Genome engineering of pluripotent cells

Abstract

The invention name is genomic engineering of pluripotent cells. Provided are methods and compositions for obtaining genomically engineered iPSCs and derivative cells, wherein stable, functional genomic editing occurs at selected loci. Also provided are cell populations or clonal cell lines derived from genomically engineered iPSCs comprising targeted integration of one or more exogenous polynucleotides and / or insertion / deletion of one or more selected endogenous genes.

Description

[0001] This application is a divisional application. The original application was filed on November 4, 2016, with application number 2016800748113 (PCT / US2016 / 060699) and the invention title "Genomic Engineering Modification of Pluripotent Cells".

[0002] Related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 251,032, filed November 4, 2015; U.S. Provisional Application No. 62 / 337,258, filed May 16, 2016; and U.S. Provisional Application No. 62 / 366,503, filed July 25, 2016, the disclosures of which are incorporated herein by reference in their entirety. Technical Field

[0004] This disclosure generally relates to the field of gene editing and genome engineering of stem cells. More specifically, this disclosure relates to the use of molecular strategies to regulate genes in clonal pluripotent stem cells, said molecular strategies targeting specific gene loci and ensuring the continuous maintenance and / or functionalization of the edited genetic material. Background Technology

[0005] As research into human induced pluripotent stem cells (hiPSCs) continues to advance, and with the emergence of clinical trials for genetically engineered hiPSC-derived cell therapies, safety concerns related to the delivery of genetically modified cells must be addressed and mitigated. To address these safety concerns, various strategies have been explored to facilitate the selective exclusion of abnormal cells, including recombinant peptides, monoclonal antibodies, small-molecule-regulated enzyme activity, and gene-specific modifications. Generally, previous studies have utilized viral vectors (e.g., lentiviruses) and short promoters to stably introduce suicide genes (including herpes simplex virus thymidine kinase or inducible caspase-9 (iCasp9)) into human cells. However, the use of viral vectors can induce random integration events that can cleave or activate disease-related genes, potentially leading to detrimental effects. Other problems with current methods include (but are not limited to) low insertion rates; random insertions; mutations; high insertion copy numbers; and difficulties in sorting cells when selecting from heterogeneous cell populations with copy number variations due to random insertions. Furthermore, during iPSC genome engineering, many artificial promoters and genomic regions are prone to epigenetic silencing under pluripotent and differentiated states, rendering the promoters or inserted genes unresponsive to events such as cell expansion, passage, reprogramming, differentiation, and / or dedifferentiation. Therefore, identifying optimal genome editing strategies, integration sites, promoters, and other factors is crucial to maintaining the responsiveness of the inserted functional pattern without compromising safety, especially in the development of genetically engineered immune cells for therapeutic purposes. Summary of the Invention

[0006] One object of the present invention is to provide methods and compositions for generating single-cell-source iPSC clones or cells derived therefrom, comprising one or more gene modifications at selected sites, including polynucleotide insertions, deletions, and substitutions, wherein the modifications are retained and remain functional in subsequently derived cells after differentiation, dedifferentiation, reprogramming, amplification, passage, and / or transplantation.

[0007] One objective of this invention is to generate single-cell-source iPSC clones comprising one or more gene modifications located at selected sites, produced by reprogramming non-pluripotent cells containing the same gene modifications, including targeted integration and / or targeted insertion / deletion. Specifically, one objective is to reprogram non-pluripotent cells before genome engineering the reprogrammed cells, thereby first obtaining iPSCs or poorly differentiated cells, followed by genome editing. Reprogramming non-pluripotent cells after genome engineering is also an objective, thereby first obtaining genome-engineered non-pluripotent cells, followed by cell reprogramming. Another objective is to enable simultaneous non-pluripotent cell reprogramming and genome engineering, thereby eliminating the need to isolate intermediate cells for reprogramming or genome engineering, and allowing both processes to occur simultaneously in a single cell pool.

[0008] One objective of this invention is to generate non-pluripotent cells derived from iPSCs (or poorly differentiated cells) containing one or more gene-modified sites located at selected loci, including (but not limited to) CD34 cells, hematopoietic endothelial cells, HSCs (hematopoietic stem cells and progenitor cells), hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells, and B cells, generated by differentiating iPSCs or poorly differentiated cells containing the same gene-modified sites located at the same selected loci. Specifically, one objective of this invention is to differentiate poorly differentiated cells (including progenitor cells and pluripotent cells) after genome engineering, thereby first obtaining genome-engineered poorly differentiated cells, followed by cell differentiation. Another objective of this invention is to enable the differentiation of poorly differentiated cells and the genome engineering of poorly differentiated cells to occur simultaneously, thereby eliminating the need to isolate intermediate cells for differentiation or genome engineering, and allowing both processes to occur simultaneously in a single cell pool.

[0009] One aspect of the invention provides a construct comprising: (1) one or more exogenous polynucleotides of interest, operatively ligated to one or more exogenous promoters contained in the construct, or to an endogenous promoter contained at a selected site, after integration; and (2) a pair of homologous arms specific to the selected site and side-attached to the exogenous polynucleotide of interest, for targeting the integration of the exogenous polynucleotide into the selected site in iPSCs; and those one or more exogenous polynucleotides remain integrated and functional in subsequently amplified iPSCs, differentiated cells derived from 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 construct further comprises at least one polynucleotide encoding a marker. In other embodiments, the polynucleotides contained in the construct encode a marker driven by an endogenous promoter at the selected site. In some embodiments, the selected site is a safe harbor locus, a high-expression locus, a time-expressed locus, or an interruption locus. In some embodiments, the safe harbor locus may be AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or a locus that meets the genomic safe harbor criteria as defined herein. In some embodiments, the disruption locus comprises TAP1, TAP2, or a TAP-associated glycoprotein (tapasin), or a disruption of other genes of interest that are associated with desired cellular function or characteristics, such as NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the 6p21 region of chromosome 6.

