematopoietic cells.

[0069] [Diagram 23] Figure 23 shows that expression of modified non-classical HLA immunosuppressive proteins is downregulated during hematopoietic differentiation of hiPSCs. A. HLA-E and HLA-G proteins are absent, while PDL1 expression is maintained. B. HLA-E and HLA-G proteins in the supernatant fraction, indicating shedding of the proteins.

[0070] [Figure 24] Figure 24 shows the design and expression of uncleavable HLAG fusion proteins in hiPSCs. A. Design of cleavage-resistant versions of fusion proteins HLA-E / B2M and HLA-G / B2M. B. HLA-G / HLA-A3 uncleavable proteins are expressed on the cell surface without affecting the quality of iPSCs.

[0071] [Diagram 25]Figure 25 shows the generation of TAP2- / - iPSCs that show reduced expression of HLA class I. A. Flow cytometry analysis of two selected clones that show a significant reduction in HLA class I expression compared to parental FTi121. B. After treatment with IFNγ, these TAP2- / - clones show reduced expression of HLA class I compared to wild-type FTi121.

[0072] [Figure 26] Figure 26 shows that host NK cells contribute to hiPSC teratoma rejection in immunocompetent recipients. A. Absence of CD4+ T cells, CD8+ T cells and NK cells 3 days after antibody injection. B. NK-depleted mice 120 hours after iPSC injection showed the highest resistance to tumor rejection compared to animals treated with IgG control.

[0073] [Figure 27A] Figure 27 shows that modified HLA-I deficient iPSCs have increased persistence and resistance in vitro. Expression of immunosuppressive proteins on hiPSC-derived cells prevents recognition and proliferation of: A. purified allogeneic human T cells; B. PBMC allogeneic human T cells; C. PBMC allogeneic human NK cells. [Figure 27B] Same as above. [Figure 27C] Same as above.

[0074] [Figure 28A] Figure 28 shows that expression of hnCD16 protein does not affect the hematopoietic differentiation potential of modified iPSCs. A. Modified iNK cells with hnCD16 cell surface expression. B. hnCD16 is constitutively expressed and persistently maintained on iNK cells derived from genetically modified iPSCs. [Figure 28B] Same as above.

[0075] [Figure 29A]Figure 29 shows the enhancement of functionality of hnCD16 expressing iNK cells. A. Enhancement of cytokine production induced by CD16 stimulation in hnCD16 expressing iNK cells. B. hnCD16 enhances antibody-dependent cell-mediated cytotoxicity (ADCC) of hnCD16 expressing iNK cells. [Figure 29B] Same as above.

[0076] [Diagram 30] FIG. 30 shows the quality of iPSC-derived iNK cells by means of iNK cell expansion.

[0077] [Diagram 31] FIG. 31 shows that iNK cells have longer telomeres compared to mature NK cells.

[0078] [Figure 32A] Figure 32 shows Tet-inducible CAR-2A-LNGFR expression in iPSC lines integrated with doxycycline-inducible CAR. A. Inducible CD19CAR expression in TetCAR iPSCs. B. iNK-mediated cytotoxicity was assessed by flow cytometry for expression of caspase-3 on target cells. iNK cytotoxicity is enhanced by inducible CD19CAR expression. [Figure 32B] Same as above.

[0079] [Diagram 33] Figure 33 illustrates Cre or doxycycline-induced expression in iPSCs. A. After a single Cre recombinase induction, the stop codon is removed and CAR expression is activated. B. After a single doxycycline induction, the stop codon is removed and CAR expression is activated.

[0080] [Diagram 34]Figure 34 illustrates the insertion of a gene of interest at the locus selected for disruption and the clonal selection of hiPSCs after gene editing. A. Construct design of target vector and donor vector for inserting a gene of interest into the B2M locus. B. Selection of hiPSCs after gene editing for a clonal population containing the optimal gene at the selected locus for insertion.

[0081] [Diagram 35] FIG. 35 shows inducible pro-T conversion (iProT conversion), cryopreservation, and expression of CD34 and CD7 by flow cytometry in pro-T cells, A. before pro-T cell isolation; B. 3 days after thawing; C. cryopreserved on days 10+13; D. not cryopreserved; E. pro-T cells are viable after thawing and show increased cell proliferation when cultured in T cell differentiation medium.

[0082] [Diagram 36] Figure 36 shows functional characterization (cytokine release and chemotaxis) of iPSC-derived and CB-derived pro-T cells. A. iPSC-derived pro-T cells can successfully migrate toward CCL21 and SDF; B. iPSC-derived pro-T cells can produce and secrete the cytokines IFNγ and TNFα in response.

[0083] [Figure 37] FIG. 37 shows that iPSC-derived pro-T cells have a reduced proportion of germline remaining from the TCRγ locus and initiate VDJ recombination at the TCR locus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] Genome modification of stem cells such as hESCs (embryonic stem cells) or iPSCs (induced pluripotent stem cells) encompasses polynucleotide insertion, deletion and replacement. Exogenous gene expression in genomically modified iPSCs often encounters problems such as gene silencing or reduced gene expression after long-term clonal expansion of the original genomically modified iPSCs, after cell differentiation, and in dedifferentiated cell types from genomically modified iPSC-derived cells. The present invention provides an efficient, reliable and targeted approach to stably integrate one or more exogenous genes, including suicide genes and other modes of function, into iPSCs and maintain the functional response of said genes in expanded iPSCs and differentiated cells derived from genomically modified iPSCs. In one embodiment, said iPSCs are clonal iPSCs derived from a single cell. In one embodiment, the present invention provides a genetically encoded inducible "suicide" system suitable for targeted integration at one or more selected sites in iPSCs, expanded iPSCs, and differentiated cells derived therefrom that are responsive to specific non-toxic chemical inducers. The present invention thus provides an efficient, reliable, and targeted approach for the elimination of administered cells without damaging surrounding cells and tissues, suitable for a variety of cell therapeutics. Furthermore, the present invention also provides methods and systems for obtaining clonal iPSCs incorporating multiple genetic modalities involved in reprogramming and dedifferentiation, iPSC differentiation, proteins that promote engraftment, trafficking, homing, migration, cytotoxicity, viability, maintenance, proliferation, longevity, self-renewal, persistence, and / or survival of iPSCs or their derived cells, including but not limited to HSCs (hematopoietic stem and progenitor cells), T cell progenitors, NK cell progenitors, T cells, NKT cells, and NK cells. Additionally, the present invention describes the identification of mutations in the inducible caspase 9 gene that render cells refractory to chemical induction and allow escape from induced cell death, as well as approaches such as dual suicide gene safety surveillance systems or biallelic suicide gene insertions to overcome or prevent such escape.

[0085] definition 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. For purposes of the present invention, the following terms are defined below.

[0086] The articles "a", "an" and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0087] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0088] It should be understood that the term "and / or" means either one or both of the alternatives.

[0089] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that differs by about 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range that is about ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0090] As used herein, the term "substantially" or "essentially" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "essentially the same" or "substantially the same" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range that is about the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0091] As used herein, the terms "substantially free of" and "essentially free of" are used interchangeably and, when used to describe a composition such as a cell population or a medium, refer to a composition that is free of a particular substance or source thereof, e.g., free of 95%, free of 96%, free of 97%, free of 98%, free of 99%, or undetectable when measured by routine methods. The term "free of" or "essentially free of" a particular component or substance in a composition means that such component or substance (1) is not present in the composition at any concentration, or (2) is present in the composition at a low concentration that is functionally inactive. The same meaning applies to the term "absence of," which refers to the absence of a particular substance or source of a composition.

[0092] Throughout this specification, unless the context requires otherwise, the words "comprising" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of other steps or elements or groups of steps or elements. In certain embodiments, the terms "comprising," "having," "containing," and "comprising" are used interchangeably.

[0093] "Consisting of" means inclusive and limited to everything listed before the phrase "consisting of." Thus, the phrase "consisting of" implies that the listed elements are required or mandatory, and that no other elements may be present.

[0094] "Consisting essentially of" is meant to include all elements that are recited in the phrase and are limited to other elements that do not intervene or contribute to the operation or function specified in the disclosure of the recited elements. That is, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the operation or function of the recited elements.

[0095] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0096] The term "ex vivo" generally refers to activities carried out outside an organism, such as experiments or measurements made in or on living tissue in an artificial environment outside the organism, preferably with minimal differences from natural conditions. In certain embodiments, "ex vivo" procedures involve living cells or tissues obtained from an organism and cultured, usually under sterile conditions, in laboratory equipment, typically for a few hours or up to about 24 hours, but up to 48 or 72 hours depending on the circumstances. In some embodiments, such tissues or cells can be harvested, frozen, and then thawed for ex vivo processing. Tissue culture experiments or procedures using living cells or tissues that last longer than a few days are typically considered "in vitro", although in some embodiments the term may be used interchangeably with ex vivo.

[0097] The term "in vivo" generally refers to activities carried out within an organism.

[0098] As used herein, the term "reprogramming" or "dedifferentiation" or "increasing cell potency" or "increasing developmental potency" refers to a method of increasing the capacity of a cell or dedifferentiating a cell to a less differentiated state. For example, a cell with increased cell potency has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in a non-initialized state. That is, a cell that is less differentiated than the same cell in a non-initialized state is a reprogrammed cell.

[0099] As used herein, the term "differentiation" refers to the process by which an unspecialized ("uncommitted") or less specialized cell acquires the characteristics of a specialized cell, such as a blood cell or muscle cell. A differentiated or differentiation-induced cell is a cell that has attained a more specialized ("committed") position within a cell's lineage. The term "committed" as applied to the process of differentiation refers to a cell that has progressed down a differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a particular cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. As used herein, the term "pluripotency" refers to the ability of a cell to form all lineages of the body or soma (i.e., embryo proper). For example, an embryonic stem cell is a type of pluripotent stem cell that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential ranging from incompletely or partially pluripotent cells that cannot give rise to a complete organism (e.g., epiblast stem cells or EpiSCs), to more primitive, more pluripotent cells that can give rise to a complete organism (e.g., embryonic stem cells).

[0100] As used herein, the term "induced pluripotent stem cell," or iPSC, refers to a stem cell generated from a differentiated mature neonatal or embryonic cell that has been induced or changed, i.e., reprogrammed, into a cell capable of differentiating into tissues of all three germ layers or layers (mesoderm, endoderm, and ectoderm). Generated iPSC does not refer to the cell as it exists in nature.

[0101] As used herein, the term "embryonic stem cell" refers to the spontaneously occurring pluripotent stem cell of the inner cell mass of embryonic blastocyst. Embryonic stem cells are pluripotent and give rise to all derivatives of the three primary germ layers (ectoderm, endoderm and mesoderm) during development. Embryonic stem cells do not contribute to extraembryonic membranes or placenta, i.e., they are not pluripotent.

[0102] As used herein, the term "multipotent stem cell" refers to a cell that has the developmental potential to differentiate into cells of one or more, but not all three, germ layers (ectoderm, mesoderm, and endoderm). Thus, a multipotent cell may also be referred to as a "partially differentiated cell". Multipotent cells are well known in the art, and examples of multipotent cells include adult stem cells, such as hematopoietic stem cells and neural stem cells. "Multipotency" refers to a cell being capable of forming many cell types in a given lineage, but not cells of other lineages. For example, a multipotent hematopoietic cell can form a wide variety of blood cells (red blood cells, white blood cells, platelets, etc.), but cannot form neurons. Thus, the term "multipotency" refers to a state of a cell that is less than totipotent or pluripotent in its developmental potential.

[0103] Pluripotency can be determined, in part, by assessing the pluripotent properties of the cells, including, but not limited to, (i) pluripotent stem cell morphology, (ii) the potential for unlimited self-renewal, (iii) expression of pluripotent stem cell markers, including, but not limited to, SSEA1 (mouse only), SSEA3 / 4; SSEA5, TRA1-60 / 81; TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30, and / or CD50, (iv) the ability to differentiate into all three somatic cell lineages (ectoderm, mesoderm, and endoderm), (v) teratoma formation consisting of the three somatic cell lineages, and (vi) formation of embryoid bodies consisting of cells from the three somatic cell lineages.

[0104] Two types of pluripotency have been described above: "primed" or "metastable" pluripotency, which resembles the epiblast stem cells (EpiSCs) of late blastocysts, and "naive" or "ground" pluripotency, which resembles the inner cell mass of early / pre-implantation blastocysts. Both pluripotent states exhibit the characteristics described above, but the naive or ground state further exhibits (i) preinactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and survival in single cell culture, (iii) a global decrease in DNA methylation, (iv) decreased localization of H3K27me3 repressive chromatin marks on developmentally regulated gene promoters, and (v) reduced expression of differentiation markers compared to primed pluripotent cells. Standard methodologies for reprogramming cells, in which an exogenous pluripotent gene is induced into a somatic cell, expressed, and then silenced or removed from the resulting pluripotent cell, are generally seen to have the pluripotency characteristics of the primed state. Under standard pluripotent cell culture conditions, such cells will remain in the primed state unless expression of the exogenous transgene is maintained, which is what is characteristic of the ground state observed.

[0105] As used herein, the term "pluripotent stem cell morphology" refers to the classical morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a small, spherical shape with a high nucleus-to-cytoplasm ratio, prominent presence of nucleoli, and typical intercellular spaces.

[0106] As used herein, the term "subject" refers to any animal, and preferably refers to a human patient, livestock, or other domesticated animal.

[0107] "Pluripotency factor" or "reprogramming factor" refers to an agent that can increase the developmental potential of a cell, either alone or in combination with other agents. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of a cell. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0108] "Culture" or "cell culture" refers to the maintenance, growth and / or differentiation of cells in an in vitro environment. "Cell culture medium", "culture medium" (or in some cases simply "medium"), "supplement" and "medium supplement" refer to nutritional components that foster cell cultures.

[0109] "Cultivate" refers to maintaining, propagating (growing) and / or differentiating cells outside a tissue or body, such as in a sterile plastic (or coated plastic) cell culture dish or flask. Cultivation may utilize a medium as a source of nutrients, hormones and / or other factors that help propagate and / or maintain the cells.

[0110] As used herein, the term "mesoderm" refers to one of three germinal layers that emerge during early embryogenesis and give rise to a variety of specialized cell types, including blood cells of the circulatory system, muscle, heart, dermis, skeleton, and other supportive and connective tissues.

[0111] As used herein, the terms "definitive hemogenic endothelium" (HE) or "pluripotent stem cell-derived definitive hemogenic endothelium" (iHE) refer to a subset of endothelial cells that give rise to hematopoietic stem and progenitor cells in a process termed endothelial-to-hematopoietic transition. Hematopoietic cell development in the embryo progresses sequentially from lateral plate mesoderm, through hemangioblasts, to committed hemogenic endothelium and hematopoietic progenitor cells.

[0112] The terms "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells", or "hematopoietic precursor cells" refer to cells that are committed to the hematopoietic lineage but are capable of further blood cell differentiation and include multipotent hematopoietic stem cells (hemocyte stem cells), myeloid progenitor cells, megakaryocyte progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (T cells, B cells, NK cells) lineages. The term "definitive hematopoietic stem cell" as used herein refers to a CD34+ hematopoietic cell that can give rise to both mature myeloid and lymphoid cell types, including T cells, NK cells, and B cells. Hematopoietic cells also encompass various undifferentiated hematopoietic cell subsets that give rise to undifferentiated erythrocytes, megakarocytes, and macrophages.

[0113] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to the major type of white blood cell that has completed maturation in the thymus and has a variety of roles in the immune system, including identifying specific foreign antigens in the body and activating and deactivating other immune cells. The T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. The T cell can be a CD3+ cell. The T cells can be any type of T cell, including but not limited to CD4+ / CD8+ double positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells, and the like. Further types of helper T cells include cells such as Th3 (Treg) cells, Th17 cells, Th9 cells, or Tfh cells. Further types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells), and the like. T cells can also refer to genetically modified T cells, such as T cells modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells can also be differentiated from stem or progenitor cells.

[0114] "CD4+ T cells" refers to a T cell subset that expresses CD4 on its surface and is associated with cellular immune responses. CD4+ T cells are characterized by a secretory profile following stimulation that may include secretion of cytokines such as IFN-γ, TNF-α, IL-2, IL-4, and IL-10. "CD4" is a 55 kD glycoprotein originally defined as a differentiation antigen on T lymphocytes, but is also found on other cells, including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin superfamily and has been implicated as an associational recognition element in MHC (major histocompatibility complex) class II restricted immune responses. On T lymphocytes, the CD4 antigen defines helper / inducer subsets.

[0115] "CD8+ T cells" refers to a T cell subset that expresses CD8 on its surface, is MHC class I restricted, and functions as a cytotoxic T cell. The "CD8" molecule is a differentiation antigen present on thymocytes, as well as on cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin superfamily and is the associative recognition element in major histocompatibility complex class I restricted interactions.

[0116] As used herein, the term "NK cells" or "natural killer cells" refers to a peripheral blood lymphocyte subset defined by expression of CD56 or CD16 and the absence of T cell receptor (CD3). As used herein, the terms "adaptive NK cells" and "memory NK cells" are interchangeable and refer to an NK cell subset that is phenotypically CD3- and CD56+, expresses NKG2C and CD57, and optionally CD16, but lacks expression of one or more of the following: PLZF, SYK, FceRγ, and EAT-2. In some embodiments, the isolated CD56+ NK cell subpopulation includes expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A, and DNAM-1. CD56+ may be dim or bright expression.

[0117] As used herein, the term "NKT cells" or "natural killer T cells" refers to CD1d-restricted T cells that express the T cell receptor (TCR). Unlike conventional T cells, which detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d, a non-classical MHC molecule. Two types of NKT cells are currently recognized. Invariant or type I NKT cells express a very limited TCR repertoire, a standard α chain (Vα24-Jα18 in humans) coupled with a limited range of β chains (Vβ11 in humans). The second NKT cell population is called non-classical or non-invariant type II NKT cells, which show a more heterogeneous use of TCRαβ. Type I NKT cells are currently considered suitable for immunotherapy. Acquired or invariant (Type I) NKT cells can be identified by expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161 and CD56.

[0118] As used herein, the term "isolated" and the like refers to a cell or cell population that is separated from its original environment, i.e., the environment of the isolated cell is substantially free of at least one component present in the environment in which the "unisolated" reference cell is present. The term encompasses a cell that has been removed from some or all components present in its natural environment (e.g., tissue, biopsy). The term also encompasses a cell that has been removed from at least one, some or all components present in a non-naturally occurring environment (e.g., medium, cell suspension). Thus, an isolated cell is partially or completely separated from at least one component, including other substances, cells or cell populations, as they are naturally present or as they are grown, stored or survived in a non-naturally occurring environment. Specific examples of isolated cells include partially pure cells, substantially pure cells, and cells cultured in a non-naturally occurring medium. An isolated cell may be obtained by isolating a desired cell or population thereof from other substances or cells in the environment, or by removing one or more other cell populations or cell subpopulations from the environment. As used herein, the term "purify" or the like refers to increasing purity. For example, purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0119] As used herein, the term "encode" refers to the inherent property of a particular nucleotide sequence within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom. That is, a gene codes for a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to the gene. Both the coding strand, which is the nucleotide sequence that is identical to the mRNA sequence and is usually shown in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to code for the protein or other product of the gene or cDNA.

[0120] A "construct" refers to a complex of molecules, including a polymer or polynucleotide, that is delivered to a host cell in vitro or in vivo. A "vector" as used herein refers to any nucleic acid construct capable of directing the delivery or transfer of foreign genetic material to a target cell capable of replicating and / or expressing the foreign genetic material. The term "vector" as used herein includes the construct that is delivered. A vector may be a linear or circular molecule. A vector may be an integrating or non-integrating vector. The main types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, and the like.

[0121] "Integration" means that one or more nucleotides of a construct are stably inserted into a cell genome, i.e., are covalently linked to a nucleic acid sequence in the chromosomal DNA of a cell. "Targeted integration" means that nucleotides of a construct are inserted into the chromosomal or mitochondrial DNA of a cell at a preselected site, i.e., "integration site". The term "integration" as used herein further refers to a process that includes the insertion of one or more foreign sequences or nucleotides of a construct, with or without the deletion of endogenous sequences or nucleotides at the integration site. When there is a deletion at the insertion site, "integration" can also include the replacement of the deleted endogenous sequences or nucleotides with the one or more inserted nucleotides.

[0122] As used herein, the term "exogenous" is intended to mean that the referenced molecule or referenced activity is one that has been introduced into the host cell. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the genetic material of the host, such as by integration into a host chromosome, or as non-chromosomal genetic material, such as a plasmid. Thus, when used in reference to expression of an encoding nucleic acid, the term refers to the introduction of the encoding nucleic acid into the cell in an expressible form. The term "endogenous" refers to a referenced molecule or activity that is present within the host cell. Similarly, when used in reference to expression of an encoding nucleic acid, the term refers to expression of an encoding nucleic acid that is contained within the cell and not exogenously introduced.

[0123] As used herein, a "gene of interest" or a "polynucleotide sequence of interest" is a DNA sequence that, when placed under the control of appropriate control sequences, is transcribed into RNA and optionally translated into a polypeptide in vivo. A gene of interest or a polynucleotide of interest can include, but is not limited to, a prokaryotic sequence, a cDNA derived from eukaryotic mRNA, a genomic DNA sequence derived from eukaryotic (e.g., mammalian) DNA, and a synthetic DNA sequence. For example, a gene of interest can code for miRNA, shRNA, a natural polypeptide (i.e., a naturally occurring polypeptide) or a fragment thereof; a variant polypeptide (i.e., a variant of a natural polypeptide having less than 100% sequence identity with the natural polypeptide) or a fragment thereof; an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selection marker, etc.

[0124] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. The sequence of a polynucleotide is composed of the four nucleotide bases adenine (A); cytosine (C); guanine (G); thymine (T), and if the polynucleotide is RNA, thymine is uracil (U). A polynucleotide can include a gene or gene fragment (e.g., a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotide also refers to both double-stranded and single-stranded molecules.

[0125] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to molecules having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, but there is no limit to the maximum number of amino acids in a polypeptide. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, and longer chains, commonly referred to in the art as polypeptides or proteins. "Polypeptides" include, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. These polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0126] "Operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is affected by the other.For example, a promoter is operably linked to a coding sequence or functional RNA if the promoter can affect the expression of the coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter).The coding sequence can be operably linked to a control sequence in either sense or antisense orientation.

[0127] As used herein, the term "genetic imprint" refers to genetic or epigenetic information that contributes to a preferential therapeutic property in a source cell or iPSC and can be retained in iPSCs derived from the source cell and / or hematopoietic cells derived from the iPSC. As used herein, a "source cell" is a non-pluripotent cell that can be used to generate iPSCs through reprogramming, and iPSCs derived from a source cell can be further differentiated into specific cell types, including any hematopoietic cell. Depending on the context, iPSCs derived from a source cell and differentiated cells therefrom may be collectively referred to as "derived cells." As used herein, genetic imprints that confer preferential therapeutic properties are incorporated into iPSCs through donor-specific, disease-specific, or treatment response-specific reprogramming of selected source cells, or through the introduction of genetic modification patterns into iPSCs using genome editing. In the context of source cells obtained from a specifically selected donor situation, disease situation or treatment situation, the genetic imprints contributing to the preferential therapeutic properties may include any situation-specific genetic or epigenetic modifications that manifest a retainable phenotype, i.e., preferential therapeutic properties, inherited by the derived cells of the selected source cells, regardless of whether the underlying molecular phenomenon has been identified. Donor-specific, disease-specific, or treatment response-specific source cells may include genetic imprints that can be retained in iPSCs and derived hematopoietic cells, including, but not limited to, those from a given single-specific TCR, such as virus-specific T cells or invariant natural killer T (iNKT) cells; traceable and desirable genetic polymorphisms, such as those homozygous for a point mutation that codes for high affinity CD16 receptor in the selected donor; and selected HLA-matched donor cells that exhibit a given HLA requirement, i.e., a population-increased haplotype.As used herein, preferential therapeutic properties include improved engraftment, improved trafficking, improved homing, improved viability, improved self-renewal, improved persistence, improved control and regulation of immune responses, improved survival, and improved cytotoxicity of derived cells. Preferential therapeutic properties may also be related to expression of antigen targeting receptors; HLA presentation or lack thereof; tolerance to the tumor microenvironment; induction of bystander immune cells and immune regulation; improved on-target specificity with reduced off-tumor effects; and resistance to treatments such as chemotherapy.

[0128] The term "enhanced therapeutic properties" as used herein refers to enhanced therapeutic properties of cells when compared to typical immune cells of the same normal cell type. For example, NK cells with "enhanced therapeutic properties" have enhanced, improved, and / or increased therapeutic properties when compared to typical, unmodified, and / or naturally occurring NK cells. Therapeutic properties of immune cells may include, but are not limited to, cell engraftment, trafficking, homing, viability, self-renewal, persistence, control and regulation of immune responses, survival, and cytotoxicity. Therapeutic properties of immune cells are also manifested by: antigen targeting receptor expression; HLA presentation or lack thereof; resistance to intratumoral microenvironment; induction of bystander immune cells and immune regulation; improved target specificity with reduced extratumoral effects; resistance to treatments such as chemotherapy.

[0129] As used herein, the term "engager" refers to a molecule, e.g., a fusion polypeptide, capable of forming a link between an immune cell, e.g., a T cell, a NK cell, a NKT cell, a B cell, a macrophage, a neutrophil, and a tumor cell; and activating the immune cell. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTEs), bispecific killer cell engagers (BiKEs), trispecific killer cell engagers, or multispecific killer cell engagers, or universal engagers that are compatible with multiple immune cell types.

[0130] As used herein, the term "surface triggering receptor" refers to a receptor that can trigger or initiate an immune response, such as a cytotoxic response. The surface triggering receptor may be engineered and expressed on effector cells, such as T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils. In some embodiments, the surface triggering receptor promotes binding between effector cells and specific target cells (e.g., tumor cells) by bispecific or multispecific antibodies, regardless of the effector cell's natural receptor and cell type. Using this approach, iPSCs containing universal surface triggering receptors may be generated, and then the iPSCs may be differentiated into a population of various effector cell types that express the universal surface triggering receptor. By "universal" it is meant that the surface triggering receptor can be expressed on and activate any effector cell, regardless of cell type, and that all effector cells expressing the universal receptor can be coupled or linked to an engager with the same epitope recognizable by the surface triggering receptor, regardless of the tumor-binding specificity of the engager. In some embodiments, engagers with the same tumor targeting specificity are used to couple with the universal surface triggering receptor. In some embodiments, engagers with different tumor targeting specificity are used to couple with the universal surface triggering receptor. Thus, binding one or more effector cell types can kill one particular type of tumor cell in some cases, and can kill more than one type of tumor in other cases. Surface triggering receptors usually contain a costimulatory domain for effector cell activation and an anti-epitope specific to the epitope of the engager. The bispecific engager is specific for an anti-epitope of a surface triggering receptor at one end and a tumor antigen at the other end.

[0131] As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent potential toxic or otherwise adverse effects of a cell therapy. In some cases, expression of the safety switch protein is conditionally controlled to address safety concerns regarding transplanted engineered cells that have permanently incorporated into their genome a gene encoding the safety switch protein. This conditional control can be variable and can include control via small molecule mediated post-translational activation and tissue-specific and / or temporal transcriptional control. The safety switch can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation, and / or antibody mediated depletion. In some cases, the safety switch protein is activated by an exogenous molecule (e.g., a prodrug) that, when activated, causes apoptosis and / or cell death of the therapeutic cell. Examples of safety-switch proteins include, but are not limited to, caspase 9 (or caspase 3 or caspase 7), thymidine kinase, cytosine deaminase, B-cell CD20, suicide genes such as modified EGFR, and any combination thereof. In this strategy, a prodrug administered in the event of an adverse event is activated by the suicide gene product and kills the transduced cell.

[0132] As used herein, the term "pharmaceutical active protein or peptide" refers to a protein or peptide capable of achieving a biological and / or pharmaceutical effect on an organism. Pharmaceutically active proteins have curative, therapeutic, or symptomatic properties against a disease and can be administered to ameliorate, alleviate, mitigate, reverse, or reduce the severity of the disease. Pharmaceutically active proteins also have prophylactic properties and are used to prevent the occurrence of a disease or to reduce the severity of such a disease or pathological condition if it does occur. Pharmaceutically active proteins include whole proteins or peptides or pharmaceutical active fragments thereof. Pharmaceutically active analogs of proteins or peptides or analogs of fragments of proteins or peptides are also included. The term pharmaceutical active proteins also refers to multiple proteins or peptides that act cooperatively or synergistically to produce a therapeutic effect. Examples of pharma- ceutical active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth suppressor proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0133] As used herein, the term "signal transduction molecule" refers to any molecule that regulates, participates in, inhibits, activates, reduces, or increases intercellular communication. Signal transduction refers to the transmission of molecular signals in the form of chemical modifications by the recruitment of protein complexes along a pathway that ultimately initiates biochemical events in cells. Signal transduction pathways are well known in the art and include, but are not limited to, G protein-coupled receptor signal transduction, tyrosine kinase receptor signal transduction, integrin signal transduction, toll gate signal transduction, ligand-gated ion channel signal transduction, ERK / MAPK signal transduction pathway, Wnt signal transduction pathway, cAMP-dependent pathway, and IP3 / DAG signal transduction pathway.

[0134] As used herein, the term "targeting modality" refers to molecules, e.g., polypeptides, genetically incorporated into cells to promote antigen and / or epitope specificity, including but not limited to: i) antigen specificity, in the case of unique chimeric antigen receptors (CARs) or T cell receptors (TCRs); ii) engager specificity, in the case of monoclonal antibodies or bispecific engagers; iii) transformed cell targeting; iv) cancer stem cell targeting; and v) other targeting strategies in the absence of a specific antigen or surface molecule.

[0135] As used herein, the terms "specific" or "specificity" can be used to refer to the ability of a molecule, such as a receptor or engager, to selectively bind to a target molecule, as opposed to non-specific or non-selective binding.

[0136] As used herein, the term "adoptive cell therapy," as used herein, refers to cell-based immunotherapy involving the transfusion of autologous or allogeneic lymphocytes, which may or may not be genetically modified and which may be T cells or B cells that have been expanded ex vivo prior to transfusion.

[0137] A "therapeutically sufficient amount," as used herein, includes within its meaning a non-toxic, but sufficient and / or effective amount of a particular therapeutic and / or pharmaceutical composition that is believed to produce a desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient's overall health, the patient's age, and the stage and severity of the condition. In certain embodiments, a therapeutically sufficient amount is sufficient and / or effective to ameliorate, reduce, and / or ameliorate at least one symptom associated with the disease or condition of the subject being treated.

[0138] Differentiation of pluripotent stem cells requires changes in the culture system, such as changing stimuli in the medium or the physical state of the cells. The most common strategies utilize the formation of embryoid bodies (EBs) as a common and critical intermediate to initiate lineage-specific differentiation. "Embryoid bodies" are three-dimensional clusters that have been shown to mimic embryonic development by giving rise to multiple lineages within their three-dimensional space. Throughout the differentiation process, typically over hours to days, simple EBs (e.g., aggregated pluripotent stem cells induced to differentiate) continue to mature and develop into cystic EBs, at which point they are further processed to continue differentiation, typically for days to weeks. EB formation is initiated by bringing pluripotent stem cells into close proximity to each other in a three-dimensional multi-layered cell cluster, typically achieved by one of several methods, including settling the pluripotent cells into droplets and settling the cells into "U"-bottom well plates, or by mechanical agitation. To promote EB development, pluripotent stem cell aggregates require additional differentiation cues, as aggregates maintained in pluripotent culture maintenance medium do not form proper EBs. Thus, pluripotent stem cell aggregates need to be transferred to differentiation medium that provides optimal lineage-directed induction cues. EB-based pluripotent stem cell medium typically results in the generation of differentiated cell populations (ectoderm, mesoderm, and endoderm) with moderate proliferation within the EB cell clumps. Although known to promote cell differentiation, EBs produce heterogeneous cells with inconsistent differentiation states due to inconsistent cell exposure to environmental differentiation cues within the three-dimensional structure. Moreover, EBs are laborious to generate and maintain. Moreover, cell differentiation via EBs is accompanied by moderate cell proliferation, which also contributes to low differentiation efficiency.

[0139] In comparison, "aggregate formation" can be used to expand cell populations derived from pluripotent stem cells, unlike "EB formation". For example, during aggregate-based pluripotent stem cell expansion, the medium is selected to maintain proliferation and pluripotency. Cell proliferation generally increases the size of the aggregates by forming larger aggregates, but these aggregates can be periodically mechanically or enzymatically separated into smaller aggregates to maintain cell proliferation and increase cell number in culture. Unlike EB culture, cells cultured in aggregates in maintenance medium maintain markers of pluripotency. Pluripotent stem cell aggregates require additional differentiation cues to induce differentiation.

[0140] As used herein, "monolayer differentiation" refers to a differentiation method that is different from differentiation through three-dimensional multi-layered cell clusters, i.e., "EB formation". Monolayer differentiation does not require EB formation to induce differentiation, although other advantages are disclosed herein. Since monolayer culture does not mimic embryonic development such as EB formation, differentiation toward specific lineages is considered minimal compared to differentiation into all three germ layers in EBs.

[0141] As used herein, "dissociated" cells refer to cells that have been substantially separated or purified from other cells or a surface (e.g., a culture dish surface). For example, cells can be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that are aggregated in a tube can be dissociated from each other, such as by enzymatic or mechanical dissociation into a suspension of clusters, single cells, or a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells are dissociated from a culture dish or other surface. Thus, dissociation may involve breaking cellular interactions with the extracellular matrix (ECM) and the culture substrate (e.g., the culture surface) or breaking the ECM between cells.

[0142] As used herein, "feeder cells" or "feeders" are terms describing one type of cell that is co-cultured with a second type of cell to provide an environment in which the second type of cell can grow, since the feeder cells provide growth factors and nutrients for the support of the second cell type. Feeder cells may be from a different species than the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts, or immortalized mouse embryonic fibroblasts. Feeder cells can typically be inactivated by irradiation or treatment with mitotic inhibitors such as mitomycin to prevent the feeder cells from outgrowing the cells they support when co-cultured with other cells. Feeder cells can include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the above, one particular feeder cell type can be a human feeder, such as human dermal fibroblasts. Another feeder cell type can be mouse embryonic fibroblasts (MEFs). In general, various feeder cells can be used in part to maintain pluripotency, direct differentiation to certain lineages, and promote maturation into specialized cell types such as effector cells.

[0143] As used herein, a "feeder-free (FF)" environment refers to an environment, such as a cell culture or medium, that is essentially free of feeder cells and / or has not been preconditioned by the growth of feeder cells. A "preconditioned" medium refers to a medium that is harvested after the growth of feeder cells in the medium for a period of time, e.g., at least one day. A preconditioned medium contains numerous mediators, including growth factors and cytokines, secreted by the feeder cells grown in the medium.

[0144] "Functional," as used in reference to genome editing or modification of iPSCs and derived non-pluripotent cells differentiated therefrom, or non-pluripotent cells and derived iPSCs reprogrammed therefrom, refers to: (1) at the genetic level, successful knock-in, knock-out, knock-down, transgenic, or controlled gene expression, such as inducible or temporal expression at a desired stage of cellular development, achieved through direct genome editing or modification, or through "passing-on" through differentiation or reprogramming from an originally genomically engineered starting cell; or (2) at the cellular level, (ii) gene expression modifications that are maintained in the cell through "propagation" from the original genomically engineered starting cell through differentiation or reprogramming thereof; (iii) downstream gene regulation in the cell as a result of gene expression modifications that are only present at an early stage in the development of the cell or only present in the starting cell that gives rise to the cell through differentiation or reprogramming; or (iv) an enhancement or acquisition of a new cellular function or property that is exhibited in the mature cellular product resulting from genome editing or modifications originally performed on an iPSC, progenitor cell or de-differentiated cellular source.

[0145] "HLA-deficient," which includes HLA class I deficiency, HLA class II deficiency, or both, refers to a cell that lacks or can no longer maintain, or has reduced levels of cell surface expression of complete MHC complexes, including HLA class I protein heterodimers and / or HLA class II heterodimers, such that the reduced or reduced levels are less than those naturally detectable by other cells or by synthetic methods. HLA class I deficiency can be achieved by functional deletion of any region of the HLA class I locus (chromosome 6p21), or by deletion or reduction of the expression levels of HLA class I-associated genes, including but not limited to the beta-2 microglobulin (B2M) gene, the TAP1 gene, the TAP2 gene, and tapasin. HLA class II deficiency can be achieved by functional deletion or reduction of HLA-II-associated genes, including but not limited to RFXANK, CIITA, RFX5, and RFXAP. Prior to the present invention, it was unclear whether iPSCs with HLA complex deletions or modifications have the ability to enter developmental stages, mature, and give rise to functional differentiated cells while retaining regulated activity. Furthermore, prior to the present invention, it was unclear whether HLA complex-deficient differentiated cells can be reprogrammed into iPSCs and maintained as pluripotent stem cells while retaining HLA complex deletions. Unexpected failures during cell reprogramming, maintenance of pluripotency, and differentiation may be related to aspects including, but not limited to, developmental stage-specific gene expression or lack thereof, requirements for HLA complex presentation, proteolytic shedding of introduced surface expression patterns, the need for proper and efficient clonal reprogramming, and the need for reconstitution of differentiation protocols.

[0146] "Modified HLA-deficient iPSCs", as used herein, refers to HLA-deficient iPSCs that have been further modified by the introduction of genes expressing proteins associated with improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to inhibition, proliferation, costimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cytotoxic activity, including, but not limited to, non-classical HLA class I proteins (e.g., HLA-E and HLA-G), chimeric antigen receptors (CARs), T cell receptors (TCRs), CD16 Fc receptors, BCL11b, NOTCH, RUNX1, IL15, 41BB, DAP10, DAP12, CD24, CD3z, 41BBL, CD47, CD113, and PDL1. "Modified HLA-deficient" cells also include cells other than iPSCs.

[0147] "Fc receptors", abbreviated as FcR, are classified based on the type of antibody they recognize. For example, those that bind the most common antibody class IgG are called Fcγ receptors (FcγR), those that bind IgA are called Fcα receptors (FcαR), and those that bind IgE are called Fcε receptors (FcεR). FcR classes are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T cells, and B cells) and the signaling properties of each receptor. Fcγ receptors (FcγR) include several members, FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b), which have different antibody affinities due to differences in their molecular structures.

[0148] CD16 has been identified as two isoforms of Fc receptors, FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells that activates NK cells and promotes antibody-dependent cell-mediated cytotoxicity (ADCC) by binding to monomeric IgG attached to target cells. "High affinity CD16", "uncleavable CD16" or "high affinity uncleavable CD16" as used herein refers to a variant of CD16. Wild-type CD16 has low affinity and undergoes extracellular domain shedding, a proteolytic process that controls the cell surface density of various cell surface molecules on leukocytes after NK cell activation. F176V and F158V are exemplary CD16 variants with high affinity; whereas, the S197P variant is an example of an uncleavable CD16.

[0149] I. Targeted editing of selected loci in iPSC cells Genome editing, or genomic editing, or gene editing, used interchangeably herein, is a type of genetic engineering in which DNA is inserted, deleted, and / or replaced in the genome of a target cell. Targeted genome editing (interchangeable with "targeted genomic editing" or "targeted gene editing") allows for insertion, deletion, and / or replacement at a preselected site in the genome. If an endogenous sequence is deleted at the insertion site during targeted editing, the endogenous gene containing the affected sequence can be knocked out or knocked down by the deletion of the sequence. Thus, targeted editing can also be used to disrupt endogenous gene expression. The term "targeted integration" is also used herein to refer to a process that includes the insertion of one or more foreign sequences with or without the deletion of endogenous sequences at the insertion site. In comparison, randomly integrated genes are subject to position effects and silencing, making their expression uncertain and unpredictable. For example, centromeric and subtelomeric regions are particularly susceptible to transgene silencing. Reciprocally, newly integrated genes may alter cell behavior or aid in cell transformation by affecting surrounding endogenous genes and chromatin. Therefore, inserting foreign DNA into safe harbor loci, or preselected loci such as intragenomic safe harbors (GSH), is important for safety, efficiency, copy number control, and reliable gene response regulation.

[0150] Targeted editing can be achieved through a nuclease-independent approach or through a nuclease-dependent approach, in which homologous sequences flanking the exogenous polynucleotide to be inserted are directed to homologous recombination through the enzymatic machinery of the host cell.

[0151] Alternatively, targeted editing can be achieved at higher frequencies through the specific introduction of double-strand breaks (DSBs) by specific rare-cutting endonucleases. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms, including non-homologous end joining (NHEJ), which occurs in response to DSBs. Without a donor vector containing exogenous genetic material, this NHEJ often results in random insertion or deletion (insertion / deletion) of a small number of endogenous nucleotides. In comparison, in the presence of a donor vector containing exogenous genetic material flanked by a pair of homology arms, this exogenous genetic material can be introduced into the genome during homology-directed repair (HDR) by homologous recombination, resulting in "targeted integration."

[0152] Available endonucleases that can introduce specific and targeted DSBs include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided CRISPR-caspase 9 nucleases (CRISPR / caspase 9; Clustered Regular Interspaced Short Palindromic Repeats Associated 9). In addition, the DICE (dual integrase cassette exchange) system, which utilizes phiC31 integrase and Bxb1 integrase, is also a promising tool for targeted integration.

[0153] ZFN is a targeted nuclease that comprises a nuclease fused to a Zn finger DNA binding domain. "Zn finger DNA binding domain" or "ZFBD" refers to a polypeptide domain that binds to DNA in a sequence-specific manner through one or more Zn fingers. Zn finger is a domain of about 30 amino acids in the Zn finger binding domain that has a structure stabilized through the coordination of zinc ions. Examples of Zn fingers include, but are not limited to, C2H2Zn finger, C3HZn finger, and C4Zn finger. A "designed" Zn finger domain is a non-naturally occurring domain whose design / composition is primarily derived from rational criteria, such as the application of substitution rules and computer algorithms to process information in a database that stores information on the design and binding data of existing ZFPs. See, for example, U.S. Patent Nos. 6,140,081; 6,453,242; and 6,534,261; also International Publication Nos. WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496. A "selected" Zn finger domain is a non-naturally occurring domain whose generation is primarily derived from experimental processes such as phage display, interaction trap or hybrid selection. ZFNs are described in more detail in U.S. Patent Nos. 7,888,121 and 7,972,854, the complete disclosures of which are incorporated herein by reference. The most recognized example of ZFN in the art is the fusion of FokI nuclease with a Zn finger DNA binding domain.

[0154] TALENs are targeted nucleases that contain a nuclease fused to a TAL effector DNA binding domain. "Transcription activation-like effector DNA binding domain", "TAL effector DNA binding domain", or "TALE DNA binding domain" refers to the polypeptide domain of a TAL effector protein that is involved in the binding of the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of plant cells, bind to effector-specific DNA sequences via their DNA binding domain, and activate gene transcription in these sequences via their transactivation domain. The specificity of the TAL effector DNA binding domain depends on the number of imperfect 34 amino acid repeats that contain polymorphisms at selected repeat positions, called repeat variable-diresidues (RVDs), that vary from effector to effector. TALENs are described in more detail in U.S. Patent Application No. 2011 / 0145940, which is incorporated herein by reference. The most recognized example of a TALEN in the art is a fusion polypeptide between FokI nuclease and a TAL effector DNA-binding domain.

[0155] Another example of a targeted nuclease useful in the present method is a targeted Spo11 nuclease, which is a polypeptide that includes a Spo11 polypeptide having nuclease activity fused to a DNA binding domain, such as a zinc finger DNA binding domain, a TAL effector DNA binding domain, etc., with specificity for a DNA sequence of interest. See, e.g., U.S. Application No. 61 / 555,857, the disclosure of which is incorporated herein by reference.

[0156] Further examples of targeted nucleases suitable for the present invention, whether used individually or in combination, include, but are not limited to, Bxb1, phiC31, R4, PhiBT1, and Wβ / SPBc / TP901-1.

[0157] Other non-limiting examples of targeted nucleases include naturally occurring and recombinant nucleases, such as CRISPR / caspase 9, restriction enzymes, meganucleases homing endonucleases, and the like.

[0158] CRISPR / caspase-9 requires two key components: (1) caspase-9 endonuclease (Casp9) and (2) crRNA-tracrRNA complex. When co-expressed, these two components form a complex and are recruited to target DNA sequences containing the PAM and a PAM-proximal seeding region. When combined, the crRNA and tracrRNA form a chimeric guide RNA (gRNA), which can guide Casp9 to target a selected sequence. These two components can then be delivered into mammalian cells via transfection or transduction.

[0159] In DICE-mediated insertion, a pair of recombinases (e.g., phiC31 and Bxb1) is used to allow unidirectional integration of foreign DNA tightly restricted by the small attB and attP recognition sites of each enzyme. These target att sites do not naturally occur in mammalian genomes and must first be introduced into the genome at the desired integration site. See, for example, U.S. Application Publication No. 2015 / 0140665, the disclosure of which is incorporated herein by reference.

[0160] One aspect of the present invention provides a construct comprising one or more exogenous polynucleotides for targeted genomic integration. In one embodiment, the construct further comprises a pair of homology arms specific to a desired integration site, and the targeted integration method comprises introducing the construct into a cell, thereby allowing site-specific homologous recombination by the host enzyme machinery of the cell. In another embodiment, the targeted integration method in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing a ZFN expression cassette comprising a DNA binding domain specific to a desired integration site into the cell, thereby allowing ZFN-mediated insertion. In another embodiment, the targeted integration method in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing a TALEN expression cassette comprising a DNA binding domain specific to a desired integration site into the cell, thereby allowing TALEN-mediated insertion. In another embodiment, the method of targeted integration in a cell includes introducing into the cell a construct comprising one or more exogenous polynucleotides, a caspase 9 expression cassette, and a gRNA comprising a guide sequence specific for a desired integration site, thereby allowing caspase 9-mediated insertion. In yet another embodiment, the method of targeted integration in a cell includes introducing into the cell a construct comprising one or more att sites for a pair of DICE recombinases at a desired integration site, introducing into the cell a construct comprising one or more exogenous polynucleotides, and introducing an expression cassette for DICE recombinase, thereby allowing DICE-mediated targeted integration.

[0161] Potential sites for targeted integration include, but are not limited to, safe harbor loci, or genomic safe harbors (GSH), which are intragenic or extragenic regions of the human genome that theoretically accommodate predictable expression of newly integrated DNA without deleterious effects on the host cell or host organism. A useful safe harbor should allow sufficient transgene expression to obtain the desired levels of vector-encoded protein or non-coding RNA. Additionally, the safe harbor should not predispose cells to malignant transformation or alter cellular function. For an integration site to be a potential safe harbor locus, it should ideally meet the following criteria, but not be limited to: no disruption of regulatory elements or genes (as judged by sequence annotation); be an intergenic region in a gene-dense region, i.e., a meeting point between two genes that are transcribed in opposite directions; maintain a distance between the vector-encoded transcriptional activator and the promoters of adjacent genes, particularly cancer-related and microRNA genes, to minimize the possibility of long-range interactions; and have apparent ubiquitous transcriptional activity (reflected by spatially and temporally widespread expressed sequence tag (EST) expression patterns indicative of ubiquitous transcriptional activity). This latter feature is particularly important in stem cells, where chromatin remodeling during differentiation typically results in the silencing of some loci and may activate others. The exact locus chosen for insertion, within a region favorable for exogenous insertion, should have no repetitive components or conserved sequences and be one for which primers for amplification of homology arms can be readily designed.