[0010] In some embodiments of the aforementioned construct, at least one exogenous polynucleotide is operatively linked to an exogenous promoter, comprising CMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters. In one embodiment, the exogenous promoter included in the construct is CAG. In some other embodiments, one or more polynucleotides encode one or more of the following: safety switch proteins; targeting mechanisms; receptors; signal transduction molecules; transcription factors; pharmaceutically active proteins or peptides; drug target candidates; and proteins that promote the transplantation, transport, homing, viability, self-renewal, retention, and / or survival of iPSCs or their derivatives integrated with the construct. In some specific embodiments, one or more polynucleotides encode a safety switch protein comprising caspase, thymidine kinase, cytosine deaminase, modified EGFR, B-cell CD20, or any combination thereof. In some embodiments, the caspase may be caspase 9, caspase 3, or caspase 7. In some other embodiments, the polynucleotide encoding the safety switch protein is operatively linked to CAG in the construct.

[0011] One aspect of the present invention provides a method for generating genome-engineered iPSCs, said iPSCs comprising at least one targeted genome editing occurring at one or more selected sites in the genome, said method comprising (I), (II), or (III):

[0012] (I): Genetically engineer iPSCs by one or both of (i) and (ii) in any order: (i) introducing one or more constructs into iPSCs to allow targeted integration at selected sites; (ii) (a) introducing one or more double-strand breaks at selected sites into iPSCs using one or more endonucleases capable of recognizing selected sites; and (b) culturing the iPSCs of steps (I) (ii) (a) to achieve endogenous DNA repair, thereby generating targeted insertions / deletions at selected sites, thereby obtaining genome-engineered iPSCs containing at least one functional targeted genome edit, wherein the genome-engineered iPSCs are capable of differentiating into partially differentiated or fully differentiated cells.

[0013] (II): Genetically engineering reprogrammed non-pluripotent cells to obtain genome-engineered iPSCs, comprising: (i) contacting non-pluripotent cells with one or more reprogramming factors and optionally a small molecule composition to initiate non-pluripotent cell reprogramming, said small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor; and (ii) introducing one or both of (a) and (b) in any order into the reprogrammed non-pluripotent cells of step (II) (i): (a) one or more constructs to allow targeted integration at selected sites; (b) generating one or more double-strand breaks at selected sites using at least one endonuclease capable of recognizing the selected sites, wherein the cells of step (II) (ii) (b) are cultured to achieve endogenous DNA repair, thereby generating targeted insertions / deletions at selected sites, thereby obtaining genome-engineered iPSCs comprising at least one functionally targeted genome editing, and wherein the genome-engineered iPSCs are capable of differentiating into partially differentiated or fully differentiated cells.

[0014] (III): Genetically engineered non-pluripotent cells to obtain genome-engineered iPSCs, comprising (i) and (ii): introducing one or both of (a) and (b) in any order into the non-pluripotent cells: (a) one or more constructs to allow targeted integration at selected sites; (b) one or more double-strand breaks generated at the selected sites using at least one endonuclease capable of recognizing the selected sites, wherein the cells of steps (III) (i) and (b) are cultured to achieve endogenous DNA repair, thereby generating targeted insertions / deletions at the selected sites; and (ii) causing the cells of steps (i) and (b) to undergo further processing. III)(i) cells are contacted with one or more reprogramming factors and optionally a small molecule composition to obtain genome-engineered iPSCs, the small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and / or a ROCK inhibitor, the iPSCs comprising targeted editing at selected sites, thereby obtaining genome-engineered iPSCs comprising at least one functional targeted genome editing, and wherein the genome-engineered iPSCs are capable of differentiating into partially differentiated or fully differentiated cells.

[0015] In one embodiment of the above method, at least one targeted genome editing occurring at one or more selected sites comprises the insertion of one or more exogenous polynucleotides encoding safety switch proteins, targeting modes, receptors, signal transduction molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote the transplantation, transport, homing, viability, self-renewal, retention, and / or survival of genome-engineered iPSCs or their derivatives. In some embodiments, the inserted exogenous polynucleotide is operatively linked to (1) one or more exogenous promoters comprising 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, comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the genome safe harbor criteria. In some embodiments, the genome-engineered iPSCs generated using the above method contain one or more different exogenous polynucleotides, the proteins encoded by the polynucleotides including caspase, thymidine kinase, cytosine deaminase, modified EGFR or B-cell CD20, wherein when the genome-engineered iPSCs contain two or more suicide genes, the suicide genes are integrated into different safe harbor loci, the safe harbor loci including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR or RUNX1.

[0016] In some other embodiments, the genome-engineered iPSCs generated using the methods provided herein contain insertions / deletions at one or more endogenous genes associated with: targeting patterns, receptors, signaling molecules, transcription factors, drug target candidates, immune response regulation and modulation, or proteins that inhibit the transplantation, transport, homing, viability, self-renewal, survival, and / or survival of iPSCs or their derived cells. In some embodiments, the endogenous gene for disruption comprises at least one of the following: B2M, TAP1, TAP2, TAP-associated glycoprotein, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and chromosomes. 6p Any gene in region 21.

[0017] In some other embodiments, the genome-engineered iPSCs produced using the methods provided herein contain an exogenous polynucleotide encoding caspase at the AAVS1 locus and an exogenous polynucleotide encoding thymidine kinase at the H11 locus.

[0018] In other embodiments, methods (I), (II), and / or (III) further include contacting the genome-engineered iPSCs with a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor to maintain the pluripotency of the genome-engineered iPSCs. In one embodiment, the resulting genome-engineered iPSCs comprising at least one targeted genome edit are functional, capable of efficient differentiation, and able to differentiate into non-pluripotent cells comprising the same functional genome edit.