[0162] Suitable sites for human genome editing, or specifically targeted integration, include, but are not limited to, the adeno-associated virus site 1 (AAVS1), the human orthologue of the chemokine (CC motif) receptor 5 (CCR5) locus, and the mouse ROSA26 locus. Additionally, the human orthologue of the mouse H11 locus may also be a suitable site for insertion using the compositions and targeted integration methods disclosed herein. Additionally, the collagen locus and the HTRP locus may also be used as safe harbors for targeted integration. However, validation of each selected site has been shown to be necessary for specific integration events, especially in stem cells, and optimization of the insertion strategy, including promoter selection, sequence and positioning of the foreign gene, and construct design, is often required.

[0163] In targeted insertion / deletion, the editing site is often included in the endogenous gene whose expression and / or function is to be deleted.In one embodiment, the endogenous gene that comprises targeted insertion / deletion is associated with immune response control and regulation.In some other embodiments, the endogenous gene that comprises targeted insertion / deletion is associated with targeting modalities, receptors, signaling molecules, transcription factors, potential drug targets, immune response control and regulation, or proteins that suppress the transplantation, transport, homing, viability, self-renewal, persistence, and / or survival of stem cells and / or progenitor cells and their derived cells.

[0164] Thus, one aspect of the present invention provides a method of targeted integration at a selected locus, including a genomic safe harbor or preselected locus known or found to be safe and well-regulated for sustained or temporal gene expression, such as the B2M locus, TAP1 locus, TAP2 locus, or tapasin locus, as provided herein. In one embodiment, the genomic safe harbor for the targeted integration method includes one or more desired integration sites including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria for a genomic safe harbor. In one embodiment, a method for targeted integration in a cell includes introducing into a cell a construct comprising one or more exogenous polynucleotides, and introducing a construct comprising a pair of homology arms and one or more exogenous sequences specific to a desired integration site, thereby allowing site-specific homologous recombination by the host enzymatic machinery of the cell, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR or RUNX1, or other locus that meets the criteria for intragenomic safe harbor.

[0165] In another embodiment, a method for targeted integration in a cell includes introducing into the cell a construct comprising one or more exogenous polynucleotides and introducing into the cell a ZFN expression cassette comprising a DNA binding domain specific for a desired integration site, thereby enabling ZFN-mediated insertion, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR or RUNX1, or any other locus that meets the criteria for intragenomic safe harbor. In another embodiment, a method for targeted integration in a cell includes introducing into the cell a construct comprising one or more exogenous polynucleotides and introducing into the cell a TALEN expression cassette comprising a DNA binding domain specific for a desired integration site, thereby enabling TALEN-mediated insertion, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR or RUNX1, or other locus that meets the criteria for intragenomic safe harbor. In another embodiment, a method for targeted integration in a cell includes introducing into the cell a construct comprising one or more exogenous polynucleotides, a caspase 9 expression cassette, and a gRNA comprising a guide sequence specific for a desired integration site, thereby enabling caspase 9 mediated insertion, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other locus that meets the criteria for intragenomic safe harbor.In yet another embodiment, a method for targeted integration in a cell comprises introducing a construct comprising one or more att sites for a pair of DICE recombinases into a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette for DICE recombinase to enable DICE-mediated targeted integration, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR or RUNX1, or any other locus that meets the criteria for intragenomic safe harbor.

[0166] Further, as provided herein, the targeted integration method in the above safe harbor may be used to insert any polynucleotide of interest, such as polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutically active proteins and peptides, potential drug targets, and proteins that promote stem cell and / or progenitor cell engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival. In some other embodiments, the construct comprising the one or more exogenous polynucleotides further comprises one or more marker genes. In one embodiment, the exogenous polynucleotide in the construct of the invention is a suicide gene encoding a safety switch protein. Suitable suicide gene systems for inducible cell death include, but are not limited to, caspase 9 (or caspase 3 or caspase 7) and AP1903; thymidine kinase (TK) and ganciclovir (GCV); cytosine deaminase (CD) and 5-fluorocytosine (5-FC). In addition, some suicide gene systems are cell type specific, for example, genetic modification of T lymphocytes with the B cell molecule CD20 allows for the elimination of T lymphocytes after administration of the mAb Rituximab. Furthermore, using a modified EGFR containing an epitope recognized by Cetuximab allows the genetically modified cells to be depleted when exposed to Cetuximab. Thus, one embodiment of the present invention provides a method for targeted integration of one or more suicide genes encoding a safety switch protein selected from caspase 9 (caspase 3 or 7), thymidine kinase, cytosine deaminase, modified EGFR, and B cell CD20.

[0167] In some embodiments, one or more exogenous polynucleotides integrated by the method herein are driven by an exogenous promoter operably linked to the construct for targeted integration. The promoter may be an inducible promoter or a constitutive promoter, and may be temporally specific, tissue specific, or cell type specific. Constitutive promoters suitable for the method of the present invention include, but are not limited to, cytomegalovirus (CMV) promoter, elongation factor 1 alpha (EF1 alpha) promoter, phosphoglycerate kinase (PGK) promoter, hybrid CMV enhancer / chicken β-actin (CAG) promoter, and ubiquitin C (UBC) promoter. In one embodiment, the exogenous promoter is CAG.

[0168] The exogenous polynucleotide integrated by the method herein may be driven by an endogenous promoter in the host genome at the integration site. In one embodiment, the method of the present invention is used for targeted integration of one or more exogenous polynucleotides at the AAVS1 locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous AAVS1 promoter. In another embodiment, the method of the present invention is used for targeted integration at the ROSA26 locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous ROSA26 promoter. In another embodiment, the method of the present invention is used for targeted integration at the H11 locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous H11 promoter. In another embodiment, the method of the present invention is used for targeted integration at the collagen locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous collagen promoter. In another embodiment, the method of the present invention is used for targeted integration at the HTRP locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous HTRP promoter. In theory, gene expression of the foreign gene driven by the endogenous promoter would be possible if it could be inserted precisely at the desired site.

[0169] In some embodiments, one or more exogenous polynucleotides included in the construct for targeted integration are driven by one promoter. In some embodiments, the construct includes one or more linker sequences between two adjacent polynucleotides driven by the same promoter to provide greater physical separation between the moieties and maximize accessibility to enzymatic machinery. The linker peptide of the linker sequence may be composed of amino acids selected to provide a more flexible or more rigid physical separation between the moieties (exogenous polynucleotides and / or their encoded proteins or peptides) depending on the function involved. The linker sequence may be cleavable by a protease or chemically cleavable to obtain the separated moieties. Examples of enzymatic cleavage sites in a linker include sites for cleavage by proteolytic enzymes such as enterokinase, activated factor X, trypsin, collagenase, and thrombin. In some embodiments, the protease is a protease naturally produced by the host or an exogenously introduced protease. Alternatively, the cleavage site in the linker may be one that is cleavable after exposure to a selected chemical, such as cyanogen bromide, hydroxylamine, or low pH. The optional linker sequence may serve a purpose other than providing a cleavage site. The linker sequence should allow for effective positioning of the moiety relative to another adjacent moiety for the moiety to function properly. The linker may be a simple amino acid sequence of sufficient length to prevent any steric hindrance between the moieties. Additionally, the linker sequence may allow for post-translational modifications, including but not limited to, for example, phosphorylation sites, biotinylation sites, sulfation sites, gamma-carboxylation sites, and the like. In some embodiments, the linker sequence is flexible so as not to hold the biologically active peptide in a single undesirable conformation. Flexibility may be provided by having the linker comprised primarily of amino acids with small side chains, such as glycine, alanine, and serine.In some embodiments, about 80 percent or about 90 percent or more of the linker sequence comprises glycine, alanine, or serine residues, particularly glycine and serine residues. In some embodiments, a G4S linker peptide separates the end processing domain and the endonuclease domain of the fusion protein. In other embodiments, a 2A linker sequence allows two separate proteins to be produced from a single translation. Suitable linker sequences can be easily identified by experiment. In addition, suitable sizes and sequences of linker sequences can also be determined by conventional computer modeling techniques. In one embodiment, the linker sequence encodes a self-cleaving peptide. In one embodiment, the self-cleaving peptide is 2A. In some other embodiments, the linker sequence provides an internal ribosome entry sequence (IRES). In some embodiments, the two consecutive linker sequences are different.

[0170] The method of introducing the construct containing the exogenous polynucleotide for targeted integration into cells can be achieved by using a method of gene transfer into cells that is known per se. In one embodiment, the construct comprises a viral vector backbone, such as an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a lentivirus vector, or a Sendai virus vector. In some embodiments, a plasmid vector (e.g., pAl-11, pXTl, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc.) is used to deliver and / or express the exogenous polynucleotide to the target cell. In some other embodiments, an episomal vector is used to deliver the exogenous polynucleotide to the target cell. In some embodiments, a recombinant adeno-associated virus (rAAV) can be used for genetic engineering to introduce insertions, deletions, or substitutions through homologous recombination. Unlike lentiviruses, rAAVs do not integrate into the host genome. In addition, episomal rAAV vectors mediate a much faster homology-directed gene targeting compared to the transfection of conventional targeting plasmids. In some embodiments, AAV6 or AAV2 vectors are used to introduce insertions, deletions or substitutions at target sites in the genome of iPSCs.

[0171] II. Methods for Obtaining and Maintaining Genome-Modified iPSCs The present invention provides a method for obtaining and maintaining genomically modified iPSCs containing one or more targeted edits at one or more desired sites, wherein the targeted edits remain intact and functional in the genomically modified iPSCs or iPSC-derived non-pluripotent cells after expansion at each selected edit site. The targeted edits introduce insertions, deletions, and / or substitutions, i.e., targeted integrations and / or insertions / deletions, at the selected sites into the genome iPSCs.

[0172] In certain embodiments, genomically modified iPSCs containing one or more targeted edits at one or more selected sites are maintained, passaged and expanded as single cells for extended periods in a cell culture medium as shown in Table 1 as Fate Maintenance Medium (FMM), wherein the iPSCs retain the targeted edits and functional modifications at the selected sites. The components of the medium may be present in the medium in amounts within the optimal ranges as shown in Table 1. iPSCs cultured in FMM have been shown to continue to maintain their undifferentiated and ground state or naive characteristics, i.e., genomic stability, without the need for culture washes or selection, and readily give rise to all three somatic cell lineages, in vitro by differentiation through embryoid bodies or monolayers (without formation of embryoid bodies); and in vivo by differentiation through teratoma formation. See, e.g., U.S. Application No. 61 / 947,979, the disclosure of which is incorporated herein by reference. [Table 1]

[0173] In some embodiments, genomically modified iPSCs containing one or more targeted integrations and / or targeted insertions / deletions are maintained, passaged and expanded in a medium containing MEKi, GSKi, and ROCKi, and free or essentially free of TGFβ receptor / ALK5 inhibitors, and the iPSCs retain intact and functional targeted edits at selected sites. In some embodiments, the sites for targeted integration include AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR or RUNX1, or other loci that meet the criteria for intragenomic safe harbor. In some embodiments, the sites for targeted insertions / deletions are selected from endogenous genes associated with targeting modalities, receptors, signaling molecules, transcription factors, potential drug targets; control and regulation of immune responses; or proteins that inhibit engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or derived cells thereof. In some embodiments, the maintained, passaged and expanded genomically modified iPSCs comprise one or more inducible suicide genes integrated at one or more desired integration sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR or RUNX1, or other loci that meet the criteria for a genomic safe harbor. In some other embodiments, the maintained, passaged and expanded genomically modified iPSCs comprise polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutical active proteins and peptides, drug target candidates, or proteins that promote stem and / or progenitor cell engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival at the same or different desired integration sites.

[0174] In some embodiments, the genomically modified iPSCs comprise one or more exogenous polynucleotides and further comprise an insertion / deletion in one or more endogenous genes. In some embodiments, the insertion / deletion in the endogenous genes results in disruption of gene expression. In some embodiments, the insertion / deletion in the endogenous genes results in knockout of the edited gene. In some embodiments, the insertion / deletion in the endogenous genes results in knockdown of the edited gene. In some embodiments, the genomically modified iPSCs comprise one or more exogenous polynucleotides at a selected site may further comprise one or more targeted edits comprising an insertion / deletion at a selected site. In some embodiments, the insertion / deletion is in one or more endogenous genes associated with controlling and mediating immune responses. In some embodiments, the insertion / deletion is in one or more endogenous checkpoint genes. In some embodiments, the insertion / deletion is in one or more endogenous T cell receptor genes. In some embodiments, the insertion / deletion is in one or more endogenous MHC class I inhibitory genes. In some embodiments, the insertion / deletion is in one or more endogenous genes associated with the major histocompatibility complex. In some embodiments, the insertion / deletion is in one or more endogenous genes, including but not limited to B2M, PD1, TAP1, TAP2, tapasin, TCR genes. In one embodiment, the genomically modified iPSCs comprising one or more exogenous polynucleotides at a selected site further comprise a targeted edit in the gene encoding B2M (beta-2-microglobulin).

[0175] Another aspect of the invention provides a method of generating genomically modified iPSCs through iPSC targeted editing; or by first generating genomically modified non-pluripotent cells by targeted editing, and then reprogramming the selected / isolated genomically modified non-pluripotent cells to obtain iPSCs containing the same targeted editing as the non-pluripotent cells. A further aspect of the invention provides a genomically modified non-pluripotent cell undergoing simultaneous reprogramming by introducing targeted integration and / or targeted insertion / deletion into the cell, where the contacted non-pluripotent cell is under conditions sufficient for reprogramming, and the conditions for reprogramming include contacting the non-pluripotent cell with one or more reprogramming factors and small molecules. In various embodiments of the method for simultaneous genomically modifying and reprogramming, targeted integration and / or targeted insertion / deletion can be introduced into the non-pluripotent cell by contacting the non-pluripotent cell with one or more reprogramming factors and small molecules prior to or essentially simultaneously with the initiation of reprogramming.

[0176] In some embodiments, to simultaneously genome modify and reprogram non-pluripotent cells, targeted integration and / or targeted insertion / deletion may be introduced into non-pluripotent cells after a multi-day reprogramming process has been initiated by contacting the non-pluripotent cells with one or more reprogramming factors and small molecules, where the vector carrying the construct is introduced before the cells being reprogrammed exhibit stable expression of one or more endogenous pluripotency genes, including but not limited to SSEA4, Tra181 and CD30.

[0177] In some embodiments, reprogramming is initiated by contacting the non-pluripotent cells with at least one reprogramming factor and, optionally, a combination of a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FRM; Table 1). In some embodiments, iPSCs genomically modified through any of the above methods are further maintained and expanded using a mixture comprising a combination of a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FMM; Table 1).

[0178] In some embodiments of the method for producing genomically modified iPSCs, the method comprises genomically modifying iPSCs at a selected site by introducing one or more targeted integrations and / or targeted insertions / deletions into the iPSCs to obtain genomically modified iPSCs. Alternatively, the method for producing genomically modified iPSCs comprises (a) introducing one or more targeted edits into a non-pluripotent cell to obtain a genomically modified non-pluripotent cell comprising a targeted integration and / or a targeted insertion / deletion at a selected site, and (b) contacting the genomically modified non-pluripotent cell with one or more reprogramming factors, and optionally a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor, to obtain a genomically modified iPSC comprising a targeted integration and / or a targeted insertion / deletion at a selected site. Alternatively, the method for producing genomically modified iPSCs includes: (a) contacting a non-pluripotent cell with one or more reprogramming factors and, optionally, a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and / or a ROCK inhibitor to induce reprogramming of the non-pluripotent cell; (b) introducing one or more targeted integrations and / or targeted insertions / deletions into the non-pluripotent cell being reprogrammed for genome modification; and (c) obtaining a clonal genomically modified iPSC comprising the targeted integration and / or targeted insertion / deletion at a selected site.

[0179] The reprogramming factor is selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, L1TD1, and any combination thereof. The one or more reprogramming factors may be in the form of a polypeptide. The reprogramming factor may be in the form of a polynucleotide, and thus is introduced into the non-pluripotent cell by a vector, such as a retrovirus, Sendai virus, adenovirus, episome, and minicircle. In certain embodiments, the one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, the one or more polynucleotides are introduced by an episomal vector. In various other embodiments, the one or more polynucleotides are introduced by a Sendai virus vector.

[0180] In some embodiments, the non-pluripotent cells are introduced with multiple constructs, including different exogenous polynucleotides and / or different promoters, by multiple vectors for targeted integration at the same or different selected sites. These exogenous polynucleotides may include suicide genes, or genes encoding targeting modalities, receptors, signaling molecules, transcription factors, pharmacoactive proteins and peptides, potential drug targets, or genes encoding proteins that promote the engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or their derived cells. In some embodiments, the exogenous polynucleotides encode RNAs, including but not limited to siRNAs, shRNAs, miRNAs, and antisense nucleic acids. These exogenous polynucleotides may be driven by one or more promoters selected from the group consisting of constitutive promoters, inducible promoters, time-specific promoters, and tissue- or cell-type-specific promoters. Thus, the polynucleotides are expressible when under conditions that activate the promoters, for example, in the presence of an inducer or in a particular differentiated cell type. In some embodiments, the polynucleotide is expressed in iPSCs and / or in cells differentiated from the iPSCs. In one embodiment, one or more suicide genes are driven by constitutive promoters, e.g., caspase-9 is driven by CAG. These constructs, including different exogenous polynucleotides and / or different promoters, can be introduced simultaneously or sequentially into non-pluripotent cells. Non-pluripotent cells that have undergone targeted integration of multiple constructs can be contacted with the one or more reprogramming factors simultaneously to trigger reprogramming with genome modification, thereby obtaining genome-modified iPSCs that contain multiple targeted integrations in the same cell pool. This robust method thus allows for simultaneous reprogramming and editing strategies to obtain clonal genome-modified hiPSCs with multiple modalities integrated into one or more selected target sites.

[0181] In some embodiments, the non-pluripotent cells are introduced with one or more insertions / deletions at selected sites comprising one or more endogenous genes. In some embodiments, the non-pluripotent cells comprising one or more insertions / deletions further comprise one or more targeted integrations as described above. In some embodiments, the insertions / deletions comprised in the endogenous genes result in disruption of gene expression. In some embodiments, the insertions / deletions comprised in the endogenous genes result in knockout of the edited gene. In some embodiments, the insertions / deletions comprised in the endogenous genes result in knockdown of the edited gene. In some embodiments, the genomically modified iPSCs comprising one or more exogenous polynucleotides at selected sites may further comprise one or more targeted edits comprising an insertion / deletion at a selected site. In some embodiments, the insertions / deletions are comprised in one or more endogenous genes associated with controlling and mediating immune responses. In some embodiments, the insertions / deletions are comprised in one or more endogenous checkpoint genes. In some embodiments, the insertions / deletions are comprised in one or more endogenous T cell receptor genes. In some embodiments, the insertion / deletion is in one or more endogenous MHC class I suppressor genes. In some embodiments, the insertion / deletion is in one or more endogenous genes associated with the major histocompatibility complex. In some embodiments, the insertion / deletion is in one or more endogenous genes, including but not limited to B2M, PD1, TAP1, TAP2, tapasin, TCR genes. In one embodiment, the genomically modified iPSCs comprising one or more exogenous polynucleotides at a selected site further comprise a targeted edit in a gene encoding B2M (beta-2-microglobulin).

[0182] III. Obtaining genetically modified non-pluripotent cells by differentiation of genome-modified iPSCs Further aspects of the invention provide methods of in vivo differentiation of genomically modified iPSCs by teratoma formation, wherein differentiated cells derived in vivo from genomically modified iPSCs retain intact and functional targeted edits comprising targeted integration and / or targeted insertion / deletion at desired sites. In some embodiments, differentiated cells derived in vivo from genomically modified iPSCs via teratomas contain one or more inducible suicide genes integrated at one or more desired sites, including AAVS1, CCR5, ROSA26, collagen, HTRP H11, beta-2 microglobulin, GAPDH, TCR or RUNX1, or other loci that meet the criteria for a genomic safe harbor. In some other embodiments, differentiated cells derived in vivo from genomically modified iPSCs via teratomas contain polynucleotides encoding targeting modalities or polynucleotides encoding proteins that promote trafficking, homing, viability, self-renewal, persistence, and / or survival of stem and / or progenitor cells. In some embodiments, differentiated cells derived in vivo from genomically modified iPSCs via teratoma containing one or more inducible suicide genes further comprise one or more insertions / deletions in endogenous genes associated with controlling and mediating immune responses. In some embodiments, the insertions / deletions are in one or more endogenous checkpoint genes. In some embodiments, the insertions / deletions are in one or more endogenous T cell receptor genes. In some embodiments, the insertions / deletions are in one or more endogenous MHC class I inhibitory genes. In some embodiments, the insertions / deletions are in one or more endogenous genes associated with major histocompatibility complex. In some embodiments, the insertions / deletions are in one or more endogenous genes, including but not limited to B2M gene, PD1 gene, TAP1 gene, TAP2 gene, tapasin gene, TCR gene. In one embodiment, the genomically modified iPSCs containing one or more exogenous polynucleotides at selected sites further comprise targeted editing in a gene encoding B2M (beta-2-microglobulin).

[0183] In certain embodiments, genomically modified iPSCs containing one or more targeted edits at selected sites as provided herein are used to derive hematopoietic cell lines or any other specific cell type in vitro, and derived non-pluripotent cells retain the functional targeted edits at the selected sites. In one embodiment, genomically modified iPSC-derived cells include, but are not limited to, mesodermal cells capable of differentiation into committed hemogenic endothelial (HE), committed HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPP), T cell progenitors, NK cell progenitors, myeloid cells, neutrophil progenitors, T cells, NKT cells, NK cells, and B cells, and these genomically modified iPSC-derived cells retain the functional targeted edits at the desired sites.

[0184] Differentiation methods and compositions applicable to obtain iPSC-derived hematopoietic cell lines include, for example, those shown in International Application No. PCT / US2016 / 044122, the disclosure of which is incorporated herein by reference. As provided, methods and compositions for generating hematopoietic cell lines through committed hemogenic endothelium (HE) derived from pluripotent stem cells containing hiPSCs, without the need for EB formation, under serum-free, feeder-free, and / or stroma-free conditions, and in an expandable and monolayer culture platform. Cells that can be differentiated according to the provided methods range from pluripotent stem cells to committed progenitor cells to specific highly differentiated cell types, and transdifferentiated cells, which are cells of various lineages that have transitioned directly to a hematopoietic fate without passing through a pluripotent intermediate. Similarly, cells resulting from differentiation of stem cells also range from pluripotent stem cells or pluripotent progenitor cells to highly differentiated stem cells and all intervening hematopoietic cell lineages.

[0185] A method for differentiating and expanding hematopoietic cells from pluripotent stem cells in monolayer culture includes contacting the pluripotent stem cells with a BMP pathway activator and, optionally, bFGF. As provided, mesodermal cells derived from pluripotent stem cells are obtained and expanded without forming embryoid bodies from the pluripotent stem cells. The mesodermal cells are then contacted with a BMP pathway activator, bFGF, and a WNT pathway activator to obtain expanded mesodermal cells with committed hemogenic endothelial (HE) differentiation potential without forming embryoid bodies from the pluripotent stem cells. Subsequent contact with bFGF and, optionally, a ROCK inhibitor and / or a WNT pathway activator differentiates the mesodermal cells with committed HE differentiation potential into committed HE cells, which also proliferate during differentiation.