[0019] Another aspect of the invention provides genome-engineered iPSCs generated by any of the methods described above. In one embodiment, the resulting genome-engineered iPSCs comprise one or more exogenous polynucleotides introduced using the constructs provided above, and said exogenous polynucleotides are integrated into the iPSCs at one or more selected sites. In one embodiment, the resulting genome-engineered iPSCs comprise one or more insertions / deletions from endogenous genes associated with: targeting patterns; receptors; signal transduction molecules; transcription factors; drug target candidates; immune response regulation and control; and proteins that inhibit the transplantation, transport, homing, viability, self-renewal, retention, and / or survival of iPSCs or their derived cells. In one embodiment, said endogenous genes comprise one or more of the following: B2M, TAP1, TAP2, TAP-associated glycoproteins, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the 6p21 region of chromosome 6. In some other embodiments, the genome-engineered iPSCs comprise the deletion or reduced expression of at least one of the following: B2M, TAP1, TAP2, TAP-associated glycoprotein, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the 6p21 region of chromosome 6. In some other embodiments, the genome-engineered iPSCs comprise the introduction or increase of the expression of at least one of the following: HLA-E, HLA-G, CD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2AR, CAR, TCR, Fc receptors, conjugates, and surface trigger receptors for coupling with bispecific, multispecific, or universal conjugates. In some embodiments, expression introduction or enhancement is performed by an integration or non-integration method to introduce a polynucleotide encoding a foreign protein. In some embodiments, the genome-engineered iPSCs lack HLA class I and / or class II. In some embodiments, the genome-engineered iPSCs comprise B2M knockout or low-content, TAP1 knockout or low-content, and / or TAP2 knockout or low-content. In some embodiments, the genome-engineered iPSCs comprise an integrated or non-integrated foreign polynucleotide encoding one or more of HLA-E, HLA-G, CD16, 41BBL, and PDL1 proteins; or wherein the genome-engineered iPSCs comprise the expression introduction of one or more of HLA-E, HLA-G, CD16, 41BBL, and PDL1 proteins. In some embodiments, the expression introduction is an increase in the expression of a non-expressed or low-expressed gene contained in the cell. In some embodiments, the non-integrated exogenous polynucleotide is introduced using Sendai virus, a free organism, or a plastid.

[0020] In some other embodiments, the genome-engineered 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 iPSC comprises one or more exogenous polynucleotides encoding proteins, said proteins comprising: safety switch proteins; targeting mechanisms; receptors; signal transduction molecules; transcription factors; pharmaceutically active proteins or peptides; drug target candidates; or proteins that promote the transplantation, transport, homing, viability, self-renewal, retention, and / or survival of iPSCs or their derived cells.

[0021] In some other embodiments, upon integration, one or more constructs contain exogenous polynucleotides operatively 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 at selected sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1. In some other embodiments, iPSCs contain at least one exogenous polynucleotide encoding a safety switch protein, said safety switch protein including caspase, thymidine kinase, cytosine deaminase, modified EGFR, or B-cell CD20. In some specific embodiments, the iPSCs comprise at least two exogenous polynucleotides encoding the same or different safety switch proteins, the exogenous polynucleotides being integrated into the same safe harbor locus, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the genomic safe harbor criteria. In one embodiment, the iPSCs comprise two exogenous polynucleotides encoding the same or different safety switch proteins, the exogenous polynucleotides being integrated into different safe harbor loci, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the genomic safe harbor criteria. In one particular embodiment, the iPSCs comprise two exogenous polynucleotides encoding the same or different safety switch proteins, the exogenous polynucleotides being integrated into different safe harbor loci, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR or RUNX1, or other loci that meet the genomic safe harbor criteria.

[0022] In some embodiments, genome-engineered iPSCs generated using the provided method achieve improved survival, enhanced resistance to immune cells, enhanced immune resistance, or enhanced resistance to T and / or NK cells compared to iPSCs that have not undergone the same genome engineering. The genome-engineered iPSCs maintain pluripotency and retain the potential to differentiate into non-pluripotent cells with the same functional genome engineering. In one embodiment, the resulting genome-engineered iPSCs are capable of differentiating into partially differentiated or fully differentiated cells, wherein the partially differentiated or fully differentiated cells contain at least one targeted genome edit contained in the iPSCs. In one embodiment, the resulting genome-engineered iPSCs are capable of differentiating into hematopoietic lineage cells, including mesodermal cells, CD34 cells, hematopoietic endothelial cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells, or B cells.

[0023] Another aspect of the present invention provides genome-engineered iPSCs comprising (i) B2M knockout or low-content; (ii) TAP1 knockout or low-content; (iii) TAP2 knockout or low-content; (iv) TAP-associated glycoprotein knockout or low-content; (v) introduction of HLA-E or non-cleavable HLA-E expression; (vi) introduction of HLA-G or non-cleavable HLA-G expression; (vii) introduction of PDL1 expression; (viii) non-cleavable high-affinity CD16 (hnCD16); (ix) Fc receptor; (x) T cell receptor (TCR); (xi) chimeric antigen receptor (CAR); (xii) one or more suicide genes expressing safety switch proteins; (xiii) bispecific, multispecific, or universal conjugates, or any combination thereof. In some embodiments, the genome-engineered iPSCs are B2M knockout, TAP1 knockout, TAP2 knockout, or TAP-associated glycoprotein knockout. In some embodiments, the genome-engineered iPSCs are B2M knockout types, wherein expression of one or more of HLA-E, HLA-G, PDL1, and hnCD16 is introduced. In some embodiments, the genome-engineered iPSCs are B2M knockout types, wherein expression of one or more of HLA-E, HLA-G, PDL1, hnCD16, and CAR is introduced. In some embodiments, the genome-engineered iPSCs are B2M knockout types, wherein expression of one or more of HLA-E, HLA-G, PDL1, hnCD16, CAR, and at least one safety switch protein is introduced. In some embodiments, the expression introduction is an increase in the expression of a non-expressed or low-expressed gene contained in the cell. In some other embodiments, the expression introduction is exogenous expression. In some embodiments, TCR, CAR, conjugate, Fc receptor, or suicide gene is inducible. In some embodiments, genome-engineered iPSCs, compared to iPSCs that have not undergone the same genome engineering, achieve improved survival, enhanced resistance to immune cells, enhanced resistance to T and / or NK cells, or enhanced immune resistance, wherein genome-engineered iPSCs maintain pluripotency; and wherein genome-engineered iPSCs maintain the potential to differentiate into non-pluripotent cells that retain the same functional genome engineering.