[0186] The methods provided herein for obtaining hematopoietic cells are superior to EB-mediated pluripotent stem cell differentiation because EB formation results in moderate to minimal cell proliferation, does not allow for monolayer culture, which is important for many applications requiring uniform proliferation and differentiation of cells within a population, and is laborious and less efficient.

[0187] The monolayer differentiation platform provided promotes differentiation towards fate-committed hemogenic endothelium resulting in induction of hematopoietic stem cells and differentiated progeny such as T cells, B cells, NKT cells and NK cells. The monolayer differentiation strategy, combining enhanced differentiation efficiency with mass expansion, allows for the delivery of therapeutically meaningful numbers of pluripotent stem cell-derived hematopoietic cells for a variety of therapeutic applications. Furthermore, monolayer culture using the methods provided herein results in functional hematopoietic lineage cells that are capable of in vitro differentiation, ex vivo regulation, and long-term hematopoietic self-renewal, reconstitution and engraftment in vivo. As provided, iPSC-derived hematopoietic lineage cells include, but are not limited to, fate-committed hemogenic endothelium, hematopoietic multipotent progenitor cells, hematopoietic stem cells and progenitors, T cell progenitors, NK cell progenitors, T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0188] A method for directing differentiation of pluripotent stem cells into committed hematopoietic cells includes: (i) contacting a pluripotent stem cell with a composition comprising a BMP activator and optionally bFGF to induce differentiation and proliferation of mesodermal cells from said pluripotent stem cells; (ii) contacting said mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally no TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of mesodermal cells having committed HE differentiation potential from said mesodermal cells. (iii) contacting said mesodermal cells having committed HE differentiation potential with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator, and optionally no TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of committed hemogenic endothelium from pluripotent stem cell-derived mesodermal cells having committed hemogenic endothelial differentiation potential.

[0189] In some embodiments, the method further comprises contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but not including a TGFβ receptor / ALK inhibitor, to seed and grow the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs, or naive iPSCs, or iPSCs comprising one or more genetic imprints; the one or more genetic imprints comprised in the iPSC are retained in hematopoietic cells differentiated from the iPSC. In some embodiments of the method for directing the differentiation of pluripotent stem cells into hematopoietic cells, the differentiation of the pluripotent stem cells into hematopoietic cells does not produce embryoid bodies and is in monolayer culture form.

[0190] In some embodiments of the above methods, the resulting pluripotent stem cell-derived committed hemogenic endothelial cells are CD34+. In some embodiments, the resulting hemogenic committed endothelial cells are CD34+CD43-. In some embodiments, the hemogenic committed endothelial cells are CD34+CD43-CXCR4-CD73-. In some embodiments, the hemogenic committed endothelial cells are CD34+CXCR4-CD73-. In some embodiments, the hemogenic committed endothelial cells are CD34+CD43-CD93-. In some embodiments, the hemogenic committed endothelial cells are CD34+CD93-.

[0191] In some embodiments of the above methods, the method further comprises: (i) contacting the pluripotent stem cell-derived committed hemogenic endothelium with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator, to induce differentiation of the committed hemogenic endothelium into a pre-T cell progenitor; and optionally (ii) contacting the pre-T cell progenitor with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but not including one or more of VEGF, bFGF, TPO, a BMP activator, and a ROCK inhibitor, to induce differentiation of the pre-T cell progenitor into a T cell progenitor or T cell. In some embodiments of the above methods, the pluripotent stem cell-derived T cell progenitor is CD34+CD45+CD7+. In some embodiments of the methods, the pluripotent stem cell-derived T cell progenitors are CD45+CD7+.

[0192] In some further embodiments of the above-described methods for directing differentiation of pluripotent stem cells towards hematopoietic cells, the method further comprises: (i) contacting the pluripotent stem cell-derived committed hemogenic endothelium with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15; and optionally, a BMP activator, to trigger differentiation of the committed hemogenic endothelium into a pre-NK cell progenitor; and optionally, (ii) contacting the pluripotent stem cell-derived pre-NK cell progenitor with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and not including one or more of VEGF, bFGF, TPO, a BMP activator, and a ROCK inhibitor, to trigger differentiation of the pre-NK cell progenitor into an NK cell progenitor or an NK cell. In some embodiments, the pluripotent stem cell-derived NK progenitor cells are CD3-CD45+CD56+CD7+. In some embodiments, the pluripotent stem cell-derived NK cells are CD3-CD45+CD56+, and optionally further characterized as NKp46+, CD57+, and CD16+.

[0193] That is, using the above differentiation methods, one or more iPSC-derived hematopoietic cell populations can be obtained: (i) CD34+ HE cells (iCD34) using one or more media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; (ii) committed hemogenic endothelial (iHE) using one or more media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; (iii) committed HSC using one or more media selected from iMPP-A, iTC-A2, iTC-B2, iNK (iv) multipotent progenitor cells (iMPP) use one or more media selected from iMPP-A; (v) T cell progenitor cells (ipro-T) use one or more media selected from iTC-A2 and iTC-B2; (vi) T cells (iTC) use iTC-B2; (vii) NK cell progenitor cells (ipro-NK) use one or more media selected from iNK-A2 and iNK-B2; and / or (viii) NK cells (iNK) and iNK-B2. In some embodiments, the media: a. iCD34-C comprises one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor, bFGF, VEGF, SCF, IL6, IL11, IGF, and EPO, and optionally a Wnt pathway activator; does not comprise a TGFβ receptor / ALK inhibitor; b. iMPP-A comprises a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11; c. iTC-A2 comprises a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, and IL7; and optionally, a BMP activator; d. iTC-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7; said composition does not comprise one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors; e. iNK-A2 comprises a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL3, IL7, and IL15; and optionally a BMP activator; f. iNK-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7 and IL15.

[0194] In some embodiments, the genomically modified iPSC-derived cells obtained from the above method comprise one or more inducible suicide genes integrated at one or more desired integration sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria for a genomic safe harbor. In some other embodiments, the genomically modified iPSC-derived cells comprise polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutical active proteins and peptides, drug target candidates, or proteins that promote stem and / or progenitor cell trafficking, homing, viability, self-renewal, persistence, and / or survival. In some embodiments, the genomically modified iPSC-derived cells comprising one or more suicide genes further comprise one or more insertions / deletions in one or more endogenous genes associated with controlling and mediating immune responses, including, but not limited to, checkpoint genes, endogenous T cell receptor genes, and MHC class I inhibitory genes. In one embodiment, the genomically modified iPSC-derived cells comprising one or more suicide genes further comprise an insertion / deletion in the B2M gene, where B2M is knocked out.

[0195] Further, dedifferentiation methods and compositions applicable for obtaining genome-modified hematopoietic cells of a first to second fate can be, for example, those shown in International Publication No. WO2011 / 159726, the disclosure of which is incorporated herein by reference. The methods and compositions provided in said International Publication allow for partial reprogramming of starting non-pluripotent cells into non-pluripotent intermediate cells by limiting expression of the endogenous Nanog gene during reprogramming; and exposing the non-pluripotent intermediate cells to conditions for differentiating the intermediate cells into the desired cell type.

[0196] IV. Compositions for targeted integration at selected loci to integrate genes of interest in iPSC cells In view of the above, the present invention provides a construct for targeted integration of one or more exogenous polynucleotides into the genome of induced pluripotent stem cells (iPSCs) at a selected site, with or without deletion of endogenous sequences at the integration site. In one embodiment, the construct comprises at least one exogenous promoter operably linked to one or more exogenous polynucleotides expressing one or more proteins of interest. In one embodiment, the construct is introduced into iPSCs by a vector for targeted integration. In another embodiment, the construct is introduced into a non-pluripotent cell by a vector for targeted integration, and the non-pluripotent cell is then reprogrammed to obtain a genomically modified iPSC. With either approach, the construct remains integrated and the exogenous polynucleotide is functional in the iPSCs obtained from reprogramming or vector introduction, in the iPSCs subsequently expanded, in differentiated cells derived from the iPSCs, and in dedifferentiated cells derived therefrom.

[0197] In some embodiments, the construct comprises at least one exogenous promoter operably linked to one or more exogenous polynucleotides, further comprises a homology arm specific to a selected site, said homology arm being adjacent to said promoter and said exogenous polynucleotide of interest in said construct. This embodiment of the construct allows for a nuclease-independent targeted integration strategy. In some embodiments, said exogenous promoter is CAG.

[0198] In some embodiments, one or more exogenous polynucleotides contained in a construct having at least one exogenous promoter are linked to each other by a linker sequence. In some embodiments, the linker sequence encodes a self-cleaving peptide. In other embodiments, the linker sequence provides an internal ribosome entry sequence (IRES). In some embodiments, one or more exogenous polynucleotides in the construct are polycistronic.

[0199] Some embodiments of the construct comprising one or more exogenous promoters and one or more exogenous polynucleotides operably linked thereto further comprise at least one polynucleotide encoding a marker. In some embodiments, the at least one polynucleotide encoding a marker is driven by an endogenous promoter at a selected site. In one embodiment, one polynucleotide encodes a fluorescent protein. In another embodiment, one polynucleotide is a puromycin resistance gene.

[0200] In some embodiments, the construct comprising the operably linked polynucleotide further comprises a component that enables targeted integration at a safe harbor locus, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other locus that meets the criteria for a genomic safe harbor. In some embodiments, the safe harbor locus is selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, and RUNX1.

[0201] A construct comprising operably linked polynucleotides may include one or more polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutical active proteins and peptides, drug target candidates, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, self-renewal, persistence, and / or survival of genomically modified iPSCs or derived cells. In some embodiments, the one or more polynucleotides are driven by the same promoter. In some embodiments, the one or more polynucleotides are driven by one or more different promoters selected from the group consisting of constitutive promoters, inducible promoters, time-specific promoters, and tissue-type or cell-type specific promoters. In some embodiments, the one or more polynucleotides are integrated into the same selected safe harbor locus. In some embodiments, the one or more polynucleotides are integrated into different selected safe harbor loci. In one particular embodiment, one or more of the exogenous polynucleotides operably linked to an exogenous or endogenous promoter encodes one or more safety switch proteins. The safety switch protein may be selected from the group consisting of caspase 9 (or caspase 3 or caspase 7), thymidine kinase, cytosine deaminase, modified EGFR, and B cell CD20. In one embodiment, the suicide gene included in the construct encodes caspase 9. In one embodiment, the suicide gene included in the construct encodes thymidine kinase. In some embodiments, the construct is for targeted integration at one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, and other loci that meet the criteria for intragenomic safe harbor. In some embodiments, the construct is for targeted integration at AAVS1.In some embodiments, the construct is for targeted integration in ROSA26. In some embodiments, the construct is for targeted integration in H11.

[0202] The present invention also provides genomically modified iPSCs comprising one or more exogenous polynucleotides and / or insertions / deletions at selected sites. In some embodiments, the genomically modified iPSCs are obtained through vector-mediated construct introduction into iPSCs and / or editing using NHEJ. In some other embodiments, the genomically modified iPSCs are obtained from non-pluripotent cells undergoing reprogramming with targeted editing, including targeted integration and / or targeted insertion / deletion. In yet some further embodiments, the genomically modified iPSCs are obtained from simultaneously reprogramming and genomically modifying non-pluripotent cells with vectors carrying constructs for targeted integration of reprogramming factors, reprogramming small molecules, and / or polynucleotides of interest.

[0203] In some embodiments, the genomically modified iPSCs comprise one or more exogenous polynucleotides encoding proteins selected from safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutically active proteins and peptides, potential drug targets; or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or derived cells. In some embodiments, the genomically modified iPSCs comprise insertions / deletions in one or more endogenous genes encoding proteins associated with targeting modalities, receptors, signaling molecules, transcription factors, potential drug targets; control and regulation of immune responses; or proteins that inhibit engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or derived cells. In some embodiments, the genomically modified iPSCs comprise one or more exogenous polynucleotides and further comprise insertions / deletions in one or more endogenous genes. In some embodiments, the insertions / deletions in the endogenous genes result in disruption of gene expression. In some embodiments, the insertion / deletion in the endogenous gene results in a knockout of the edited gene. In some embodiments, the insertion / deletion in the endogenous gene results in a knockdown of the edited gene. In some embodiments, the genomically modified iPSCs comprising one or more exogenous polynucleotides at a selected site may further comprise one or more targeted edits comprising an insertion / deletion at a selected site. In some embodiments, the insertion / deletion is in one or more endogenous genes associated with controlling and mediating an immune response. In some embodiments, the insertion / deletion is in one or more endogenous checkpoint genes. In some embodiments, the insertion / deletion is in one or more endogenous T cell receptor genes. In some embodiments, the insertion / deletion is in one or more endogenous MHC class I inhibitory genes. In some embodiments, the insertion / deletion is in one or more endogenous genes associated with the major histocompatibility complex.In some embodiments, the insertion / deletion is in one or more endogenous genes, including but not limited to B2M, PD1, TAP1, TAP2, tapasin, TCR genes. In one embodiment, the genomically modified iPSCs comprising one or more exogenous polynucleotides at a selected site further comprise a targeted edit in a gene encoding B2M (beta-2-microglobulin). In one embodiment, the genomically modified iPSCs comprising a targeted edit in the B2M gene are B2M null or low expressing and HLA-I deficient.

[0204] In some embodiments, the genomically modified iPSCs comprise at least one suicide gene integrated at a selected site. In some embodiments, the genomically modified iPSCs comprise at least two suicide genes, each integrated at the same or different integration sites. In some embodiments, the exogenous polynucleotide is driven by one or more promoters selected from the group consisting of a constitutive promoter, an inducible promoter, a time-specific promoter, and a tissue-type or cell-type specific promoter. In some embodiments, the genomically modified iPSCs comprise one or more suicide genes at a selected integration site and further comprise an insertion / deletion in one or more selected endogenous genes. As described herein, non-pluripotent cells with a desired pattern of multiple targeted edits are suitable for reprogramming using the culture platform of Table 1 to obtain derived genomically modified iPSCs. In some embodiments, genomic modification of non-pluripotent cells for targeted integration or targeted insertion / deletion occurs simultaneously with reprogramming of the non-pluripotent cells, thereby obtaining genomically modified iPSCs in a robust, efficient, accurate, and reliable manner.

[0205] The present invention further provides non-pluripotent cells derived from said genomically modified iPSCs, wherein said genomically modified iPSCs are obtained through targeted editing of iPSCs or through sequential (first obtaining reprogrammed iPSCs followed by genomically modified; or first obtaining genomically modified non-pluripotent cells followed by reprogramming) or simultaneous (simultaneous reprogramming and genomically modified of the same pool of cells) reprogramming of genomically modified non-pluripotent cells with site-specific integration or insertion / deletion. In some embodiments, said genomically modified iPSC-derived non-pluripotent cells are progenitor cells or fully differentiated cells. In some embodiments, said genomically modified iPSC-derived cells are mesoderm cells, CD34 cells, hemogenic endothelial cells, hematopoietic stem or progenitor cells, hematopoietic pluripotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, or B cells.

[0206] The present invention also provides a composition for obtaining genomically modified iPSCs. In one embodiment, the composition comprises: a genomically modified non-pluripotent cell; one or more reprogramming factors; and a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor for reprogramming the genomically modified non-pluripotent cell into a genomically modified iPSC comprising the same targeted edit. In some embodiments of the composition, the genomically modified non-pluripotent cell for reprogramming comprises one or more polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutically active proteins and peptides, drug target candidates, or proteins that promote transplantation, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or derived cells, wherein the one or more polynucleotides are driven by one or more promoters selected from the group consisting of constitutive promoters, inducible promoters, time-specific promoters, and tissue- or cell-type specific promoters. In some embodiments, the one or more polynucleotides are included in different constructs capable of targeted integration at the same or different selected sites. In some embodiments, the one or more selected sites include AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria for a genomic safe harbor.

[0207] In some other embodiments, the composition for obtaining genomically modified iPSCs comprises a non-pluripotent cell, one or more site-specific endonucleases, one or more reprogramming factors; and a combination of TGFβ receptor / ALK inhibitors, MEK inhibitors, GSK3 inhibitors and / or ROCK inhibitors, and optionally one or more constructs comprising a polynucleotide operably linked to homology arms for targeted integration, the composition being useful for simultaneous genomic modification and reprogramming of a non-pluripotent cell into a genomically modified iPSC comprising targeted integration and / or insertion / deletion at a selected site. In some embodiments, the different constructs and / or insertion / deletion are introduced into the non-pluripotent cell simultaneously or sequentially with the reprogramming factors. In some embodiments, the different constructs and / or insertion / deletion are introduced into the non-pluripotent cell during reprogramming, such that genomic modification and reprogramming occur simultaneously or concurrently.

[0208] In some other embodiments, the composition for obtaining genomically modified iPSCs comprises a non-pluripotent cell, one or more reprogramming factors; and a combination of TGFβ receptor / ALK inhibitor, MEK inhibitor, GSK3 inhibitor and ROCK inhibitor, and one or more constructs comprising a polynucleotide operably linked to homology arms for targeted integration, the composition is useful for simultaneous genomic modification and reprogramming of a non-pluripotent cell into a genomically modified iPSC comprising targeted integration and / or insertion / deletion at a selected site. In some embodiments, the different constructs and / or insertion / deletion are introduced into the non-pluripotent cell simultaneously or sequentially with the reprogramming factors. In some embodiments, the different constructs and / or insertion / deletion are introduced into the non-pluripotent cell during reprogramming, such that genomic modification and reprogramming occur simultaneously or in parallel.

[0209] Further, a composition for maintaining genomically modified iPSC obtained from reprogramming genomically modified non-pluripotent cell is also provided herein.In one embodiment, said composition comprises genomically modified iPSC reprogrammed from genomically modified non-pluripotent cell; and a combination of MEK inhibitor, GSK3 inhibitor and ROCK inhibitor.In one embodiment, said iPSC obtained from reprogramming retains pluripotency and target editing during long-term passage and proliferation.

[0210] Also provided are compositions comprising genomically modified iPSCs and a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor and a ROCK inhibitor, wherein said genomically modified iPSCs are obtained from: (a) reprogramming of genomically modified non-pluripotent cells, wherein the resulting iPSCs contain the same target edits contained in said genomically modified non-pluripotent cells; or (b) genomically modifying a clonal iPSC or iPSC pool by incorporating one or more target edits at selected sites; or (c) genomically modifying a pool of non-pluripotent cells undergoing reprogramming by introduction of one or more target edits at selected sites into said pool of non-pluripotent cells being contacted with one or more reprogramming factors and optionally a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and / or a ROCK inhibitor. In some embodiments, the genomically modified iPSCs comprise one or more polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutically active proteins and peptides, potential drug targets; or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or their derived cells reprogrammed from non-pluripotent cells. In some embodiments, the genomically modified iPSCs comprise an insertion / deletion in one or more endogenous genes encoding proteins associated with targeting modalities, receptors, signaling molecules, transcription factors, potential drug targets; control and regulation of immune responses; or proteins that inhibit engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or their derived cells. In some embodiments, the genomically modified iPSCs comprise one or more exogenous polynucleotides and further comprise an insertion / deletion in one or more endogenous genes.

[0211] V. Therapeutic Applications of Genetically Modified iPSCs and Derived Immune Cells with Functional Modalities The invention provides compositions comprising genetically modified iPSCs and / or isolated populations or subpopulations of immune cells derived from said iPSCs using the disclosed methods and compositions, said immune cells being genetically modified and suitable for cell-based adoptive therapy. In one embodiment, said isolated population or subpopulation of genetically modified immune cells comprises iPSC-derived HSC cells. In one embodiment, said isolated population or subpopulation of genetically modified immune cells comprises iPSC-derived HSC cells. In one embodiment, said isolated population or subpopulation of genetically modified immune cells comprises iPSC-derived pro-T cells or T cells. In one embodiment, said isolated population or subpopulation of genetically modified immune cells comprises iPSC-derived pro-NK cells or NK cells. In some embodiments, said iPSC-derived genetically modified immune cells are further conditioned ex vivo for improved therapeutic potential. In one embodiment, the isolated population or subpopulation of genetically modified immune cells derived from iPSCs has an increased number or percentage of naive T cells, stem cell memory T cells, and / or central memory T cells. In one embodiment, the isolated population or subpopulation of genetically modified immune cells derived from iPSCs has an increased number or percentage of type I NKT cells. In another embodiment, the isolated population or subpopulation of genetically modified immune cells derived from iPSCs has an increased number or percentage of acquired NK cells. In some embodiments, the isolated population or subpopulation of genetically modified CD34 cells, HSC cells, T cells, or NK cells derived from iPSCs is allogeneic. In some other embodiments, the isolated population or subpopulation of genetically modified CD34 cells, HSC cells, T cells, or NK cells derived from iPSCs is autologous.

[0212] In some embodiments, iPSCs for differentiation contain a genetic imprint that conveys a desired therapeutic property in an effector cell, and said genetic imprint is retained and functional in differentiated hematopoietic cells derived from said iPSCs.

[0213] In some embodiments, the genetic imprint of the pluripotent stem cell comprises (i) one or more genetically modified modalities obtained through intragenomic insertions, deletions or substitutions in the genome of the pluripotent cell during or after reprogramming from a non-pluripotent cell to an iPSC; or (ii) one or more retainable therapeutic properties of source-specific immune cells that are donor-specific, disease-specific, or therapeutic response-specific, and the pluripotent cell was reprogrammed from the source-specific immune cell, and the iPSC retains the source therapeutic property, and the source therapeutic property is also contained in hematopoietic cells derived from the iPSC.

[0214] In some embodiments, the genetically modified modalities include one or more of the following: safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmacologic active proteins and peptides, drug target candidates; or proteins that promote the engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and regulation, and / or survival of iPSCs or derived cells. In some other embodiments, the genetically modified modalities include one or more of the following: (i) deletion or reduction in expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and any genes within the chromosome 6p21 region; (ii) introduction or increased expression of HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, CAR, TCR, Fc receptor, or a surface triggering receptor for binding to a bispecific or multispecific or universal engager.

[0215] In yet some further embodiments, the hematopoietic cells comprise therapeutic properties of source-specific immune cells associated with one or more of the following: (i) expression of antigen targeting receptors; (ii) HLA presentation or lack thereof; (iii) tolerance to the tumor microenvironment; (iv) induction of bystander immune cells and immunomodulation; (iv) improved target specificity with reduced extratumoral effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, persistence, and cytotoxicity.

[0216] In some embodiments, the iPSCs and derived hematopoietic cells contain one or more of null or low expression of B2M, null or low expression of HLA-E / G, PDL1, A2AR, CD47, LAG3, null or low expression of TIM3, null or low expression of TAP1, null or low expression of TAP2, null or low expression of tapasin, null or low expression of NLRC5, null or low expression of PD1, null or low expression of RFKANK, null or low expression of CIITA, null or low expression of RFX5, and null or low expression of RFXAP. These cells with modified HLA class I and / or modified HLA class II show improved in vivo persistence due to increased resistance to immune detection. Moreover, such cells can circumvent HLA matching requirements in adoptive cell therapy, thus providing a source of universal, off-the-shelf therapeutic regimens.

[0217] In some embodiments, the iPSCs and derived hematopoietic cells are characterized by the expression of hnCD16 (high affinity non-cleavable CD16), HLA-E, HLA-G, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, CAR, or TCR. Such cells have enhanced immune effector capabilities.

[0218] In some embodiments, the iPSCs and their derived hematopoietic cells are antigen-specific.

[0219] By introducing the immune cells of the present invention into a subject suitable for adoptive cell therapy, various diseases may be ameliorated. Examples of diseases include, but are not limited to, various autoimmune disorders including alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), certain types of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, certain types of myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic erythroderma, lupus erythematosus, certain types of thyroiditis, certain types of uveitis, vitiligo, (Wegener's) granulomatosis with polyangiitis; acute leukemia, and hematopoietic malignancies, including but not limited to diffuse leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome; solid tumors, including but not limited to tumors of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testis, bladder, kidney, head, neck, stomach, cervix, rectum, larynx, or esophagus; and infectious diseases, including but not limited to HIV (human immunodeficiency virus)-associated disorders, RSV (respiratory syncytial virus)-associated disorders, EBV (Epstein-Barr virus)-associated disorders, CMV (cytomegalovirus)-associated disorders, adenovirus-associated disorders, and BK polyomavirus-associated disorders.

[0220] Certain embodiments of the present invention are directed to methods of treating a subject in need of treatment by administering to the subject a composition comprising any of the cells described herein. In certain embodiments, the terms "treat", "treatment" and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of a complete or partial prevention of the disease, and / or therapeutic, in terms of a partial or complete cure of the disease and / or adverse effects caused by the disease. "Treatment", as used herein, encompasses any treatment of a disease in a mammal, including preventing the disease from arising in a subject susceptible to, but not yet diagnosed as suffering from, the disease; inhibiting the disease, i.e., arresting its development; or relieving the disease, i.e., causing regression of the disease. The therapeutic agent or composition may be administered before, during, or after the onset of the disease or injury. Treatment of ongoing disease, stabilizing or reducing undesirable clinical symptoms in the patient, is particularly important.

[0221] In certain embodiments, the subject has a disease, condition, and / or injury that can be treated, ameliorated, and / or improved by cell therapy. In some embodiments, it is contemplated that the subject in need of cell therapy is a subject with an injury, disease, or condition, whereby cell therapy (e.g., a therapy in which cellular material is administered to the subject) can treat, ameliorate, improve, and / or reduce the severity of at least one symptom associated with the injury, disease, or condition. Certain embodiments contemplate that the subject in need of cell therapy includes, but is not limited to, bone marrow or stem cell transplant candidates, subjects who have undergone chemotherapy or radiation therapy, subjects suffering from or at risk of suffering from a hyperproliferative disease or cancer (e.g., a hematopoietic hyperproliferative disease or cancer), subjects having or at risk of developing a tumor (e.g., a solid tumor), subjects suffering from or at risk of suffering from a viral infection or a disease associated with a viral infection.

[0222] Thus, the present invention further provides a pharmaceutical composition comprising a pluripotent cell-derived hematopoietic cell produced by the methods and compositions disclosed herein, and further comprising a pharma- ceutically acceptable medium. In one embodiment, the pharmaceutical composition comprises a pluripotent cell-derived T cell produced by the methods and compositions disclosed herein. In one embodiment, the pharmaceutical composition comprises a pluripotent cell-derived NK cell produced by the methods and compositions disclosed herein. In one embodiment, the pharmaceutical composition comprises a pluripotent cell-derived CD34+ HE cell produced by the methods and compositions disclosed herein. In one embodiment, the pharmaceutical composition comprises a pluripotent cell-derived HSC cell produced by the methods and compositions disclosed herein.

[0223] In addition, the present invention provides therapeutic uses of the above pharmaceutical compositions by introducing the composition into a subject suitable for adoptive cell therapy having an autoimmune disorder; a hematopoietic tumor; a solid tumor; or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0224] The isolated pluripotent stem cell-derived hematopoietic cells may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% T cells, NK cells, NKT cells, pro-T cells, pro-NK cells, CD34+HE cells, or HSCs. In some embodiments, the isolated pluripotent stem cell-derived hematopoietic cells comprise about 95% to about 100% T cells, NK cells, NKT cells, pro-T cells, pro-NK cells, CD34+HE cells, or HSCs. In some embodiments, the invention provides pharmaceutical compositions comprising purified T cells, NK cells, NKT cells, CD34+HE cells, pro-T cells, pro-NK cells, or HSCs, such as compositions comprising an isolated population of about 95% T cells, NK cells, NKT cells, pro-T cells, pro-NK cells, CD34+HE cells, or HSCs, for treating a subject in need of cell therapy.

[0225] In some embodiments, the pharmaceutical composition comprises an isolated population of pluripotent stem cell derived hematopoietic cells that comprises less than about 0.1%, less than about 0.5%, less than about 1%, less than about 2%, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, or less than about 30% of iPSC-derived T cells, NK cells, NKT cells, pro-T cells, pro-NK cells, CD34+HE cells, or HSC. The isolated population of derived hematopoietic cells may in some embodiments comprise more than about 0.1%, more than about 0.5%, more than about 1%, more than about 2%, more than about 5%, more than about 10%, more than about 15%, more than about 20%, more than about 25%, or more than about 30% of T cells, NK cells, NKT cells, pro-T cells, pro-NK cells, CD34+HE cells, or HSC. In other embodiments, the isolated population of derived hematopoietic cells may comprise about 0.1% to about 1%, about 1% to about 3%, about 3% to about 5%, about 10% to about 15%, about 15% to about 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 60% to 70%, about 70% to 80%, about 80% to 90%, about 90% to 95%, or about 95% to about 100% T cells, NK cells, NKT cells, pro-T cells, pro-NK cells, CD34+ HE cells or HSCs.

[0226] In certain embodiments, the derived hematopoietic cells may comprise about 0.1%, about 1%, about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% T cells, NK cells, NKT cells, pro-T cells, pro-NK cells, CD34+ HE cells or HSCs.

[0227] As will be understood by those skilled in the art, both autologous and allogeneic immune cells can be used for cell therapy. Autologous cell therapy may have reduced infection, low probability of GvHD, and fast immune reconstitution. Allogeneic cell therapy may have immune-mediated graft-versus-malaria (GVM) effect and low recurrence rate. Based on the specific condition of the patient or subject who needs cell therapy, those skilled in the art can determine which specific type of therapy to administer.

[0228] In certain embodiments, the derived hematopoietic cells of the pharmaceutical composition of the invention are allogeneic to the subject. In certain embodiments, the derived hematopoietic cells of the pharmaceutical preparation of the invention are autologous to the subject. In an autologous transplant, the isolated population of derived hematopoietic cells is fully or partially HLA-matched to the patient. In another embodiment, the derived hematopoietic cells are not HLA-matched to the subject.

[0229] In some embodiments, the number of derived hematopoietic cells in the pharmaceutical composition is at least 0.1×10 5 Cells, at least 0.5 x 10 5 Cells, at least 1 x 10 5 Cells, at least 5 x 10 5 Cells, at least 10 x 10 5 Cells, at least 0.5 x 10 6 Cells, at least 0.75 x 10 6 Cells, at least 1 x 10 6 Cells, at least 1.25 x 10 6 Cells, at least 1.5 x 10 6 Cells, at least 1.75 x 10 6 Cells, at least 2 x 10 6 Cells, at least 2.5 x 10 6 Cells, at least 3 x 10 6 Cells, at least 4 x 10 6 Cells, at least 5 x 10 6 Cells, at least 10 x 10 6 Cells, at least 15 x 10 6 Cells, at least 20 x 106 Cells, at least 25 x 10 6 cells, or at least 30 × 10 6 It is a cell.

[0230] In some embodiments, the number of derived hematopoietic cells in the pharmaceutical composition is about 0.1×10 5 Cells ~ approx. 10×10 5 Cells; approx. 0.5×10 6 Cells ~ approx. 5 x 10 6 Cells; approx. 1 x 10 6 cells ~ approx. 3 x 10 6 Cells; approx. 1.5 x 10 6 Cells ~ approx. 2.5×10 6 cells; or approximately 2 x 10 6 Cells ~ approx. 2.5×10 6 It is a cell.

[0231] In some embodiments, the number of derived hematopoietic cells in the pharmaceutical composition is about 1×10 6 cells ~ approx. 3 x 10 6 Cells; approx. 1.0×10 6 Cells ~ approx. 5 x 10 6 Cells; approx. 1.0×10 6 Cells ~ approx. 10×10 6 cells, approximately 10 x 10 6 Cells ~ approx. 20×10 6 cells, approximately 10 x 10 6 Cells ~ approx. 30 x 10 6 cells, or approximately 20 x 10 6 Cells ~ approx. 30 x 10 6 It is a cell.

[0232] In some other embodiments, the number of derived hematopoietic cells in the pharmaceutical composition is about 1×10 6 Cells ~ approx. 30 x 10 6 Cells; approx. 1.0×10 6 Cells ~ approx. 20×10 6 Cells; approx. 1.0×10 6 Cells ~ approx. 10×10 6 cells, approximately 2.0 x 10 6 Cells ~ approx. 30 x 10 6 cells, approximately 2.0 x 10 6 Cells ~ approx. 20×10 6cells, or approximately 2.0 x 10 6 Cells ~ approx. 10×10 6 It is a cell.

[0233] In yet another embodiment, the number of derived hematopoietic cells in the pharmaceutical composition is about 1×10 6 cells, approximately 2 x 10 6 cells, approximately 5 x 10 6 cells, approximately 7 x 10 6 cells, approximately 10 x 10 6 cells, approximately 15 x 10 6 cells, approximately 17 x 10 6 cells, approximately 20 x 10 6 Cells approximately 25×10 6 cells, or approximately 30 x 10 6 It is a cell.

[0234] In one embodiment, the number of derived hematopoietic cells in the pharmaceutical composition is the number of immune cells in a partial or complete umbilical cord blood, i.e., at least 0.1×10 5 At least 0.5 x 10 cells / kg body weight 5 At least 1 x 10 cells / kg body weight 5 At least 5 x 10 cells / kg body weight 5 Cells / kg body weight, at least 10 x 10 5 At least 0.5 x 10 cells / kg body weight 6 At least 0.75 x 10 cells / kg body weight 6 At least 1 x 10 cells / kg body weight 6 At least 1.25 x 10 cells / kg body weight 6 At least 1.5 x 10 cells / kg body weight 6 At least 1.75 x 10 cells / kg body weight 6 At least 2 x 10 cells / kg body weight 6 At least 2.5 x 10 cells / kg body weight 6 At least 3 x 10 cells / kg body weight 6 At least 4 x 10 cells / kg body weight 6 At least 5 x 10 cells / kg body weight 6 Cells / kg body weight, at least 10 x 10 6 At least 15 x 10 cells / kg body weight 6At least 20 x 10 cells / kg body weight 6 At least 25 x 10 cells / kg body weight 6 cells / kg body weight, or at least 30 x 10 6 Cells / kg body weight.

[0235] The derived genetically modified hematopoietic cells provided by the present invention can be administered to a subject without ex vivo or in vitro expansion prior to administration. In certain embodiments, an isolated population of derived genetically modified hematopoietic cells is conditioned and treated ex vivo with one or more agents to obtain immune cells with improved therapeutic potential. The conditioned population of derived genetically modified hematopoietic cells can be washed to remove the treatment agents, and the improved population is administered to a patient without further in vitro expansion of the population.

[0236] In other embodiments, the invention provides isolated populations of derived genetically modified hematopoietic cells that are expanded prior to modulation with one or more agents. The isolated populations of derived hematopoietic cells can be recombinantly produced to express a TCR, CAR or other protein using the methods and compositions provided herein.

[0237] With respect to genetically modified derived hematopoietic cells expressing a recombinant TCR or CAR, either before or after the cells have been genetically modified, the cells may be modified using methods described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; Activation and proliferation can be achieved using the methods described in U.S. Patent Application Publication Nos. 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0238] In certain embodiments, the primary and costimulatory signals for the derived genetically modified hematopoietic cells can be provided by a variety of protocols. For example, the agents providing each signal can be in solution or bound to a surface. If bound to a surface, the agents can be bound to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one agent can be bound to a surface and the other in solution. In one embodiment, the agent providing the costimulatory signal can be bound to a cell surface and the agent providing the primary activation signal can be in solution or bound to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents can be in soluble form and then cross-linked to a surface, such as a cell expressing an Fc receptor or an antibody or other binding agent that binds to the agents, e.g., artificial antigen presenting cells (aAPCs) disclosed in US Patent Publication Nos. 20040101519 and 20060034810, are contemplated for use in activating and expanding T lymphocytes of the present invention.

[0239] The compositions comprising the population of derived hematopoietic cells of the present invention can be sterile and suitable for administration to a human patient and can be in an administrable state (i.e., can be administered without further processing). In some embodiments, the therapeutic composition is in a state ready for infusion into a patient. An administrable cell-based composition refers to a composition that does not require any additional processing or manipulation prior to implantation or administration into a subject.

[0240] A sterile, therapeutically acceptable composition suitable for administration to a patient may contain one or more pharma- ceutically acceptable carriers (additives) and / or diluents (e.g., a pharma- ceutically acceptable medium, e.g., cell culture medium), or other pharma- ceutically acceptable components. Pharmaceutically acceptable carriers and / or diluents are determined, in part, by the particular composition being administered and the particular method used to administer the therapeutic composition. Thus, there are a wide variety of suitable formulations of the therapeutic compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed. 1985, the disclosure of which is incorporated herein by reference in its entirety).

[0241] In certain embodiments, the therapeutic cell composition comprising the isolated population of derived genetically modified hematopoietic cells also comprises a pharma- ceutically acceptable cell culture medium. The therapeutic compositions comprising the derived genetically modified hematopoietic cell populations disclosed herein can be administered enterally or parenterally, alone or together with other suitable compounds to achieve the desired therapeutic purpose.

[0242] The pharma- ceutically acceptable carriers and / or diluents should be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the human subject being treated, and should also maintain or increase the stability of the therapeutic composition.

[0243] Such carrier solutions may also contain buffers, diluents and other suitable additives. A buffer refers to a solution or liquid that has a chemical makeup that neutralizes acids or bases without significantly changing the pH. Examples of buffers contemplated by the present invention include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), and saline (0.9% NaCl).

[0244] These pharma- ceutically acceptable carriers and / or diluents may be present in an amount sufficient to maintain a pH of the therapeutic composition of about 3 to about 10. That is, the buffering agent may be as much as about 5% of the total composition on a weight-by-weight basis. Electrolytes such as, but not limited to, sodium chloride and potassium chloride may also be included in the therapeutic composition. In one aspect, the pH of the therapeutic composition is in the range of about 4 to about 10. Alternatively, the pH of the therapeutic composition is in the range of about 5 to about 9, about 6 to about 9, or about 6.5 to about 8. In another embodiment, the therapeutic composition comprises a buffer having a pH within one of the above pH ranges. In another embodiment, the therapeutic composition has a pH of about 7. Alternatively, the therapeutic composition has a pH within the range of about 6.8 to about 7.4. In yet another embodiment, the therapeutic composition has a pH of about 7.4.

[0245] The sterile composition of the present invention can be a sterile solution or a sterile suspension in a non-toxic pharma- ceutically acceptable medium. A suspension can refer to a non-adherent state in which cells are not attached to a solid phase. For example, cells maintained in a suspension can be stirred and are not attached to a support such as a culture dish.

[0246] A suspension is a dispersion (mixture) of a finely divided chemical species combined with another chemical species, the former being so finely divided and mixed that it does not settle rapidly. A suspension can be prepared using a vehicle such as a liquid medium that contains a solution. In some embodiments, the therapeutic composition of the present invention is a suspension in which stem cells and / or progenitor cells are dispersed in an acceptable liquid medium or solution (e.g., saline or serum-free medium) and are not attached to a solid phase. In daily life, the most common suspension is a suspension of solids in liquid water. Acceptable diluents (e.g., vehicles and solvents) that can be used include water, Ringer's solution, isotonic sodium chloride (saline) solution, and serum-free cell culture medium. In some embodiments, hypertonic solutions are used to make suspensions. In addition, sterile, non-volatile oils are conventionally used as solvents or suspending media. In parenteral applications, particularly suitable vehicles consist of solutions, preferably oily or aqueous solutions, and suspensions, emulsions, or implants. Aqueous suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. In some embodiments, the infusion solution is isotonic with the target tissue. In some embodiments, the infusion solution is hypertonic with respect to the target tissue.

[0247] The pharma- ceutically acceptable carriers, diluents, and other components of the pharmaceutical compositions of the present invention ready for administration are obtained from US pharmaceutical grade reagents that allow the therapeutic compositions to be used in clinical therapy. Typically, these final reagents, including any media, solutions, or other pharma- ceutically acceptable carriers and / or diluents, are sterilized by methods conventional in the art, such as sterile filtration, and are tested for various undesirable contaminants, such as mycoplasma, endotoxin, or viral contamination, prior to use. The pharma- ceutical acceptable carriers, in one embodiment, are substantially free of natural proteins of human or animal origin and are suitable for preserving the cell population of the pharmaceutical composition, including hematopoietic stem and progenitor cells. The pharmaceutical compositions are intended to be administered to human patients, and therefore are substantially free of cell culture medium components, such as bovine serum albumin, horse serum, and fetal bovine serum.

[0248] The invention also provides, in part, the use of the pharma- ceutically acceptable cell culture medium, particularly the composition and / or medium, of the invention. Such compositions are suitable for administration to human subjects. In general, any medium that supports the maintenance, growth, and / or health of the derived hematopoietic cells of the invention is suitable for use as a pharmaceutical cell culture medium. In certain embodiments, the pharma-ceutically acceptable cell culture medium is serum-free and / or feeder-free.

[0249] The pharmaceutical composition may comprise a serum-free medium suitable for the storage of an isolated population of regulated derived hematopoietic cells. In various embodiments, the serum-free medium is animal-free and may optionally be protein-free. Optionally, the medium may contain biopharmaceutical acceptable recombinant proteins. Animal-free medium refers to a medium whose components are derived from non-animal sources. Recombinant proteins replace the animal proteins originally contained in the animal-free medium, and these nutrients are obtained from synthetic, plant or microbial sources. Protein-free medium, in contrast, is defined as being substantially free of protein.

[0250] Those of skill in the art will appreciate that the above examples of media are illustrative and not limiting of media formulations suitable for use in the present invention, and that there are many suitable media known and available to those of skill in the art.

[0251] The pharmaceutical composition is substantially free of mycoplasma, endotoxin, and microbial contamination. In certain embodiments, the therapeutic composition contains less than about 10, about 5, about 4, about 3, about 2, about 1, about 0.1, or about 0.05 μg / ml of bovine serum albumin.

[0252] With respect to mycoplasma contamination and microbial contamination, "substantially free of" as used herein means a negative reading in a commonly accepted test known to those skilled in the art. For example, mycoplasma contamination is determined by subculturing a sample of the therapeutic composition in broth medium with appropriate positive and negative controls and plating on agar plates at 37°C on days 1, 3, 7, and 14. The appearance of the sample is compared to that of the positive and negative controls under a microscope at 100x. Additionally, an inoculum of indicator cell culture is incubated for 3 and 5 days and examined for the presence or absence of mycoplasma by epifluorescence microscopy using a DNA-binding fluorescent dye at 600x. If the agar and / or broth medium procedures and the indicator cell culture procedures do not show any evidence of mycoplasma contamination, the sample is deemed satisfactory.

[0253] Organic solvent or suitable organic solvent generally refers to a liquid or gas containing carbon that dissolves solid, liquid, or gaseous solutes to produce a solution. A suitable organic solvent is an organic solvent that is suitable for ex vivo administration to mammalian cells or incubation with mammalian cells, and may also be suitable for in vivo administration to a subject, such as by having minimal toxicity or other inhibitory effects under ex vivo conditions (e.g., cell culture) or in vivo at the selected concentration, during the incubation or administration time. A suitable organic solvent should also be suitable for storage stability and handling of the agent described herein.

[0254] Examples of suitable organic solvents include, but are not limited to, dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME), and dimethylacetamide, including mixtures or combinations thereof. In certain embodiments, the composition or organic solvent is substantially free of methyl acetate, meaning that only trace amounts of methyl acetate should be present in the composition or solvent, preferably in undetectable amounts (e.g., as measured by high pressure liquid chromatography (HPLC), gas chromatography (GC), etc.).

[0255] An endotoxin-free container or composition means that the container or composition contains at most trace amounts of endotoxin (i.e., amounts that have no adverse physiological effects on a subject) or an undetectable amount of endotoxin. "Substantially endotoxin-free" means less endotoxin per dose of cells than the 5 EU / kg body weight / day total endotoxin allowed by the FDA for biologics, or 350 EU / total dose of cells for an average 70 kg person.

[0256] In one embodiment, the endotoxin-free container and / or composition is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% endotoxin-free. Endotoxins are toxins associated with certain bacteria, typically gram-negative bacteria, although endotoxins can be present in gram-positive bacteria such as Listeria monocytogenes. The most common endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS), which are present in the outer membrane of various gram-negative bacteria and represent a central pathogenic property in the disease-causing ability of these bacteria. Small amounts of endotoxins can cause fever, lowering of blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects in humans. Thus, it is often desirable to remove most or all trace amounts of endotoxins from the formulation container, since even small amounts can cause adverse effects in humans. Endotoxins can be removed from the containers using methods known in the art, for example, the containers can be washed in a HEPA sterilizing washer with endotoxin-free water, depyrogenated at 250°C, and clean packaged in a HEPA sterilizing workstation located in a Class 100 / 10 cleanroom (e.g., a Class 100 cleanroom has no more than 100 particles larger than 0.5 microns in one cubic foot of air). EXAMPLES

[0257] The following examples are offered by way of illustration and not by way of limitation.

[0258] Example 1: Materials and Methods To efficiently select and test suicide systems under the control of various promoters in combination with various safe harbor loci integration strategies, Applicant's proprietary hiPSC platform was used, which allows single cell passaging and high-throughput 96-well plate-based flow cytometry sorting, and allows the derivation of clonal hiPSCs by single or multiple gene modulation.

[0259] Maintenance of hiPSCs in small molecule culture medium: Once cultures reached 75%–90% confluency, hiPSCs were routinely passaged as single cells. For single cell dissociation, hiPSCs were washed once with PBS (Mediatech) and treated with Accutase (Millipore) for 3–5 min at 37°C, followed by pipetting to ensure single cell dissociation. This single cell suspension was then mixed with equal volumes of regular medium, centrifuged at 225 × g for 4 min, resuspended in FMM, and plated onto Matrigel-coated surfaces. Cells were routinely passaged 1:6–1:8 and transferred to tissue culture plates precoated with Matrigel for 2–4 h at 37°C and fed with FMM every 2–3 days. Cell cultures were maintained in a humidified incubator set at 37°C and 5% CO2.

[0260] ZFN- and CRISPR-mediated human iPSC genome editing for targeted insertion of inducible caspase-9 into the genomic safe harbor: 2×10 for ZFN-mediated genome editing 6 iPSCs were transfected with a mixture of 2.5ug ZFN-L (FTV893; SEQ ID NOs: 2 and 4), 2.5ug ZFN-R (FTV894; SEQ ID NOs: 3 and 5) and 5ug AAVS1 inducible caspase 9 donor construct (FTV895, FTV930, FTV921, FTV931, FTV932, FTV952 or FTV953; inducible caspase 9 SEQ ID NOs: 7 and 8) plasmid DNA. For CRISPR-mediated genome editing, 2x10 6iPSCs were transfected with a mixture of 5ug ROSA26-gRNA / caspase 9 (FTV922; SEQ ID NO: 6) and 5ug ROSA26 inducible caspase 9 donor construct (FTV955 or FTV956). Transfection was performed using the Neon Transfection System (Life Technologies) with the parameters 1500V, 10ms, 3 pulses. Transfection efficiency was measured 2 or 3 days after transfection using flow cytometry if the plasmid contained GFP and / or RFP expression cassettes driven by artificial promoters. Target cells were selected 4 days after transfection by adding puromycin to the medium at a concentration of 0.1ug / ml for the first 7 days and 0.2ug / ml after 7 days. During puromycin selection, cells were passaged on fresh matrigel-coated wells on day 10. From day 16 of puromycin selection, viable cells were analyzed by flow cytometry for the percentage of GFP+ iPS cells.