[0024] Additionally, HLA-deficient modified iPSCs are provided, lacking HLA class I and / or II, but additionally containing one or more of (1) exogenous HLA-E, HLA-G, CD16, 4lBBL, CD47, CD113, and PDL1; or (2) expression introduction of one or more of HLA-E, HLA-G, CD16, 4lBBL, CD47, CD113, and PDL1 proteins; and optionally, one or more of (3) TCR, CAR, conjugate, Fc receptor, and single or dual safety switch proteins. In some embodiments, the expression introduction is an increase in the expression of a non-expressed or low-expressed gene contained in the cell. In one embodiment, the TCR, CAR, conjugate, Fc receptor, or suicide gene contained in the HLA-deficient modified iPSCs is inducible. In some embodiments, HLA-deficient modified iPSCs achieve improved survival, enhanced resistance to immune cells, enhanced resistance to T and / or NK cells, or enhanced immune resistance compared to iPSCs that have not undergone the same genomic engineering, wherein the HLA-deficient modified iPSCs maintain pluripotency; and wherein the HLA-deficient modified iPSCs maintain differentiation potential to generate HLA-deficient non-pluripotent cells with the same genomic engineering function.

[0025] Another aspect of the present invention provides a method for generating genome-engineered non-pluripotent cells derived from genome-engineered iPSCs, the method comprising: (i) obtaining genome-engineered iPSCs or HLA-deficient modified iPSCs as provided above, wherein the iPSCs contain at least one functional genome edit; and (ii) differentiating the genome-engineered iPSCs or HLA-deficient modified iPSCs to obtain derived non-pluripotent cells containing the same functional targeted genome edits contained in the genome-engineered iPSCs. In some embodiments, the differentiation step does not require the formation of embryoid bodies. In some embodiments, differentiation is performed without a feeder layer, matrix, or serum. In some embodiments, the derived non-pluripotent cells comprise hematopoietic lineage cells. In some embodiments, the derived non-pluripotent cells comprise mesodermal cells, CD34 cells, hematopoietic endothelial cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells, or B cells.

[0026] Another aspect of the present invention provides a method for generating genome-engineered non-pluripotent cells from iPSCs, the method comprising (i) subjecting iPSCs to conditions sufficient to induce lineage-specific differentiation; and (ii) genetically engineering the differentiated cells of step (i) by one or both of (a) and (b) in any order to obtain genome-engineered non-pluripotent cells: (a) introducing one or more constructs into the differentiated cells of step (i) to allow targeted integration at selected sites; (b)(1) introducing one or more double-strand breaks at selected sites into the differentiated cells of step (i) using one or more endonucleases capable of recognizing the selected sites; and (2) culturing the cells obtained from steps (ii)(b)(1) to achieve endogenous DNA repair, thereby generating targeted insertions / deletions at selected sites, thereby obtaining genome-engineered non-pluripotent cells comprising one or more functionally targeted edits at one or more selected sites. In some embodiments, the genome-engineered non-pluripotent cells comprise hematopoietic lineage cells. In some other embodiments, the genome-engineered non-pluripotent cells include mesodermal cells, CD34 cells, hematopoietic endothelial cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells, or B cells.

[0027] This invention further provides genome-engineered iPSC-derived non-pluripotent cells generated using the above-described general methods, wherein the generated non-pluripotent cells contain one or more functionally targeted genome editing features. In one embodiment, the genome-engineered iPSC-derived non-pluripotent cells comprise mesodermal cells, hematopoietic endothelial cells, CD34 cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells, or B cells. In some embodiments, the genome-engineered iPSC-derived non-pluripotent cells comprise one or more exogenous polynucleotides encoding proteins including safety switch proteins; targeted pattern receptors; signal transduction molecules; transcription factors; pharmaceutically active proteins or peptides; drug target candidates; or proteins that promote non-pluripotent cell transplantation, transport, homing, viability, self-renewal, retention, and / or survival. In some other embodiments, the genome-engineered iPSC-derived non-pluripotent cells contain one or more insertions / deletions of endogenous genes associated with: targeting patterns; receptors; signal transduction molecules; transcription factors; drug target candidates; immune response regulation and control; and proteins that inhibit non-pluripotent cell transplantation, transport, homing, viability, self-renewal, retention, and / or survival. In some specific embodiments, the genome-engineered iPSC-derived non-pluripotent cells contain one or more endogenous genes with insertions / deletions, and said endogenous genes include B2M, TAP1, TAP2, TAP-associated glycoproteins, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, or any gene in the 6p21 region of chromosome 6. In some embodiments, the genome-engineered derived non-pluripotent cells comprise the deletion or reduced expression of at least one of the following: B2M, TAP1, TAP2, TAP-associated glycoprotein, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the 6p21 region of chromosome 6. In some other embodiments, the genome-engineered derived non-pluripotent cells comprise the introduction or increase of the expression of at least one of the following: HLA-E, HLA-G, CD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, CAR, TCR, Fc receptors, conjugates, and surface-triggered receptors for coupling with bispecific, multispecific, or universal conjugates.

[0028] In some other embodiments, the genome-engineered iPSC-derived non-pluripotent cells comprise: (i) at least two exogenous polynucleotides each integrated into the same safe harbor locus, wherein the exogenous polynucleotides encode the same or different safety switch proteins; or (ii) at least two exogenous polynucleotides each integrated into different safe harbor loci, wherein the exogenous polynucleotides encode the same or different safety switch proteins. In some embodiments of the genome-engineered iPSC-derived non-pluripotent cells, the exogenous polynucleotides encode safety switch proteins selected from caspase, thymidine kinase, cytosine deaminase, modified EGFR, B-cell CD20, and any combination thereof. In some embodiments of the genome-engineered iPSC-derived non-pluripotent cells, the safe harbor locus comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the genome safe harbor criteria. In some embodiments, genome-engineered iPSC-derived non-pluripotent cells can be reprogrammed into iPSCs, wherein the iPSCs contain exogenous polynucleotides with the same function as those found in non-pluripotent cells. In some embodiments of genome-engineered iPSC-derived non-pluripotent cells, the reprogrammed iPSCs can differentiate into partially differentiated or fully differentiated cells. In other embodiments, genome-engineered iPSC-derived non-pluripotent cells can transdifferentiate into non-pluripotent cells of different fates.

[0029] Another aspect of the present invention provides genomically engineered iPSC-derived hematopoietic lineage cells comprising (i) B2M knockout or low-content cells; (ii) TAP1 knockout or low-content cells; (iii) TAP2 knockout or low-content cells; (iv) TAP-associated glycoprotein knockout or low-content cells; (v) expression of HLA-E or non-cleavable HLA-E; (vi) expression of HLA-G or non-cleavable HLA-G; (vii) expression of PDL1; (viii) non-cleavable high-affinity CD16 (hnCD16); (ix) Fc receptor; (x) T cell receptor (TCR); (xi) chimeric antigen receptor (CAR); (xii) one or more suicide genes expressing safety switch proteins; (xiii) bispecific, multispecific, or universal conjugates, or any combination thereof. In some embodiments, the genome-engineered iPSC-derived hematopoietic lineage cells are B2M knockout, TAP1 knockout, TAP2 knockout, or TAP-related glycoprotein knockout types. In some embodiments, the genome-engineered iPSC-derived hematopoietic lineage cells are B2M knockout types, wherein expression of one or more of HLA-E, HLA-G, PDL1, and hnCD16 is introduced. In some embodiments, the genome-engineered iPSC-derived hematopoietic lineage cells are B2M knockout types, wherein expression of one or more of HLA-E, HLA-G, PDL1, hnCD16, and CAR is introduced. In some embodiments, the genome-engineered iPSC-derived hematopoietic lineage cells are B2M knockout types, wherein expression of one or more of HLA-E, HLA-G, PDL1, hnCD16, CAR, and at least one safety switch protein is introduced. In some embodiments, the expression introduction is an increase in the expression of a non-expressed or low-expressed gene contained in the cell. In some other embodiments, the expression introduction is exogenous expression. In some embodiments, the TCR, CAR, conjugate, Fc receptor, or suicide gene contained in the genome-engineered iPSC-derived hematopoietic lineage cells are inducible. In some embodiments, the genome-engineered iPSC-derived hematopoietic lineage cells achieve improved survival, enhanced resistance to immune cells, enhanced resistance to T and / or NK cells, or enhanced immune resistance; and the genome-engineered low-differentiation hematopoietic lineage cells maintain the potential to differentiate into non-pluripotent cells that retain the same functional genome-engineered genes.

[0030] The present invention further provides a composition comprising iPSC-derived hematopoietic cells, the composition comprising (a) genome-engineered iPSC-derived CD34 cells; (b) genome-engineered iPSC-derived hematopoietic stem cells and progenitor cells; (c) genome-engineered iPSC-derived pluripotent hematopoietic progenitor cells; (d) genome-engineered iPSC-derived T cell progenitor cells; (e) genome-engineered iPSC-derived NK cell progenitor cells; (f) genome-engineered iPSC-derived T cells; (g) genome-engineered iPSC-derived NK cells, and any combination thereof.

[0031] Further, genome-engineered hematopoietic lineage cells are provided, comprising (i) B2M knockout or low-content cells; (ii) TAP1 knockout or low-content cells; (iii) TAP2 knockout or low-content cells; (iv) TAP-associated glycoprotein knockout or low-content cells; (v) expression introduction of HLA-E or non-cleavable HLA-E; (vi) expression introduction of HLA-G or non-cleavable HLA-G; (vii) expression introduction of PDL1; (viii) non-cleavable high-affinity CD16 (hnCD16); (ix) Fc receptor; (x) T cell receptor (TCR); (xi) chimeric antigen receptor (CAR); (xii) one or more suicide genes expressing safety switch proteins; (xiii) bispecific, multispecific, or universal conjugates, or any combination thereof. In some embodiments, the genome-engineered hematopoietic lineage cells are B2M knockout, TAP1 knockout, TAP2 knockout, or TAP-associated glycoprotein knockout cells. In some embodiments, the genome-engineered hematopoietic lineage cells are B2M knockout cells, wherein expression of one or more of HLA-E, HLA-G, PDL1, and hnCD16 is introduced. In some embodiments, the genome-engineered hematopoietic lineage cells are B2M knockout cells, wherein expression of one or more of HLA-E, HLA-G, PDL1, hnCD16, and CAR is introduced. In some embodiments, the genome-engineered hematopoietic lineage cells are B2M knockout cells, wherein expression of one or more of HLA-E, HLA-G, PDL1, hnCD16, CAR, and at least one safety switch protein is introduced. In some embodiments, the expression introduction is an increase in the expression of a non-expressed or low-expressed gene contained in the cell. In some other embodiments, the expression introduction is exogenous expression. In some embodiments, the TCR, CAR, or suicide gene contained in the genome-engineered hematopoietic lineage cells is inducible. In some embodiments, genome-engineered hematopoietic lineages achieve improved survival, enhanced resistance to immune cells, enhanced resistance to T and / or NK cells, or enhanced immune resistance; and genome-engineered low-differentiation hematopoietic lineages maintain the potential to differentiate into non-pluripotent cells that retain the same functional genome-engineered cells.