[0261] Bulk and clonal selection of genome-edited iPSCs: For iPSCs targeted by AAVS1 EF1α or CAG promoter-driven inducible caspase 9-2A-GFP via ZFNs, bulk and clonal sorts of GFP+SSEA4+TRA181+iPSCs were performed after 20 days of puromycin selection. Pools of targeted iPSCs dissociated into single cells were resuspended in chilled staining buffer containing Hanks Balanced Salt Solution (Mediatech), 4% fetal bovine serum (Invitrogen), 1x penicillin / streptomycin (Mediatech) and 10 mM HEPES (Mediatech), freshly made for optimal performance. Conjugated primary antibodies, including SSEA4-PE, TRA181-Alexa Fluor-647 (BD Biosciences), were added to the cell solution and incubated on ice for 15 min. All antibodies were diluted at 10 67 μL per cell was used in 100 μL staining buffer. Cells were washed once with staining buffer, spun down at 225g for 4 min, resuspended in staining buffer containing 10 μM Thiazovivn, and kept on ice for flow cytometry sorting. Flow cytometry sorting was performed on a FACS Aria II (BD Biosciences). For bulk sorting, GFP+SSEA4+TRA181+ cells were gated and sorted into 15 ml standard tubes filled with 7 ml FMM. For clonal sorting, sorted cells were ejected directly into 96-well plates using a 100 μM nozzle at a concentration of 3 events / well. Each well was pre-filled with 200 μL FMM supplemented with 5 μg / mL fibronectin and 1× penicillin / streptomycin (Mediatech) and pre-coated with 5× Matrigel overnight. Pre-coating with 5x Matrigel involves adding an aliquot of Matrigel to 5mL of DMEM / F12, then incubating overnight at 4°C, resuspending appropriately, and adding 50μL / well to a 96-well plate followed by overnight incubation at 37°C. This 5x Matrigel is aspirated immediately prior to adding medium to each well. After sorting is complete, the 96-well plate is centrifuged at 225g for 1-2 minutes and then incubated. The plate is left undisturbed for 7 days. On day 7, 150μL of medium is removed from each well and replaced with 100μL of FMM. On day 10 after sorting, an additional 100μL of FMM is added back to the wells. Colony formation was observed as early as day 2, with most colonies growing between days 7-10 after sorting. For the first passage, wells were washed with PBS and detached with 30μL of Accutase for approximately 10 minutes at 37°C. The need for extended Accutase treatment represents clumping of colonies that have remained untreated in culture for a long period of time. Once the cells appear to have detached, add 200 μL of FMM to each well and pipette several times to break apart the colonies. The detached colonies are transferred to separate wells of a 96-well plate pre-coated with 5× Matrigel, then centrifuged at 225 g for 2 minutes and incubated.This 1:1 passaging was done to allow the initial colonies to spread laterally before expanding. Subsequent passaging was performed routinely after 75-90% confluence with 3-5 min of Accutase treatment and 1:4-1:8 expansion in FMM in larger wells pre-coated with 1x Matrigel. Each clonal cell line was analyzed for GFP fluorescence and TRA1-81 expression levels. Clonal lines that were close to 100% GFP+ and TRA1-81+ were selected for further PCR screening and analysis. Flow cytometry analysis was performed on a Guava EasyCyte 8 HT (Millipore) and analyzed using Flowjo (FlowJo, LLC).

[0262] Inducible death of iPSCs containing inducible caspase-9 in vitro: Chemical inducer of dimerization (CID; AP1903) was purchased from Medchemexpress (Mammoth Junction, NJ). 24 hours after CID exposure, cells were harvested and stained with 7-aminoactinomycin D (7-AAD) according to the manufacturer's instructions (BD Biosciences). The percentage of 7-AAD positive cells was quantified as dead cells by flow cytometry (Guava, EMD Millipore, Billerica, MA) and analyzed with flowjo software.

[0263] To examine whether AP1903 induces complete cell death of inducible caspase-9 targeted iPSC clones, 3 wells of each targeted iPSC clone were treated with AP1903 for 48 hours, and then the wells were washed twice with PBS before adding fresh iPSC medium. The wells were allowed to recover by changing the medium every 2 days for a total of 5 days. The wells were then stained with alkaline phosphatase staining reagent (Sigma).

[0264] Tri-germ layer directed differentiation and inducible death of inducible caspase-9 targeted clones in vitro: For directed monolayer triploid differentiation, hiPSCs were seeded in FMM on Matrigel-coated wells the day before differentiation initiation (e.g., 2 × 10 for endoderm). 5 Cells / well, 5 x 10 for ectoderm 4 Cells / well, 1 x 10 for mesoderm 4 and 5×10 4Cells / well). Four replicate wells were set up for each germ layer differentiation. For endoderm differentiation, FMM medium was replaced with endoderm induction medium (RPMI-1640, ascorbic acid (50 μg / ml), monothioglycerol (450 μM), 1× glutamine, Knockout serum replacement (0.20%), activin A (100 ng / ml), CHIR99021 (Biovision) (3 μM), 1× penicillin / streptomycin). After 2 days, medium was replaced with (RPMI-1640, ascorbic acid (50 μg / ml), monothioglycerol (450 μM), 1× glutamine, Knockout serum replacement (0.50%), bFGF (5 ng / ml), activin A (100 ng / ml), 1× penicillin / streptomycin). On day 3, one well was fixed and stained for Sox17 (R&D Systems). On day 3, one well was stained with crystal violet. Two other wells were treated with AP1903 for 48 hours, then washed and replenished with fresh differentiation medium, and culture was continued for 5 days before staining with crystal violet. For mesoderm differentiation, the medium was replaced with DMEM / F12 (Media Tech) supplemented with ITS, 10 ng / ml bFGF, 20 μM forskolin, and 3 μM CHIR99021. The medium was changed every other day, and on day 4, one well was fixed and stained for αSMA (Sigma). On day 4, one well was stained with crystal violet. Two other wells were treated with AP1903 for 48 hours, then washed and replenished with fresh differentiation medium, and culture was continued for 5 days before staining with crystal violet. For ectodermal differentiation, FMM medium was replaced with neural induction medium (DMEM / F12 (Mediatech) supplemented with 1x B27 medium supplement (Life Technologies), 1x N2 medium supplement (Life Technologies), 10 μM SB431542 and 100 nM LDN-193189). Medium was changed every other day and on day 7, one well was fixed and stained for nestin (Abcam). On day 7, one well was stained with crystal violet. Two other wells were treated with AP1903 for 48 h, then washed and replenished with fresh differentiation medium and culture was continued for 5 days before staining with crystal violet.

[0265] Crystal violet staining: A 0.5% crystal violet solution was prepared by dissolving 0.5 g of crystal violet stain (Sigma) in 100 mL of deionized water. Filter and store at room temperature. For staining, wash cells twice with PBS. Replace with PBS containing 4% paraformaldehyde and leave at room temperature for 15 min. Rinse twice with PBS. Stain with 0.1% crystal violet (prepared in 10% ethanol) for 15 min at room temperature. Discard CV. Gently wash cells with deionized water from a wash bottle until the water is no longer dark. Remove deionized water and allow stained cells to dry before photographing.

[0266] In vivo induced death of teratomas derived from iPSCs engineered with inducible caspase-9: All mice were housed in the applicant's facility. All experimental protocols involving mice were approved by the applicant's Institutional Animal Care and Use Committee. All mice used in this study were 7-10 week-old female NSG mice (NOD / SCID / γ null For teratoma assays, 1–2 × 10 cells were cultured in 50% FMM medium and 50% Matrigel. 6200 μl of iPSCs were introduced subcutaneously in the dorsolateral region. Each mouse received two cell injections on each side, with iPSCs with inducible caspase 9 integrated on the left side and the unmodified parental iPSC line on the right side. Mice were administered AP1903 (125 μg / mouse) by intraperitoneal (ip) injection daily for 5 or 7 days to trigger in vivo death at the indicated time points. Mice were monitored for teratoma formation and volumetric measurements were taken starting at week 3 or as indicated. Teratomas were harvested at the indicated time points and processed for histological examination or molecular assays. AP1903 stock was prepared in DMSO (25 μg / ml). For one i.p. injection, 5 μl of stock solution was added to 200 μl of PBS and then sonicated in a water bath for 10 min. For histological examination, teratomas were harvested in PBS and fixed in 4% paraformaldehyde overnight at room temperature, then kept in 70% ethanol at room temperature for processing. Samples were submitted to the UCSD Histology Core Facility for sectioning and hematoxylin-eosin staining. Sections were examined, interpreted, and photographed using a Nikon Eclipse TS100 microscope equipped with a Nikon DS-Fi1 camera.

[0267] In vivo visualization of inducible death of transplanted luciferase-expressing iPSCs: To visualize AP1903-induced death in vivo, iPSCs with inducible caspase 9 and unmodified parental iPSCs were infected with a lentivirus (Biosettia) containing a polycistronic cassette consisting of an IRES-linked firefly luciferase gene and RFP, expressed under the control of EF1α. Infected cells were sorted for RFP+ cells and injected as described above. 2×10 6NSG mice were injected subcutaneously with inducible caspase-9-transduced iPSCs on the left side and the same number of parental iPSCs on the right side. AP1903 intraperitoneal injections were administered on the indicated days. Immediately after iPSC transplantation (day 0), engraftment and teratoma volume were monitored weekly using a Xenogen IVIS imaging system. For imaging, mice were injected intraperitoneally with D-luciferin (4 mg / mouse, Thermo Fisher) and bioluminescence was recorded 10 min later. Analysis was performed using Xenogen Living Image software.

[0268] Example 2: AAVS1 safe harbor targeted inducible caspase-9 suicide gene controlled by the CMV promoter induces heterogeneous responses when activated by AP1903 To perform high-throughput analysis of suitable integration and expression strategies in iPSCs, we selected an inducible caspase-9 suicide gene platform, where rapid caspase-9-mediated cell death can be induced by small molecule chemical inducers of dimerization such as AP1903. Note that the inducible caspase-9 gene needs to be continuously expressed at all stages of pluripotent stem cell maintenance and differentiation for small molecule induction to be effective at each developmental stage toward the end product. One hiPSC per well was used to efficiently and precisely integrate and maintain various suicide gene expression cassettes at safe harbor loci, including but not limited to the AAVS1 locus, ROSA26 locus, and H11 locus. Several integration vectors containing suicide gene expression cassettes downstream of various exogenous and endogenous promoters, including the endogenous AAVS1 or ROSA26, cytomegalovirus (CMV) promoter, elongation factor 1α (EF1α) promoter, phosphoglycerate kinase (PGK) promoter, hybrid CMV enhancer / chicken β-actin (CAG) promoter and ubiquitin C (UBC) promoter, respectively, were tested to systematically analyze and compare the activities of various suicide systems in the therapy of hiPSCs and hiPSC-derived differentiated cells.

[0269] A donor construct containing CMV-driven inducible caspase 9 (AAVS1-G1.0) was designed to target the AAVS1 locus (Figure 1A) and integrate the Casp9 suicide gene into 293T cells. This donor construct also contained the GFP and puromycin genes, whose expression is driven by the AAVS1 endogenous promoter after targeted insertion (Figure 1A). 293T cells were transfected with ZFNs specific for the AAVS1 locus and the donor construct AAVS1-G1.0. Puromycin selection was initiated 3 days after transfection and continued for 12 days. The puromycin-selected population was analyzed for GFP expression by flow cytometry to observe a range of GFP expression levels. It was found that approximately 82% of the puromycin-selected cell population expressed GFP (Figure 1B). Puromycin-selected 293T cells were then exposed to 10 nM or 120 nM AP1903 (DMSO was used as a control) treatment for 24 h. Treated cells were then harvested, stained with 7AAD, which selectively stains dying cells with compromised membranes, and analyzed by flow cytometry. The percentage of 7AAD-negative cells (presumably live cells) was plotted against each treatment (DMSO in control; AP1903 10 nM; and AP1903 120 nM) (n=2). Significant cell death was detected after AP1903 treatment at both concentrations (Figure 1C). The dose of AP1903 did not appear to affect the percentage of dying cells. Targeted insertion of the donor vector into the AAVS1 locus in puromycin-selected 293T cells was further verified using junction PCR of genomic DNA obtained from these puromycin-selected 293T cells (Figure 1D). The presence of a PCR amplification product is specific to the junction formed by the donor vector insertion.

[0270] Similar to 293T cells, hiPSCs were transfected with a donor construct (AAVS1-G1.0) containing ZFNs specific for the AAVS1 locus and CMV-driven inducible caspase 9. Puromycin selection was initiated 3 days after transfection and continued for 16 days. Puromycin-resistant cells were analyzed by flow cytometry for GFP expression. Three populations based on GFP intensity, GFP(negative), GFP(low) and GFP(high), were sorted by flow cytometry (Figure 1E). Sorted GFP(negative), GFP(low) and GFP(high) populations were expanded and then exposed to AP1903 treatment (10 nM or 100 nM) or DMSO as a control for 24 hours. Treated cells were harvested, stained with 7AAD and analyzed by flow cytometry. The percentage of 7AAD-negative cells (presumably live cells) was plotted for each treatment. However, in contrast to 293T cells with targeted Casp9 insertion at the AAVS1 locus, AP1903-mediated cell death was not detected in iPSCs with targeted Casp9 insertion at the AAVS1 locus in all treatments (Figure 1F). Junction PCR of genomic DNA obtained from hiPSCs subjected to puromycin selection and sorting showed targeted insertion of the donor vector at the AAVS1 locus only in the GFP(low) population, but not in the GFP(negative) and GFP(high) populations (Figure 1G). That is, the lack of cell death in the GFP(high) population was thought to be associated with an incorrect (non-targeted) insertion in the genome and a putative lack of inducible caspase 9 expression under the CMV promoter (Figure 1G, lane 4; lane 1 is a marker). On the other hand, the lack of cell death in GFP(low) was thought to be associated with a reduced expression of the inducible caspase 9 gene under the CMV promoter, despite the correct targeted insertion in iPSCs (Figure 1G, lane 5). Thus, the AAVS1 safe-harbor targeted inducible caspase-9 suicide gene controlled by the CMV promoter elicits heterogeneous responses when activated by AP1903, likely depending on the cell type for integration and the integration site in the genome.

[0271] Example 3: Insertion of inducible caspase-9 targeted to the safe harbor locus under various endogenous and exogenous promoters in hiPSCs As previous studies have strongly demonstrated that puromycin selection under the AAVS1 promoter maintains viability and proliferation during selection, we next designed a donor construct containing inducible caspase-9 to target the AAVS1 locus, with the endogenous AAVS1 promoter (and GFP, and puromycin marker genes) driving inducible caspase-9 gene expression after insertion (Figure 2A). hiPSCs were transfected with ZFNs specific for the AAVS1 locus and a donor construct containing inducible caspase-9 under the control of the AAVS1 endogenous promoter. Puromycin selection was initiated 3 days after transfection and continued for 20 days. Puromycin-resistant cells were then analyzed by flow cytometry and shown to have GFP expression (Figure 2B). Expanded puromycin-resistant iPSCs were then exposed to AP1903 (or DMSO control) treatment for 24 hours. Treated cells were harvested, stained with 7AAD, and analyzed by flow cytometry. The percentage of 7AAD-negative cells (i.e., viable cells) was plotted for each treatment. However, in iPSCs with targeted insertion of Casp9 at the AAVS1 locus and controlled Casp9 expression by the endogenous AAVS1 promoter, AP1903-mediated cell death was not detected in all treatments (Figure 2C). Junction PCR of genomic DNA obtained from the puromycin-resistant pool demonstrated targeted insertion of the donor vector at the AAVS1 locus in this population (Figure 2D). Thus, it appears that AAVS1 safe-harbor-targeted inducible caspase-9 suicide gene controlled by the endogenous AAVS1 promoter did not induce inducible caspase-9 expression despite precise insertion at the AAVS1 locus. Based on the GFP expression intensity, again, low-level expression does not appear to be sufficient for inducible caspase-9-mediated cell death.

[0272] Unlike other gene transfer strategies, such as lentivirus-mediated transgene integration, where multiple gene copies are introduced per individual cell, in locus-specific targeting methods, the properties of different promoters appear to play a key role in the functional output. To determine which promoters maintain robust expression at the desired levels, constructs were now designed to target the AAVS1 locus, with various exogenous promoters driving inducible caspase-9 expression and the AAVS1 endogenous promoter driving GFP and puromycin marker genes (Figure 2E). Exogenous promoters tested included CMV, UBC, PGK, EF1α, and CAG. hiPSCs were transfected with a donor construct containing a ZFN specific for the AAVS1 locus and inducible caspase-9 under the control of one of the selected exogenous promoters. Puromycin selection was initiated 3 days after transfection and continued for 20 days, after which puromycin-resistant cells were analyzed by flow cytometry for GFP expression. It was found that hiPSCs transfected with a construct containing EF1α and inducible caspase 9 under the control of CAG (see SEQ ID NO: 1) showed high GFP expression after transfection, and CAG alone showed robust expression (Figure 2F and Table 1). These two strategies were chosen for further analysis.

[0273] Example 4: Targeted insertion of EF1α promoter-driven inducible caspase-9 into the AAVS1 safe harbor in iPSCs A donor construct containing EF1α-driven inducible caspase-9 was designed to target the AAVS1 locus according to FIG. 2E. hiPSCs transfected with AAVS1 locus-specific ZFNs and donor construct containing EF1α-driven inducible caspase-9 were subjected to puromycin selection for 20 days and then AP1903 treatment. The gated area in FIG. 3A showing the flow cytometry analysis highlights GFP-positive iPSC cells with targeted insertion, but few responded to AP1903 cell death induction. To see if these GFP-positive populations could be enriched and maintained, TRA181-positive (a marker of pluripotency and undifferentiated state) and GFP-positive (indicative of inducible caspase-9 expression) hiPSCs were bulk-sorted or individually sorted in 96-well plates around day 16 after selection (FIG. 3B). GFP-positive bulk-sorted hiPSCs were maintained in puromycin-free medium for an additional time to confirm long-term population characteristics (Figure 3C). A decrease in the percentage of cells with high expression of both TRA181 and GFP was observed over a long period of time (e.g., from day 2 post-sort to day 16 post-sort). This data suggests that EF1a showed high levels of expression but was unable to maintain its expression, most likely due to silencing in the pluripotent state. To confirm whether individual hiPSCs with robust EF1a-mediated expression could exist, individual hiPSC-sorted cells were screened. Bright field images of clonal hiPSCs derived from single-cell sorted TRA181 and GFP cells showed the morphology of hiPSC colonies derived from single-sorted cells (Figure 3D). Clonal hiPSCs with targeted insertion EF1α-driven inducible caspase 9 at the AAVS1 locus were then expanded and evaluated for GFP expression. However, all colonies lost GFP expression to various degrees after expansion, suggesting that the EF1α promoter was unable to maintain expression in hiPSCs during expansion after insertion (see Figure 3E, e.g., EF1α clones C1 and C23).Taken together, at the bulk and individual sorting levels, EF1a was unable to robustly maintain GFP expression and, therefore, inducible caspase-9 expression.

[0274] Example 5: Targeted insertion of CAG promoter-driven inducible caspase-9 into the AAVS1 safe harbor in hiPSCs Although several endogenous promoters were found to drive sustained expression of inducible caspase-9 during clonal expansion of hiPSCs, the measured expression levels were too low to effectively respond to AP1903 treatment. Expression of inducible caspase-9 under the control of various exogenous promoters was also shown to be lost during long-term hiPSC clonal expansion and failed to drive AP1903-induced cell death. hiPSCs transfected with donor constructs containing ZFNs specific for the AAVS1 locus and CAG-driven inducible caspase-9 were next evaluated (Figure 2E and Table 1).

[0275] Puromycin selection was started 3 days after transfection and continued for 16 days. Puromycin-resistant cells were then bulk-sorted for GFP expression or individually sorted in 96-well plates. To determine whether high GFP expression could be maintained during iPSC expansion, GFP-positive bulk-sorted iPSCs after targeting and puromycin selection were maintained in puromycin-free medium for 21 days and then analyzed by flow cytometry. Figure 4B shows that not only was the percentage of GFP-positive cells maintained during long-term expansion, but that the percentage of GFP-positive cells was greater than 99%. This is the first finding that a promoter targeted to a specific locus could maintain robust expression, similar to that maintained by lentiviral methods where multiple copies are inserted into the genome. We next examined the inducible caspase 9 gene response of bulk sorts that maintained 99% GFP expression. Figure 4C shows that when treated with AP1903, GFP-positive cells underwent inducible cell death and became 7AAD-staining positive after treatment. As the CAG bulk population showed robust expression leading to efficient inducible caspase 9-mediated cell death, individual clones were then evaluated. This time, puromycin-resistant iPSCs clonally sorted into matrigel-coated 96-well plates were evaluated. Bright-field images of typical colonies after individual sorting show high-quality pluripotent morphology (Figure 4D). Colonies derived from single cells were individually expanded and evaluated for GFP and TRA181 expression by flow cytometry. It was shown that all selected colonies (e.g., CAG-C5, CAG-C8, CAG-C12, CAG-C38) maintained a high percentage of GFP and TRA181 expression during expansion (Figure 4E). The mean fluorescence intensity (MFI) of the selected clones is shown in Figure 4F. From Figure 4F, CAG-C5 has the highest MFI (1500), while CAG-C38 has the relatively lowest MFI (858) among the four clones. All selected GFP+ clones were used to perform junction PCR of genomic DNA to detect homologous recombination between the donor construct and the AAVS1 locus.Parental hiPSCs refers to the original hiPSC line before transfection.

[0276] CAG hiPSC population pool refers to the population after targeted transfection and selection with puromycin for 16 days. To ensure the quality of genomic DNA, GAPDH genomic sequence was amplified. All selected GFP-positive clones showed correct insertion at AAVS1 locus (Figure 4G). Thus, the above data suggested that CAG is the only promoter that is capable of robustly maintaining high-level expression of the genes under its control during iPSC proliferation after being targeted to the AAVS1 locus of hiPSC cells.

[0277] The performance of additional promoters was similarly evaluated during transfection (2-3 days post-transfection), maintenance after puromycin selection (20 days post-puromycin selection), and functional response after AP1903 treatment. Table 1 below summarizes the performance of the exogenous promoters CMV, UBC, EF1α, CAG, and PGK, and the endogenous promoter AAVS1. In Table 1, the suicide gene response is quantified based on the number of GFP-positive cells responsive to AP1903 treatment. [Table 2]

[0278] That is, among all the selected promoters, CAG shows the highest performance in all respects. The CAG-iPSC clones were further evaluated below.