[0032] Additionally, modified hematopoietic lineage cells lacking HLA are provided, lacking HLA class I and / or II, and additionally containing one or more of (1) exogenous HLA-E, HLA-G, CD16, 4lBBL, CD47, CD113, and PDL1; or (2) expression introduction of one or more of HLA-E, HLA-G, CD16, 4lBBL, CD47, CD113, and PDL1 proteins; and optionally, one or more of (3) TCR, CAR, conjugate, Fc receptor, and single or dual safety switch proteins. In some embodiments, the expression introduction is an increase in the expression of a non-expressed or low-expressed gene contained in the cells. In some embodiments, the TCR, CAR, conjugate, Fc receptor, or suicide gene contained in the HLA-deficient modified hematopoietic lineage cells is inducible. In some embodiments, HLA-deficient modified hematopoietic lineages, as provided herein, achieve improved survival, enhanced resistance to immune cells, enhanced resistance to T and / or NK cells, or enhanced immune resistance; wherein the HLA-deficient modified poorly differentiated hematopoietic lineages maintain the potential to differentiate into genomically engineered non-pluripotent cells that retain the same functions.

[0033] Another aspect of the present invention provides a composition for obtaining genome-engineered iPSCs, the composition comprising: (i) a genome-engineered non-pluripotent cell as provided herein, the cell comprising at least one targeted genome edit performed at one or more selected sites of the 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, wherein the composition is suitable for obtaining iPSCs comprising the same targeted genome edit contained in genome-engineered non-pluripotent cells. In some embodiments, the at least one targeted genome edit performed on the cells at one or more selected sites comprises the insertion of one or more exogenous polynucleotides encoding safety switch proteins, targeting patterns, receptors, signal transduction molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote the transplantation, transport, homing, viability, self-renewal, retention, and / or survival of non-pluripotent cells and iPSCs reprogrammed therein.

[0034] In some embodiments of the composition, the one or more exogenous polynucleotides are operatively linked to (1) one or more exogenous promoters comprising 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 comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1. In some embodiments of the composition, the one or more exogenous polynucleotides are operatively linked to one or more endogenous promoters contained at a selected insertion site comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1. In some other embodiments of the composition, the genome-engineered non-pluripotent cells comprise one or more exogenous polynucleotides encoding safety switch proteins, said safety switch proteins comprising caspase, thymidine kinase, cytosine deaminase, modified EGFR, B-cell CD20, or any combination thereof. In some embodiments of the composition, the genome-engineered non-pluripotent cells comprise two or more exogenous polynucleotides encoding the same or different safety switch proteins, said exogenous polynucleotides being integrated into different safe harbor loci, said safe harbor loci comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the genomic safe harbor criteria. In a particular embodiment of the composition, the genome-engineered non-pluripotent cells comprise a polynucleotide encoding caspase at the AAVS1 locus and a polynucleotide encoding thymidine kinase at the H11 locus. In some other embodiments of the composition, the genome-engineered non-pluripotent cells contain one or more insertions / deletions in endogenous genes associated with: targeting patterns; receptors; signal transduction molecules; transcription factors; drug target candidates; immune response regulation and modulation; and proteins that inhibit the transplantation, transport, homing, viability, self-renewal, retention, and / or survival of non-pluripotent cells and iPSCs derived therefrom.

[0035] In some embodiments of the composition, one or more endogenous genes interrupted by insertion / deletion are selected from B2M, TAP1, TAP2, TAP-associated glycoprotein, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the 6p21 region of chromosome 6. In some embodiments of the composition, the genome-engineered non-pluripotent cells comprise the deletion or reduced expression of at least one of the following: B2M, TAP1, TAP2, TAP-associated glycoprotein, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the 6p21 region of chromosome 6. In some other embodiments of the composition, the genome-engineered non-pluripotent cells comprise the introduction or increase of the expression of at least one of the following: HLA-E, HLA-G, CD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2AR, CAR, TCR, Fc receptors, conjugates, and surface trigger receptors for coupling with bispecific, multispecific, or universal conjugates. In some embodiments of the composition, the expression introduction or enhancement is achieved by an integrated or non-integrated method of introducing a polynucleotide encoding a foreign protein. In some embodiments, the non-integrated foreign polynucleotide is introduced using Sendai virus, a free organism, or a plastid. In one embodiment of the composition, the genome-engineered non-pluripotent cells lack HLA class I and / or II. In one embodiment, the genome-engineered non-pluripotent cells comprise B2M knockout or low-content, TAP1 knockout or low-content, TAP2 knockout or low-content, and / or TAP-associated glycoprotein knockout or low-content. In some other embodiments of the composition, the genome-engineered non-pluripotent cells comprise integrated or non-integrated foreign polynucleotides encoding one or more of HLA-E, HLA-G, CD16, 41BBL, and PDL1 proteins. In some embodiments, the non-integrated foreign polynucleotide is introduced using Sendai virus, a free organism, or a plastid. In some embodiments, the genome-engineered non-pluripotent cells comprise the expression introduction of one or more of the proteins HLA-E, HLA-G, CD16, 41BBL, and PDL1, wherein the expression introduction is an increase in the expression of a gene that is not expressed or is poorly expressed in the cell. In some embodiments of the composition, the genome-engineered 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 composition, the genome-engineered non-pluripotent cells comprise mesodermal cells, CD34 cells, hematopoietic endothelial cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells, or B cells. In some embodiments of the composition, genome-engineered non-pluripotent cells achieve improved survival, enhanced resistance to immune cells, or enhanced immune resistance; or wherein the genome-engineered non-pluripotent cells exhibit enhanced resistance to T and / or NK cells.

[0036] Another aspect of the present invention provides a composition for obtaining genome-engineered iPSCs, the composition comprising: (i) a non-pluripotent cell; (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, wherein the composition is suitable for obtaining iPSCs comprising targeted genome editing, wherein the genome-engineered iPSCs thereby achieve improved survival, enhanced resistance to immune cells, or enhanced immune resistance; or wherein the genome-engineered iPSCs exhibit enhanced resistance to T and / or NK cells.

[0037] In some embodiments, the above composition further comprises one or more endonucleases capable of recognizing a selected site to introduce a double-strand break at the selected site; and / or a construct comprising one or more exogenous polynucleotides encoding a safety switch protein, a targeting pattern, a receptor, a signal transduction molecule, a transcription factor, a pharmaceutically active protein and peptide, a drug target candidate, or a protein that promotes the transplantation, transport, homing, viability, self-renewal, retention, and / or survival of iPSCs or their derivatives that are reprogrammed into non-pluripotent cells. In some embodiments, the one or more exogenous polynucleotides are operatively linked to (1) one or more exogenous promoters comprising 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, the selected site comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1. In some other embodiments of the composition, the resulting genome-engineered iPSCs comprise one or more exogenous polynucleotides encoding safety switch proteins, said safety switch proteins comprising caspase, thymidine kinase, cytosine deaminase, modified EGFR, B-cell CD20, or any combination thereof. In some other embodiments, the resulting genome-engineered iPSCs comprise two or more exogenous polynucleotides encoding the same or different safety switch proteins, said exogenous polynucleotides being integrated into different safe harbor loci, said safe harbor loci comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the genomic safe harbor criteria.

[0038] In some other embodiments, the genome-engineered iPSCs comprise a foreign polynucleotide encoding caspase at the AAVS1 locus and a foreign polynucleotide encoding thymidine kinase at the H11 locus. In still other embodiments, the genome-engineered iPSCs comprise one or more insertions / deletions from endogenous genes associated with: targeting patterns; receptors; signal transduction molecules; transcription factors; drug target candidates; immune response regulation and control; and proteins that inhibit the transplantation, transport, homing, viability, self-renewal, retention, and / or survival of iPSCs or their derived cells. In some embodiments, the genome-engineered iPSCs comprising one or more insertions / deletions comprise one or more of the following: B2M, TAP1, TAP2, TAP-associated glycoproteins, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the 6p21 region of chromosome 6.

[0039] In one embodiment, the non-pluripotent cells in the composition comprise mesodermal cells, CD34 cells, hematopoietic endothelial cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, 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 for maintaining the pluripotency of genome-engineered iPSCs.

[0040] Another aspect of the invention includes a composition for maintaining the pluripotency of genome-engineered iPSCs, said composition comprising (i) genome-engineered iPSCs, and (ii) a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor. In some embodiments, the genome-engineered iPSCs are obtained by reprogramming genome-engineered non-pluripotent cells, wherein the resulting iPSCs contain the same targeted integrations and / or insertions / deletions at selected sites as the genome-engineered non-pluripotent cells. In some embodiments, the genome-engineered iPSCs are obtained by genome engineering a cloned iPSC or iPSC pool by introducing one or more targeted integrations and / or insertions / deletions at one or more selected sites. In some other embodiments, genome-engineered iPSCs are obtained using genome engineering by introducing one or more targeted integrations and / or insertions / deletions at one or more selected sites into a pool of reprogrammed non-pluripotent cells in contact with one or more reprogramming factors and optionally present small molecule compositions comprising TGFβ receptor / ALK inhibitors, MEK inhibitors, GSK3 inhibitors and / or ROCK inhibitors.

[0041] The genome-engineered iPSCs in the composition comprise one or more exogenous polynucleotides encoding safety switch proteins, targeting mechanisms, receptors, signal transduction molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote the transplantation, transport, homing, viability, self-renewal, retention, and / or survival of non-pluripotent cell reprogrammed iPSCs or their derivatives; and / or insertions / deletions at one or more endogenous genes associated with: targeting mechanisms, receptors, signal transduction molecules, transcription factors, drug target candidates, immune response regulation and control, or proteins that inhibit the transplantation, transport, homing, viability, self-renewal, retention, and / or survival of non-pluripotent cell reprogrammed iPSCs or their derivatives. In some embodiments, the one or more exogenous polynucleotides are operatively linked to (1) one or more exogenous promoters comprising 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 comprising 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 the selected site to introduce double-strand breaks at the selected site.

[0042] In some embodiments, the genome-engineered iPSCs in the composition comprise one or more exogenous polynucleotides encoding safety switch proteins selected from caspase, thymidine kinase, cytosine deaminase, modified EGFR, B-cell CD20, and any combination thereof. In some embodiments, the genome-engineered iPSCs comprise two or more exogenous polynucleotides encoding the same or different safety switch proteins, the exogenous polynucleotides being integrated into different safe harbor loci, the safe harbor loci comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the genomic safe harbor criteria. In some embodiments, the genome-engineered iPSCs comprise a gene encoding caspase located at the AAVS1 locus and a gene encoding thymidine kinase located at the H11 locus. In some embodiments, the genome-engineered iPSCs comprising one or more insertions / deletions include any gene from the 6p21 region of chromosome B2M, TAP1, TAP2, TAP-associated glycoprotein, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and chromosome 6p21. In some embodiments, the genome-engineered iPSCs comprise at least one deletion or reduced expression of the following genes: B2M, TAP1, TAP2, TAP-associated glycoprotein, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and chromosome 6p21. In some embodiments, the genome-engineered iPSCs in the composition comprise at least one introduced or increased expression of the following genes: HLA-E, HLA-G, CD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, CAR, TCR, Fc receptors, conjugates, and surface-triggered receptors for coupling with bispecific, multispecific, or universal conjugates. In some embodiments, the genome-engineered iPSCs comprise expression introduction or augmentation achieved through integration or non-integration methods of introducing polynucleotides encoding exogenous proteins.