[0279] Example 6: Pluripotency evaluation of CAG-hiPSC clones All selected CAG-hiPSC clones were analyzed for pluripotency evaluation. Figure 5A shows an image of CAG clone C38 maintained in feeder-free culture, showing a homogenous culture of hiPSCs with individual cells composed of a high nuclear-to-cytoplasmic ratio. Fluorescent immunostaining of pluripotency markers NANOG and OCT4 in Figure 5B indicates that the CAG-hiPSC clones are pluripotent and undifferentiated. In addition, quantitative RT-PCR was performed for the expression of various endogenous pluripotency markers, including NANOG, OCT4, SOX2, REX1, DPPA2, and MYC, to show the pluripotency status of the selected CAG-hiPSC clones (CAG-C5, CAG-C8, CAG-C12, CAG-C38, and CAG-C40). It is shown that NANOG expression is lower in all CAG-hiPSC clones than the parental iPSCs. Flow cytometry analysis of TRA181, SSEA3 and CD30 marker expression was also performed. The selected CAG clones were then directed to germ layer specific differentiation and evaluated for germ layer markers. Figure 5D shows the expression of ectodermal marker nestin; mesodermal marker αSMA; and endodermal marker SOX17 after the selected CAG clones were directed to differentiation. One of the CAG-iPSC clones, CAG-C38, was further evaluated for its ability to generate teratomas of various lineages. Figure 5E shows a teratomas harvested 6 weeks after injection, but showing three germ layers (endoderm, mesoderm or ectoderm). These data indicate that the CAG-iPSC clones retained pluripotency and the ability to differentiate into non-pluripotent cells of all lineages.

[0280] Example 7: Inducible suicide gene-mediated killing of hiPSC clones in both pluripotent and differentiated states with CAG-driven inducible caspase 9 targeted to the AAVS1 locus Twenty CAG-inducible caspase 9-targeted hiPSC clones were treated with AP1903 (or DMSO control) for 24 hours, then stained with 7AAD and analyzed for GFP and 7AAD staining by flow cytometry. All clones showed more than 95% positive 7AAD staining after AP1903 treatment (Figure 6A). Flow cytometry plots obtained from two representative clones (CAG-C5 and CAG-C38) were also shown to represent the Casp9 gene response in the pluripotent state of CAG-iPSC clones (Figure 6A). Fifteen CAG-inducible caspase 9-targeted iPSC clones were cultured in triplicate wells in a 12-well plate. After confluence, one well was stained for alkaline phosphatase and photographed, while the other two wells were treated with AP1903 (10 nM) for 48 hours. The treated wells were washed with PBS and fresh medium was added to recover residual viable cells. Wells recovered for 8 days were stained for alkaline phosphatase and photographed to document the survival of iPSC clones. Of the 15 clones, only the CAG-C14 clone contained surviving clones, i.e., avoided induced death (Figure 6B). The other 14 clones showed no staining, indicating that no cells survived after induced expression of Casp9. Figure 6C shows the kinetics of coverage of CAG-C5 clones by live cells on a 10 cm dish after treatment with AP1903 (10 nM). Live cells began to die approximately 8 minutes after treatment, and almost all live cells were dead 12 minutes after AP1903 treatment. The CAG-induced caspase 9-targeted hiPSC clones were then induced to differentiate into cells from three germ layers (endoderm, mesoderm, or ectoderm), each of which was treated with AP1903 for 48 hours and allowed to recover in differentiation medium for 5 days. Replicate wells pre-treatment and post-recovery were stained with crystal violet, resulting in the images shown in Figure 6D. Representative images from the CAG-C5 clone are shown, demonstrating that cells of all lineages died after AP1903 treatment. Finally, CAG-inducible caspase 9-targeted hiPSC clones were induced to differentiate into CD34+ cells, treated with AP1903 for 48 hours, and analyzed for cell death by flow cytometry.CD34+ cell death after AP1903 treatment was indicated by enhanced 7AAD staining as shown in Figure 6E. The data indicate that CAG targeted to the AAVS1 locus robustly maintained inducible caspase-9 expression regardless of epigenetic cell state (i.e., pluripotent state or cell type-specific state such as ectoderm, endoderm or mesoderm, or CD34+ cells).

[0281] Example 8: In vivo demonstration of AP1903-induced regression of teratomas derived from inducible caspase-9-iPSC clones The location of subcutaneous injection in the NSG mouse study to demonstrate inducible caspase-9 expression and induction-mediated cell death in vivo is shown in Figure 7A. Parental hiPSC lines and CAG-C38 clones were injected with 4E6 cells (day 0) and treated with AP1903 (i.p., 200 μg total) on days 7–11. On day 34, teratomas were harvested and imaged to show their size (Figure 7B). Among the CAG-C38 harvests, one injection site contained no tumor, while the others consisted mostly of adipoid cells. Harvested teratomas were also volumetrically measured (Figure 7C). Among the harvested tumors, teratomas sourced from CAG-38 hiPSCs were either not detectable or appeared significantly smaller than their parental controls. Harvested teratomas were stained with hematoxylin and eosin. The staining results show differences in histology between the parental iPSC clones and the CAG-hiPSC clones (Figure 7D). While the parental hiPSC line showed numerous three germ layer differentiated cell types, CAG-C38 appears to be composed mostly of highly organized adipoid cells, likely of mouse origin. The parental hiPSC line and the CAG clones were then injected with 4E6 cells and treated with AP1903 (intraperitoneally, 200 μg total) on days 40-46. Periodic volumetric measurements of all tumors showed that the CAG-iPSCs were still responsive to Casp9 gene expression, and tumor volume was reduced in mice injected with any of the selected CAG-iPSC clones. Collectively, the data show robust expression of the CAG promoter targeted to the AAVS1 locus in vitro and in vivo.

[0282] Example 9: Genome editing of the human ROSA26 locus using CRISPR / caspase 9 for targeted or nuclease-independent insertion of inducible caspase 9 To investigate the synergy of other targeting strategies with the safe harbor locus, in this experiment, different endogenous loci in the iPSC genome were examined for the possibility of nuclease-independent targeted insertion of foreign genes driven by the CAG promoter. Constructs were designed to harbor CRISPR / caspase 9 and target the ROSA26 locus, as shown in Figure 9A. hiPSCs were transfected with a donor construct containing a gRNA / caspase 9-RFP plasmid specific for the ROSA26 locus and CAG-driven inducible caspase 9. Two days after transfection, the cell population was analyzed by flow cytometry for intracellular GFP expression to determine the transfection efficiency, which was shown to be about 80% to about 85% (Figure 9B). Puromycin selection was started 3 days after transfection and continued for 20 days, after which puromycin-resistant cells were analyzed by flow cytometry for GFP expression. GFP expression was maintained during iPSC expansion (Figure 9B). Table 2 below summarizes the expression levels and functional responses of iPSCs with the constructs shown in Figure 9A. [Table 3]

[0283] Table 2 shows that CAG-driven targeted foreign gene insertion is suitable not only for the AAVS1 locus but also for the ROSA26 locus, and can be mediated by both ZFN and CRISPR strategies. We also employed a further construct design to target the ROSA26 locus by a nuclease-independent strategy by utilizing homology arms to investigate the feasibility of the strategy. The construct for the nuclease-independent ROSA26 locus consists of a CAG promoter driving GFP and inducible caspase 9, while RFP shows non-specific integration (Figure 9C). 293T cells and hiPSCs transfected with the construct in Figure 9C and selected with puromycin were enriched for targeted cells, respectively (Figure 9D and Figure 9E). The data indicate that the nuclease-independent strategy is also applicable to the current platform for efficiently generating targeted hiPSCs.

[0284] In summary, the CAG promoter maintained high levels of inducible caspase-9 expression during long-term clonal expansion of hiPSCs, regardless of targeting strategy, locus, or different differentiation states. Moreover, these clonal lines integrated with inducible caspase-9 underwent rapid cell death in the presence of AP1903, and no residual cell survival was observed when cultures were allowed to recover in the absence of the dimerization molecule. Furthermore, hiPSC clones were found to be fully susceptible to AP1903-induced cell death when differentiated in vitro into three somatic cell lineages. The clones were also specifically differentiated into hematopoietic cells and showed full induction of cell death by AP1903 treatment. A similar cell death-mediated response was observed in vivo when injected into NSG mice to form teratomas. Similar results were seen with nuclease-free CAG-driven targeted integration in iPSCs. That is, in addition to being efficient, precise, and sustainable, the CAG-driven targeted integration system provided herein is also reliable, both in vitro and in vivo, as it does not appear to cause alterations in the epigenetic landscape that are often seen with other promoters and / or constructs that would abrogate suicide gene-mediated responses in iPSCs, expanded iPSCs, or iPSC-derived differentiated cells. Other foreign genes and / or at different selected loci can also be applied to this platform. For example, in addition to creating constructs containing GFP and inducible caspase 9, genes with different functionality can be integrated into the construct. Targeting modalities such as chimeric antigen receptors or modified T cell receptors can also be introduced into specific loci. In another example, targeting of various genes to specific loci can be performed at the time or during reprogramming. As mentioned above, the hiPSC platform allows for the selection of individual cells with various characteristics, which may include successfully reprogrammed hiPSCs that contain desired genes introduced into specific loci.

[0285] In particular, one hiPSC clone contained certain rare cells and escaped induced cell death, and this clone and these rare cells were characterized to evaluate the molecular mechanism of escape.

[0286] Example 10: Characterization of escape clones reveals a single point mutation in all expanding clones Picking and expanding clones that survived AP1903 treatment enhanced the procedure used to find surviving clones after AP1903 treatment (Figure 8A). As shown, all escape clones have the correct target insertion as shown by junction PCR. To confirm whether sequence mutations were found in the escape clones, PCR amplification of the inducible caspase 9 transgene was performed, followed by purification and genomic sequencing (Figure 8B). The sequence of the inducible caspase 9 transgene of those escape clones showed a single point mutation K189E in all clones sequenced (Figure 8C). To confirm whether K189E was the direct cause of the refractory clones, an inducible caspase 9 mutant was generated according to Figure 2E, used for transfection, and the resulting targeted iPSC cells were examined. FIG. 8E shows that CAS-inducible caspase-9 mutant iPSC cells expressing GFP were not responsive to AP1903 treatment in comparison to CAS-inducible caspase-9 wild-type iPSC cells, and all cells survived after treatment with identical high-level GFP expression.

[0287] Example 11: Approaches to rescue escape clones containing common single point mutations The above examples demonstrated that in vitro treatment-resistant clones derived from a single refractory line contained a common inactivating point mutation in inducible caspase-9 in all tested clones. However, selection of hiPSC clones with biallelic inducible caspase-9 insertions improved killing kinetics and reduced resistance rates in vitro and in vivo. Therefore, to avoid the possibility of resistance to a single inducible safety system, we investigated the generation and selection of hiPSC clones containing multiple conditional suicide genes, including inducible caspase-9 and herpes simplex virus thymidine kinase, each integrated into a unique safe harbor locus. The dual safe guard system was compared to a single iCasp9 safety switch to evaluate the safety and efficient elimination of transplanted cells in vitro and in vivo.

[0288] Example 12: Copy number of targeted insertion of CAG promoter-driven inducible caspase-9 into the AAVS1 safe harbor in hiPSCs Quantitative PCR-based assessment of transgene (inducible caspase 9-GFP) copy number in CAG promoter-driven inducible caspase 9 clones revealed that a small number of clones had more than one copy of the transgene. As shown in Figure 10A, a copy number of 1 indicates monoallelic inducible caspase 9 integration, a copy number of 2 indicates biallelic inducible caspase 9 integration, while anything above 2 indicates possible random integration. The mean fluorescence intensity (MFI) of inducible caspase 9-GFP in selected pooled cultures or clones with monoallelic or biallelic inducible caspase 9 target insertions, shown in Figure 10B, indicates that transgene expression levels parallel copy number, with higher copy numbers resulting in higher MFI readings. PCR analysis of transgene integration using primers overlapping the AAVS1-transgene junction in targeted pool cells and clones confirmed specific integration of the inducible caspase 9-GFP transgene into the AAVS1 locus (Figure 10C). Primers specific for the AAVS1 integration site were used to determine the number of alleles with targeted integration. The absence of a band suggests that the integration site in both AAVS1 alleles was disrupted, i.e., biallelic insertion, whereas the presence of a band suggests that one or both alleles are not disrupted (Figure 10D). As shown by Figures 10C and 10D, CAG-C5 and CAG-C35 clones have biallelic insertion of two copies of the transgene, whereas CAG-C38 clones have one copy inserted at the targeted location of one allele. Live cell imaging was used to assess plate surface coverage immediately after addition of 10 nM AP1903. As shown in FIG. 10E, unsorted targeted pool cells (integrated and non-integrated) had the least inducible killing potential, whereas targeted pool cells sorted for inducible caspase-9-GFP (targeted random integration, variable copy number) showed slightly better inducible killing potential.The clonal CAG-C38 cell line (monallelic targeted insertion) and CAG-C5 cell line (biallelic targeted insertion) have significantly higher killing rates and efficiencies when compared to pooled cells.

[0289] Example 13: Comparison of inducible killing and teratoma regression of transplanted biallelic and monoallelic inducible caspase 9 clones in vivo NSG mice studies with subcutaneous injections were shown in Figure 11A to show in vivo cell death via expression and induction of inducible caspase 9 in CAG-C38 clones, CAG-pools, PGK-pools (PGK-inducible caspase 9 transgene), UBC-pools (UBC-inducible caspase 9 transgene) and CMV-pools (CMV-inducible caspase 9 transgene). CAG-C38 clones and pools were injected into three groups of NSG mice (n=2) on day 0 with 4E6 cells and mice were treated with AP1903 (125 μg intraperitoneally) on days 19-25. On day 28, teratomas were harvested and imaged to show size. Bioluminescence imaging of the three groups of NSG mice before AP1903 administration (FIG. 11B, top panel) and 3 days after the end of AP1903 administration (FIG. 11B, bottom panel) showed that only the exogenous CAG promoter allowed inducible caspase 9-mediated elimination of established iPSC-derived teratomas. This finding was further shown in FIG. 11C-FIG. 11G. The light output (photons / sec) indicates the tumor cell mass in teratomas from the indicated cell types, shown as mean and SD in FIG. 11C, which confirmed the teratoma size findings in FIG. 11B. The ratio of light output after treatment to light output before treatment (mean ± SD) for the indicated cell types is shown in FIG. 11D. Both results for mice injected with clonal CAG-C38 and CAG pool cells are plotted separately in FIG. 11E, with higher resolution. End-stage volume measurements of teratomas from the indicated cell types at day 28 are shown in FIG. 11F. Figure 11G shows images of teratomas from the indicated cell types at day 28. No teratomas were detected in the sides injected with CAG-C38 clones or CAG-inducible caspase 9 pool cells. Collectively, these data confirmed the in vitro studies of various promoters driving the expression of an inducible caspase 9-GFP transgene and showed that the CAG promoter was the most effective among those tested in vitro and in vivo.These data also show that the CAG-C38 clone is more effective than the CAG pooled cells at eliminating teratomas after suicide gene induction, despite the fact that inducible caspase-9-GFP is monoallelic.

[0290] Additionally, the inducible death of transplanted biallelic inducible caspase 9 clones was tested and compared to that of monoallelic inducible caspase 9 clones. NSG mice studies with subcutaneous site injection were shown in Figure 12A to show in vivo cell death via expression and induction of inducible caspase 9 in parental cells, CAG-C5 clones, and CAG-C38 clones. Each cell type was injected into four groups of NSG mice (n=2) on day 0, and mice were treated with AP1903 (125 μg intraperitoneally) on days 7-13. Teratomas were imaged on days 7, 14, and 42. On day 42, teratomas were harvested and imaged to show size.

[0291] Bioluminescence imaging of NSG mice transplanted with the indicated cell types (biallelic CAG-C5 clones, monoallelic CAG-C38 clones and parental iPSCs) at days 7, 14 and 42 after iPSC transplantation (FIG. 12B). Imaging at day 7 was performed immediately prior to AP1903 administration (125 μg i.p.; days 7-13). The middle panel shows images of harvested teratomas, if present, at day 14, and the bottom panel shows images of harvested teratomas, if present, at day 42. As shown, both CAG-C5 and CAG-C38 are capable of eliminating teratomas at day 14 in all mice tested. However, teratomas regressed in mice transplanted with CAG-C38 by day 42, whereas eliminated teratomas in mice transplanted with AG-C5 did not regrow. Figure 12C shows images of teratomas from the indicated cell types at day 42. Luminescence (photons / sec) of teratomas from the indicated groups shown as mean and SD in Figure 12D, confirmed by image data in Figures 12B and 12C. Collectively, these data demonstrated more effective inducible killing of transplanted biallelic inducible caspase 9 clones compared to monoallelic inducible caspase 9 clones.

[0292] Example 14: Creation of a safety surveillance system with dual suicide genes targeted to separate safe harbor loci To demonstrate the performance and efficacy of high-resolution precision engineering at the single-cell level, we generated clonal iPSCs with a safety monitoring system with dual suicide genes targeted to separate safe harbor loci. Using the targeted insertion method described herein, we integrated CAG-sr39TK into the H11 safe harbor in addition to the CAG-inducible caspase 9 integrated into the AAVS1 locus in CAG-C38 clonal iPSCs. As shown in Figure 13A, PCR analysis confirmed the specific genomic integration of sr39TK into the H11 safe harbor locus using primers specific for sr39TK, sr39TK-blasticidin (Bsd) junction or endogenous H11 sequence-transgene junction, with GAPDH as a loading control. Inducible caspase-9 iPSC clones (not clonal for sr39TK) carrying a targeted insertion of sr39TK at the H11 locus were treated with 25 μg / mL ganciclovir (GCV) for 9 days with or without 10 nM AP1903 treatment for the first 2 days. Treatment of the CAG-C38 inducible caspase-9 clone (monoallelic for inducible caspase-9) with AP1903 alone for 2 days resulted in the inducible death of most cells, but a few cells remained (Figure 13B(ii)). Treatment of the CAG-C38 inducible caspase-9 clone with GCV treatment alone for 9 days resulted in the proliferation of GCV-refractory cells (Figure 13B(iii)). However, combined treatment with both AP1903 and GCV effectively eliminated all refractory cells, indicating that the use of a dual suicide safety surveillance system is more effective than the use of the inducible caspase-9 suicide gene alone by killing the remaining cells that escaped under either inducible suicide gene.

[0293] Also provided herein is the design of iPSCs containing an inducible cassette at a safe harbor locus for controllable expression of a gene of interest. Locus-specific insertion of the inducible cassette occurs at the iPSC level. Expression of the gene of interest (here shown as an example CAR) is inactivated by a translation stop codon flanked by two loxP sites. After a single induction with Cre recombinase (Figure 33A) or doxycycline (Figure 33B), the stop codon is consequently removed and CAR expression is activated. The inducer can be administered at any selected stage during ex vivo iPSC differentiation, facilitating optimization of the differentiation procedure. CAR is driven at high levels by a strong constitutive promoter to sustain expression in the end product, such as iT or iNK.

[0294] A novel strategy was also developed to simultaneously insert a new gene of interest in a hiPSC population while uniquely targeting the optimal locus for gene-specific disruption, and then select hiPSC clones with all the required criteria, including only one copy number of the inserted gene, functional disruption of the target gene, and no non-specific effects, by high-throughput single-cell sorting. Figure 34A shows the construct design of a target vector containing negative and positive strand-directed nuclease binding, and a donor vector containing homology arms for B2M, an example of a gene for disruption, and a gene of interest for insertion at the disrupted locus (e.g., CD16, HLA-G, and PDL1). Figure 34B shows an example of hiPSC selection after gene editing for a clonal population containing the optimal gene at the optimal locus.

[0295] Example 15: B2M null Generation of (B2M- / -) HLA-I-deficient iPSCs and their modifications To demonstrate the feasibility of single-cell gene editing to achieve HLA complex modification, B2M-targeting gRNA pairs in a plasmid expressing caspase 9 nickase, which has been modified to have less non-specific action compared to wild-type caspase 9, were transfected into iPSC lines. Figure 14A shows flow cytometry analysis of GFP expression and B2M / HLA-I expression before (left panel) and after (middle panel) transfection. B2M and HLA-I were detected simultaneously using B2M-specific and HLA-I-specific antibodies conjugated to the same dye. Cells negative for both B2M and HLA-I (boxed population) were bulk-sorted (Figure 14A) or clonally sorted in 96-well plates to generate clonal lines (Figure 14B). The B2M- / - and HLA-I-deficient clones that had been subjected to targeted editing were further analyzed by clonal genomic DNA sequencing and confirmed to have small deletions or insertions that conferred the B2M knockout phenotype.

[0296] The HLA class I-deficient iPSCs (B2M- / -iPSCs, also known as HLA-I null iPSCs) were then genetically modified, for example by lentivirus-mediated introduction of HLA-E / B2M fusion protein, with the aim of further modifying the HLA-I-deficient cells. The quality (i.e., pluripotency status) of the modified HLA-I-deficient iPSCs (B2M- / -HLA-E iPSCs) was assessed by flow cytometry for expression of pluripotency markers TRA-181 and SSEA4, as well as HLA-E. Figure 16A shows that the modified HLA-I-deficient iPSCs express HLA-E on the cell surface and maintain a pluripotency phenotype. Similar results were seen when nucleotides expressing HLA-E / B2M fusion protein were incorporated into the HLA-I-deficient iPSCs, in comparison with the results obtained from lentiviral transduction.

[0297] Next, B2M- / -HLA-E iPSCs were genetically modified to contain PDL1 protein to generate modified HLA-I-deficient iPSCs (B2M- / -HLA-E PDL1 iPSCs). In addition, B2M- / - iPSCs were genetically modified to contain HLA-G / B2M fusion protein, and then genetically modified to contain PDL1 protein to generate another HLA-I modified B2M null iPSC (B2M- / -HLA-G PDL1 iPSCs). Figure 21A shows the expression of HLA-E, HLA-G and PDL1 on the cell surface of modified HLA-I-deficient iPSCs. Similar results were observed when nucleotides expressing HLA-G / B2M fusion protein were incorporated into HLA-I-deficient iPSCs.

[0298] To determine whether modified HLA-I-deficient iPSCs maintain blood cell differentiation potential, various modified HLA-I-deficient iPSCs (B2M- / -, B2M- / -HLA-E, B2M- / -HLA-E / PDL1, B2M- / -HLA-G and B2M- / -HLA-G / PDL1) were differentiated into CD34+HE using the iCD34 differentiation protocol used herein. Figure 21B shows that all modified HLA-I-deficient iPSCs can be differentiated into CD34+HE as seen by flow cytometry 10 days after differentiation. CD34+ cells were isolated and seeded into iNK or iT lymphoid differentiation cultures. Figure 22 shows that modification of HLA-I-deficient iPSCs via genetically modified immune resistance modalities (e.g., HLA-E, HLA-G, or PDL1) does not perturb the ability of iCD34 cells to give rise to pro-NK cells (NK precursors; CD45+CD56) and pro-T cells (T precursors; CD45+CD34+CD7+) after an additional 10 days of differentiation, as seen by flow cytometry. Similar results were seen when nucleotides expressing a PDL1 fusion protein were incorporated into HLA-I-deficient iPSCs.

[0299] To determine whether the expression of the genetically modified immune resistance protein is maintained during hematopoietic differentiation, iNK cells differentiated from modified HLA-I-deficient CD34+ cells for 20 days were evaluated for the expression of HLA-E, HLA-G, and PDL1 on the cell surface by flow cytometry. Figure 23A shows that HLA-E and HLA-G proteins are absent while PDL1 expression is maintained. To determine whether HLA-E and HLA-G proteins are cleaved from the cell surface during hematopoietic differentiation, protein lysates obtained from day 20 iNK cells and day 20 culture supernatant were examined for the expression of HLA-E and HLA-G proteins by Western blot analysis. Figure 23B shows that HLA-E and HLA-G proteins are present in the supernatant fraction, and therefore it is concluded that HLA-E and HLA-G proteins are cleaved from the cell surface of iPSC-derived NK cells.

[0300] To maintain the enhanced persistence of modified HLA-I-deficient iPSC-derived lymphoid effector cells, cleavage-resistant versions of HLA-E / B2M and HLA-G / B2M fusion proteins were designed (Figure 24A), and B2M- / - iPSCs were genetically modified to contain uncleavable HLA-G / HLA-A3 fusion proteins. Figure 24B shows that the HLA-G / HLA-A3 uncleavable proteins are expressed on the cell surface by flow cytometry for HLA-G expression, and further indicates that the proteins do not affect the quality of iPSCs, as shown by the expression of pluripotent proteins (TRA-181, SSEA4, NANOG, and OCT3 / 4).

[0301] Another method to generate HLA class I-deficient iPSCs is to disrupt the peptide loading complex (PLC), which is involved in loading peptides onto HLA class I molecules. The transporter associated with antigen processing (TAP) is an essential member of the PLC, and disruption of its gene results in a marked reduction in stable HLA class I molecules on the cell surface. However, disruption of TAP2 null It is unknown whether (TAP2- / -) HLA-I-deficient iPSCs affect the differentiation potential or persistence of iPSCs. null To generate iPSC lines, FTi121 iPSCs were transfected with TAP2 gRNA pairs in a plasmid expressing caspase 9 nickase. HLA-I negative / low expressing cells were clonally sorted into 96-well plates to generate clonal lines. Figure 25A shows flow cytometry analysis of two selected clones that show a significant decrease in HLA class I expression compared to parent FTi121. Treatment of iPSCs with IFNγ induces upregulation of HLA class I molecules on the surface. Figure 25B shows that after IFNγ treatment, TAP2- / - clones showed a decrease in HLA class I expression compared to wild-type FTi121, suggesting a unique strategy to identify the optimal level of HLA complex expression to escape detection by both T cells and NK cells.

[0302] Example 16: B2M- / - and HLA-I-deficient iPSCs have improved persistence To determine whether the absence of HLA-I allows B2M- / - hiPSCs to escape T lymphoid responses, peripheral blood T cells were pre-stimulated by co-culturing with hiPSCs for 10 days. After 10 days, expanded pre-stimulated T cells were harvested and used as cytotoxic effector cells. Wild-type hiPSCs (WT) or B2M- / - (modified) hiPSC target cells were seeded alone or in a co-culture with pre-stimulated T cells at a ratio of 1:3. The number of target cells per well was measured over a 5-day period using an Incucyte Zoom. As shown in Figure 15A, WT hiPSC target cells were recognized and killed by pre-stimulated T cells, whereas modified hiPSCs were unaffected.

[0303] Although it was shown that B2M- / - hiPSCs can avoid recognition and killing by T cells, there was concern that these cells might be killed more efficiently by NK cells, because NK cells are known to be activated by cells lacking MHC class I (in this case, the cells lack B2M), and if so, could contribute to their rejection in vivo. To examine whether B2m- / - hiPSCs have increased recognition by NK cells, we cultured them in CD45+ cells differentiated from K562 cells, wild-type iPSCs (WT), or B2M- / - iPSCs (B2M - / -CD45+ cells differentiated from hiPSCs were separately labeled with CFSE, e670 proliferation dye (eBioscience), or e450 proliferation dye (eBioscience), respectively. All three labeled target cells were mixed in equal ratios and added to the NK cell effector in a single well at the indicated ratios (Figure 15B). Alternatively, the three labeled target cells were each added to the NK cell effector in separate wells (Figure 15C). Cells were incubated for 4 hours, after which quantification of cytotoxic activity based on flow cytometry was performed. As shown in Figure 15B and Figure 15C, compared to the killing of K562 cells, a known NK cell target included as a positive control, both B2M- / - and WT hiPSC CD45+ cells were killed at the same rate and both at relatively low levels, suggesting no increased NK cell recognition in vitro. Thus, the data suggest that this B2M- / - iPSC line can evade T cell-mediated killing without reducing NK cell recognition, indicating a universally donor / recipient compatible hiPSC clonal line with improved persistence.

[0304] The improved persistence of the universal B2M- / -hiPSC clonal line was further evaluated in vivo. To determine whether the absence of HLA-A increases persistence in the presence of a responsive immune system in vivo, luciferase-transduced WT hiPSCs (WT) and B2M- / -hiPSCs (modified) were injected subcutaneously and bilaterally into immunocompromised NSG recipient mice (Figure 15D left) or immunocompetent WT C57BL / 6 recipient mice (Figure 15D right) to form teratomas. After 96 hours, the mice were subjected to bioluminescence IVIS imaging to detect teratomas. Both WT and modified teratomas were detected with equal intensity in NSG recipients, while modified B2M- / -hiPSCs showed increased persistence compared to WT hiPSCs in WT C57BL / 6 mice.

[0305] Example 17: Modulation of HLA class I on iPSCs increases persistence of iPSCs in vivo To determine whether modified HLA-I-deficient iPSCs have increased persistence in vivo, luciferase-transduced wild-type and B2M- / -HLA-E iPSCs were subcutaneously injected into opposing flanks of fully immunocompetent C57BL / 6 recipients in a teratoma assay. Mice were analyzed daily by IVIS imaging in conjunction with luciferin injections to visualize developing teratomas. Figure 16B shows that at 72-144 hours post-injection, B2M- / -HLA-E iPSCs exhibit an approximately six-fold increase in quantitative persistence compared to wild-type iPSCs. Three representative mice bearing B2M- / -HLA-E iPSC teratomas, showing enhanced luciferin imaging, are also shown in Figure 16B. Similar improved persistence was observed when HLA-G was used instead of HLA-E. In addition, as mentioned above, the application of modified forms of HLA-E or HLA-G to avoid cleavage further enhances the in vivo persistence of HLA class I modified iPSCs. Interestingly, in some scenarios where NK cells obtained from CMV+ donors show NKG2C expression, i.e., are activated, it was observed that HLA-E cell surface expression rather activates NKG2C, thereby resulting in adverse effects including NK cell recognition and the resulting killing of HLA-E expressing cells. In contrast to the case where NK cells express NKG2A, these NK cells can be inactivated by iPSCs and differentiated progeny expressing HLA-E on their surface, thereby enhancing the persistence of these HLA-E surface expressing cells.

[0306] To determine which components of the host immune response were responsible for the enhanced rejection of modified HLA-I-deficient iPSCs in wild-type recipient mice, CD4+ T cells, CD8+ T cells, and NK cells were depleted by injection of anti-CD4, anti-CD8a, and anti-NK1.1 antibodies, respectively. Figure 26A shows the absence of CD4+ T cells, CD8+ T cells, and NK cells 3 days after antibody injection. Three days after antibody-mediated depletion, luciferase-transduced B2M- / -HLA-I modified iPSCs were subcutaneously injected into the flanks of immunocompetent C57BL / 6 mice to allow teratomas to form. Mice were analyzed daily by IVIS imaging in conjunction with luciferin injection to visualize the developing teratomas. Figure 26B shows that NK cell-depleted mice showed the highest resistance to tumor rejection compared to IgG control-treated animals at 120 hours after iPSC injection.

[0307] To determine whether genetically modified HLA-I modified iPSCs have increased persistence and resistance in vitro, B2M- / -PDL1 HLA-I modified iPSCs were cultured in suspension for 3 days and then treated with IFNγ for 24 hours to induce HLA class I expression. Treated iPSCs were then seeded in co-culture with purified allogeneic T cells (Figure 27A), allogeneic T cells and NK cells contained in total peripheral blood mononuclear cells (PBMCs, Figures 27B and 27C) pre-labeled with Cell Trace Violet (CTV) to monitor cell proliferation. T cells / PBMCs alone (unstimulated) and T cells / PBMCs stimulated with anti-CD3 / CD28 beads to induce activation and proliferation (stimulated) were used as controls. Twelve days after the initiation of co-culture, the proliferation of CD4+ T cells, CD8+ T cells and CD56+ NK cells was determined by flow cytometric evaluation of the cells as an indicator of their ability to recognize and respond to HLA-I modified iPSC-derived cells. Figures 27A and 27B show that compared to T cells co-cultured with wild-type iPSC-derived cells, T cells co-cultured with B2M- / - and even B2M- / -HLA-I-modified iPSC-derived cells showed less proliferation as indicated by dilution of the CTV proliferation dye. Figure 27C shows a similar effect on NK cell proliferation, collectively enhancing the ability of HLA class I modifications to enhance immune tolerance to T cells and NK cells.

[0308] Example 18: Creation of iPSCs with enhanced properties through additional editing Molecules modified or modulated at the iPSC level may be used to enhance desired properties in immunotherapy using induced lymphocytes obtained through current differentiation platforms. These molecules may include safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmacologic active proteins and peptides, potential drug targets; or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and regulation, and / or survival of iPSCs or their derived cells. In addition to B2M / HLA-I and HLA-E / G, target molecules that contribute to desired properties include CD16 receptor and 41BBL costimulatory molecule, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A, and B. 2A Further included are, but are not limited to, surface triggering receptors for binding R, CAR (chimeric antigen receptor), TCR (T cell receptor), TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, any gene in the chromosome 6p21 region, engagers, and bispecific or multispecific or universal engagers. More specifically, genetic modification of target molecules in iPSCs includes one or more of the following: deletion or reduction of expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene within the chromosome 6p21 region; HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A Introduction or increased expression of a surface triggering receptor for binding to an R, CAR, TCR, Fc receptor, engager, or bispecific or multispecific or universal engager. The increased or decreased expression of the target molecule may be permanent, transient, temporal, or inducible, and may be endogenous or exogenous promoter controlled.

[0309] These desired modalities can be introduced into iPSCs using various delivery methods known in the art. As a typical example of this, iPSCs were genetically modified to contain high affinity non-cleavable CD16 receptor (hnCD16) and 41BBL costimulatory molecules to generate iPSCs with enhanced cytotoxicity. Figure 17A shows the efficient expression of hnCD16 and 41BBL on the surface of iPSCs after lentiviral transduction by flow cytometry analysis. Figure 17B shows that after 10 days of differentiation, the expression of hnCD16 and 41BBL is maintained and does not disrupt the ability of iPSCs to generate CD34+HE cells.

[0310] To assess whether expression of hnCD16 (high affinity non-cleavable CD16 receptor) protein affects blood cell differentiation of modified iPSCs, a pool of hnCD16 / 41BBL gene modified iPSCs was clonally sorted into 96-well plates. Three positive clones were selected and differentiated for 10 days to generate iCD34+ cells. Figure 28A shows homogenous expression of hnCD16 and 41BBL on CD34+ HE cells as shown by flow cytometry. CD34+ cells were isolated and further differentiated for 10 days under NK promoting conditions to generate pro-NK cells. Figure 28A shows that CD56+ NK progenitor cells maintained high levels of hnCD16 protein expression, indicating that expression of hnCD16 and 41BBL does not perturb the blood cell differentiation potential of modified iPSCs.

[0311] The endogenous CD16 receptor expressed by NK cells is cleaved from the cell surface after activation of NK cells. To confirm that the hnCD16 variant protein is not cleaved, iNK cells and peripheral blood (PB)-derived NK cells expressing hnCD16 were cultured under the following conditions: 1) in homeostatic culture, 2) in homeostatic culture wit...

Claims

1. 1. A method for generating derivative cells from iPSCs comprising performing at least one targeted genome edit at one or more selected sites within a genome, the method comprising: (I), (II) or (III) (I): Genetically modifying iPSCs, comprising one or both of the following (i) and (ii) in any order: (i) introducing into iPSCs one or more constructs, each containing one or more exogenous polynucleotides of interest, for targeted integration at selected sites; (ii) (a) introducing into the iPSCs one or more double strand breaks at the selected sites using one or more endonucleases capable of recognizing the selected sites; and (b) culturing the iPSCs of step (I)(ii)(a) to allow endogenous DNA repair to occur and generate a targeted insertion / deletion at said selected site; (II): Genetically modifying non-pluripotent cells undergoing reprogramming to obtain genomically modified iPSCs, comprising: (i) contacting a non-pluripotent cell with one or more reprogramming factors and, optionally, a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and / or a ROCK inhibitor to initiate reprogramming of said non-pluripotent cell; and (ii) introducing, in any order, one or both of (a) and (b) into the non-pluripotent cell being reprogrammed in step (II)(i): (a) one or more constructs, each containing one or more exogenous polynucleotides of interest, that allow targeted integration at a selected site; (b) one or more double-strand breaks at the selected sites using at least one endonuclease capable of recognizing the selected sites; wherein the cells of step (II)(ii)(b) are cultured to allow endogenous DNA repair to occur and generate a targeted insertion / deletion at said selected site; and, (III): Genetically modifying non-pluripotent cells for reprogramming to obtain genomically modified iPSCs, comprising the following (i) and (ii): (i) introducing into a non-pluripotent cell one or both of (a) and (b), in any order: (a) one or more constructs, each containing one or more exogenous polynucleotides of interest, that allow targeted integration at a selected site; (b) one or more double-strand breaks at the selected sites using at least one endonuclease capable of recognizing the selected sites; wherein the cells of step (III)(i)(b) are cultured to allow endogenous DNA repair to occur and generate a targeted insertion / deletion at said selected site; and (ii) contacting the cells of step (III)(i) with one or more reprogramming factors, and optionally a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and / or a ROCK inhibitor, to obtain genomically modified iPSCs containing a targeted edit at the selected site; thereby obtaining genomically modified iPSCs that contain at least one functional targeted genome edit; At least one of the targeted genome editing methods (I), (II), and (III) includes disruption of a gene that results in an HLA deficiency; The methods of (I), (II) and (III) further include differentiating the genomically modified iPSCs to obtain genomically modified hematopoietic cells containing at least one targeted genome edit.

2. The method of claim 1 , wherein the hematopoietic cells comprise immune cells.

3. The method of claim 2 , wherein the immune cells comprise effector cells.

4. The method of claim 3 , wherein the effector cells comprise T cells or NK cells.

5. The method of any one of claims 1 to 4, wherein the disruption of a gene that results in an HLA deficiency comprises disruption of a gene encoding a protein of the MHC class I complex or the MHC class II complex.

6. 5. The method of any one of claims 1 to 4, wherein the gene disruption resulting in an HLA deficiency comprises a disruption of one or more of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5 and RFXAP.

7. 2. The method of claim 1, wherein the at least one targeted genome edit at the one or more selected sites comprises (i) the insertion of one or more exogenous polynucleotides, or (ii) the insertion / deletion of one or more endogenous polynucleotides encoding immune response and regulation, safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutical active proteins and peptides, drug target candidates, or proteins that promote or inhibit transplantation, trafficking, homing, viability, self-renewal, persistence, and / or survival of genomically modified iPSCs or their derived cells.

8. (i) the exogenous polynucleotide is operably linked to (1) one or more exogenous promoters, including CMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, time-specific, tissue-specific, or cell-type specific promoters; or (2) one or more endogenous promoters contained at a selected site, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1; (ii) the exogenous polynucleotide encodes a protein comprising a caspase, a thymidine kinase, a cytosine deaminase, a modified EGFR, a B cell CD20, or any combination thereof, and the exogenous polynucleotide is integrated into a different safe harbor locus comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1; (iii) the endogenous gene comprises at least one of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region; and / or (iv) the genomically modified iPSCs comprise an exogenous polynucleotide encoding a caspase at the AAVS1 locus and an exogenous polynucleotide encoding a thymidine kinase at the H11 locus.

9. 2. The method of claim 1, wherein group (I), group (II) and / or group (III) further comprises contacting the genomically modified iPSCs with a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor and a ROCK inhibitor.

10. Features: (i) one or more exogenous polynucleotides of interest are operably linked to one or more exogenous promoters contained in the construct or to an endogenous promoter contained at a selected site after integration; (ii) the construct further comprises a pair of homology arms specific to the selected site and flanking the exogenous polynucleotide of interest; (iii) the one or more exogenous polynucleotides remain integrated and functional in the subsequently expanded iPSCs, differentiated cells derived from the iPSCs, and / or de-differentiated cells derived therefrom; (iv) the one or more exogenous polynucleotides are linked to each other by a linker sequence, or the one or more exogenous polynucleotides are expressed under the same or different promoters; (v) the construct further comprises at least one polynucleotide encoding a marker; (vi) the construct further comprises a polynucleotide encoding a marker driven by an endogenous promoter at the selected site; and / or (vii) the one or more exogenous polynucleotides encode a safety switch protein and are operably linked to a CAG; The method of claim 1 , further comprising at least one of:

11. Features: (i) the linker sequence (a) encodes a self-cleaving peptide, or (b) provides an internal ribosome entry sequence (IRES); (ii) the selected site is a safe harbor locus, a highly expressed locus, a time-expressed locus, or a locus for interference, or the one or more exogenous polynucleotides are operably linked to an exogenous promoter, including a CMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, time-specific, tissue-specific, or cell type-specific promoter; (iii) the selected sites include AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, RUNX1, or loci meeting the criteria for a genomic safe harbor, B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene within the chromosome 6p21 region; The method of claim 10, comprising:

12. The method of claim 1 , wherein the differentiation step does not include the formation of embryoid bodies.

13. The method of claim 1, wherein the genomically modified hematopoietic cells comprise CD34 cells, hemogenic endothelial cells, hematopoietic stem / progenitor cells, hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, or B cells.

14. 1. A method for generating genomically modified non-pluripotent cells from iPSCs, comprising: (i) exposing the iPSCs to conditions sufficient to initiate lineage-specific differentiation; and (ii) Genetically modifying the differentiating cells of step (i) to obtain genomically modified non-pluripotent cells by either or both of (a) and (b) in any order: (a) introducing into the differentiating cells of step (i) one or more constructs comprising one or more exogenous polynucleotides of interest for targeted integration at selected sites; (b) (1) introducing into the differentiating cells of step (i) one or more double-stranded breaks at the selected sites using at least one endonuclease capable of recognizing said selected sites; and (2) culturing the cells resulting from step (ii)(b)(1) to allow endogenous DNA repair to occur and generate targeted insertions / deletions at said selected sites; Including, thereby obtaining a genomically modified non-pluripotent cell containing one or more functional targeted edits at one or more selected sites; and (iii) differentiating the genomically modified differentiated cell to obtain a genomically modified hematopoietic cell containing one or more functional targeted genome edits; The one or more functional targeted genomic modifications include disruption of a gene that results in an HLA deficiency; method.

15. The method of claim 14, wherein the genomically modified hematopoietic cells comprise CD34 cells, hemogenic endothelial cells, hematopoietic stem / progenitor cells, hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, or B cells.

16. A genome-modified hematopoietic cell produced by the method of any one of claims 1 to 13.

17. The genome modified hematopoietic cell is (i) one or more insertions / deletions in endogenous genes related to targeting modalities, receptors, signaling molecules, transcription factors, potential drug targets, immune response control and regulation, or encoding proteins that inhibit the engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or their derived cells; (ii) deletion or reduced expression of at least one of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene within the chromosome 6p21 region; (iii) HLA-E, HLA-G, CD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A ２Ａ Introduction or upregulation of at least one of the following surface triggering receptors for binding to an R, a CAR, a TCR, an Fc receptor, an engager, and a bispecific or multispecific or universal engager; (iv) deletion of HLA class I and / or class II molecules; (v) an integrated or non-integrated exogenous polynucleotide encoding one or more of the HLA-E protein, HLA-G protein, CD16 protein, 41BBL protein, and PDL1 protein; or an expression introduction of one or more of the HLA-E protein, HLA-G protein, CD16 protein, 41BBL protein, and PDL1 protein; (vi) at least one exogenous polynucleotide encoding a high affinity CD16 receptor; at least one exogenous polynucleotide encoding a non-cleavable CD16 receptor; at least one exogenous polynucleotide encoding a high affinity and non-cleavable CD16 receptor (hnCD16); at least one exogenous polynucleotide encoding a non-cleavable HLA-E; or at least one exogenous polynucleotide encoding a non-cleavable HLA-G; (vii) null or low expression of B2M, null or low expression of TAP1, null or low expression of TAP2, null or low expression of tapasin; transfection of HLA-E or uncleaved HLA-E protein; transfection of HLA-G or uncleaved HLA-G protein; transfection of PDL1; high affinity uncleaved CD16 (hnCD16); Fc receptor; T cell receptor (TCR), chimeric antigen receptor (CAR); one or more suicide genes expressing a safety-switching protein; bispecific, multispecific or universal engager, or any combination thereof; and (viii) improved persistence, increased resistance to immune cells, increased resistance to T cells and / or NK cells compared to the same hematopoietic cells that are not genomically modified; The genomically modified hematopoietic cell of claim 16 .

18. A genomically modified hematopoietic cell generated using the method of claim 14 or 15, wherein the hematopoietic cell comprises one or more functional targeted genome edits.

19. The non-pluripotent cells are: (i) comprises a hemogenic endothelial cell, a CD34 cell, a hematopoietic stem or progenitor cell, a hematopoietic multipotent progenitor cell, a T cell progenitor cell, a NK cell progenitor cell, a T cell, a NKT cell, a NK cell, or a B cell; (ii) one or more insertions / deletions in endogenous genes related to targeting modalities, receptors, signaling molecules, transcription factors, potential drug targets, immune response control and regulation, or encoding proteins that inhibit the engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or their derived cells; (iii) deletion or reduced expression of at least one of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene within the chromosome 6p21 region; (iv) HLA-E, HLA-G, CD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A ２Ａ Introduction or upregulation of at least one of the following surface triggering receptors for binding to an R, a CAR, a TCR, an Fc receptor, an engager, and a bispecific or multispecific or universal engager; (v) deletion of HLA class I and / or class II molecules; (vi) an integrated or non-integrated exogenous polynucleotide encoding one or more of the HLA-E protein, HLA-G protein, CD16 protein, 41BBL protein, and PDL1 protein; or an expression introduction of one or more of the HLA-E protein, HLA-G protein, CD16 protein, 41BBL protein, and PDL1 protein; (vii) at least one exogenous polynucleotide encoding a high affinity CD16 receptor; at least one exogenous polynucleotide encoding a non-cleavable CD16 receptor; at least one exogenous polynucleotide encoding a high affinity and non-cleavable CD16 receptor (hnCD16); at least one exogenous polynucleotide encoding a non-cleavable HLA-E; or at least one exogenous polynucleotide encoding a non-cleavable HLA-G; (viii) null or low expression of B2M, null or low expression of TAP1, null or low expression of TAP2, null or low expression of tapasin; transfection of HLA-E or uncleaved HLA-E protein; transfection of HLA-G or uncleaved HLA-G protein; transfection of PDL1; high affinity uncleaved CD16 (hnCD16); Fc receptor; T cell receptor (TCR), chimeric antigen receptor (CAR); one or more suicide genes expressing a safety-switching protein; bispecific, multispecific or universal engagers, or any combination thereof; (ix) improved persistence, increased resistance to immune cells, increased resistance to T cells and / or NK cells compared to the same hematopoietic cell that is not genomically modified; (x) at least two exogenous polynucleotides, each integrated into the same safe harbor locus and encoding the same or different safety switch proteins; (xi) At least two exogenous polynucleotides, each integrated into the same safe harbor locus and encoding the same or different safety switch proteins; (xii) capable of being reprogrammed into iPSCs, the iPSCs containing the same functional exogenous polynucleotide contained in the non-pluripotent cell; (xiii) capable of transdifferentiating into non-pluripotent cells of a different fate; and / or (xiv) having improved persistence, increased resistance to immune cells, increased resistance to T cells and / or NK cells, or increased immune tolerance; The genomically modified hematopoietic cell of claim 18, comprising at least one of the following:

20. A pharmaceutical composition comprising a pharma- ceutically acceptable vehicle and the genome modified hematopoietic cell according to any one of claims 16 to 19.

21. Therapeutic use of the pharmaceutical composition of claim 20 by introducing the pharmaceutical composition of claim 20 into a subject in need of adoptive cell therapy, said subject having an autoimmune disorder; a hematopoietic tumor; a solid tumor; cancer, or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.