[0043] In some embodiments, the genome-engineered iPSCs in the composition lack HLA class I and / or class II. In some embodiments, the genome-engineered iPSCs in the composition comprise B2M knockout or low-content, TAP1 knockout or low-content, and / or TAP2 knockout or low-content. In some embodiments, the genome-engineered iPSCs in the composition comprise integrated or non-integrated exogenous polynucleotides encoding one or more of HLA-E, HLA-G, CD16, 41BBL, and PDL1 proteins; or wherein the genome-engineered iPSCs comprise the expression introduction of one or more of HLA-E, HLA-G, CD16, 41BBL, and PDL1 proteins. In some embodiments, the genome-engineered iPSCs in 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 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.

[0044] In some embodiments, the genome-engineered iPSCs in the composition achieve improved survival, enhanced resistance to immune cells, or enhanced immune resistance; or wherein the genome-engineered iPSCs enhance resistance to T and / or NK cells. In some embodiments, the genome-engineered iPSCs in the composition have the potential to differentiate into non-pluripotent cells comprising hematopoietic lineage cells with the same functionally targeted genome editing. In some embodiments, the genome-engineered iPSCs in the composition have the potential to differentiate into mesodermal cells, CD34 cells, hematopoietic endothelial cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells, or B cells.

[0045] The present invention further provides a pharmaceutical composition comprising (i) one or more of the following groups as provided by the methods and compositions disclosed herein: genome-engineered iPSCs, HLA-deficient modified iPSCs, genome-engineered non-pluripotent cells, genome-engineered hematopoietic lineage cells, HLA-deficient modified hematopoietic lineage cells, or any combination thereof; and (ii) a pharmaceutically acceptable medium. In some embodiments, the hematopoietic lineage cells are mesodermal cells, CD34 cells, hematopoietic endothelial cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells, or B cells.

[0046] Additionally, the above-mentioned pharmaceutical composition is provided for therapeutic use, which is achieved by introducing the composition into a subject suitable for adoptive cell therapy, wherein the subject suffers from an autoimmune disease; a blood malignancy; a solid tumor; cancer; or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0047] Another aspect of the present invention provides a method for manufacturing genome-engineered iPSC-derived non-pluripotent cells using the methods and compositions provided herein.

[0048] The multiple objectives and advantages of this use will become apparent from the following description, taken in conjunction with the accompanying drawings, which are provided for the purpose of illustrating and demonstrating certain embodiments of the invention. Attached Figure Description

[0049] Figure 1The diagram illustrates the targeted insertion of the iCasp9 suicide gene, driven by the CMV promoter, into the AAVS1 safe harbor, and the cellular response when the suicide gene is activated by AP1903. A. Diagram illustrating the donor construct AAVS1-G1.0 designed to target the AAVS1 locus. B. Flow cytometry analysis of GFP expression in puromycin-selected 293T cells transfected with ZFNs and the donor construct specific to the AAVS1 locus. C. Puromycin-selected 293T cells treated with AP1903 (or DMSO control) for 24 hours. Treated cells were collected, stained with 7AAD (to stain membrane-damaged dead cells), and analyzed by flow cytometry. The percentage of 7AAD-negative cells (potentially viable cells) for each treatment (n=2) is plotted. Significant cell death was detected after AP1903 treatment. D. Ligation PCR of genomic DNA from puromycin-selected 293T cells showed that the donor vector was targeted into the AAVS1 locus. E. hiPSCs were transfected with ZFNs specific to the AAVS1 locus and a donor construct (AAVS1-G1.0) containing CMV-driven iCasp9. GFP expression in puromycin-resistant cells was analyzed by flow cytometry. Three populations were sorted and amplified based on GFP intensity for further analysis. F. The sorted and amplified GFP (negative), GFP (low), and GFP (high) populations were treated with AP1903 (or DMSO control) for 24 hours. Treated cells were collected, stained with 7AAD, and analyzed by flow cytometry. The percentage of 7AAD-negative cells (viable cells) for each treatment was plotted. G. Ligation PCR of genomic DNA from puromycin-selected and sorted hiPSCs showed that the donor vector was targeted and inserted into the AAVS1 locus of the GFP (low) group, but not into the AAVS1 locus of the GFP (negative) or GFP (high) group.

[0050] Figure 2The diagram illustrates the targeted insertion of iCasp9 into the safe harbor locus of hiPSCs under the control of various endogenous and exogenous promoters. A. Diagram illustrating the construct designed to target the AAVs1 locus via the AAVS1 promoter, which drives gene expression. B. hiPSCs were transfected with ZFNs specific to the AAVS1 locus and a donor construct containing iCasp9 under the control of the endogenous AAVS1 promoter; GFP expression in puromycin-resistant cells was analyzed by flow cytometry. C. Amplified puromycin-resistant iPSCs were treated with AP1903 (or DMSO control) for 24 hours. Treated cells were collected and stained with 7AAD. The percentage of 7AAD-negative cells for each treatment was plotted. D. Ligation PCR of genomic DNA from the puromycin-resistant pool showed that the donor vector targeted the AAVS1 locus in this population. E. Diagram illustrating the construct designed to target the AAVs1 locus via various promoters. F. hiPSCs were transfected with ZFNs specific to the AAVS1 locus and donor constructs containing iCasp9 under the control of various endogenous and exogenous promoters, and GFP expression in puromycin-resistant cells was analyzed by flow cytometry.

[0051] Figure 3 The diagram illustrates the targeted insertion of EF1α-promoted iCasp9 into the AAVS1 safe harbor of iPSCs. A. HiPSCs were selected with puromycin and subsequently treated with AP1903 via ZFNs specific to the AAVS1 locus and a donor construct containing EF1α-promoted iCasp9 (see [link to diagram]). Figure 2 A. Transfection. The gated region highlights GFP-positive cells responding to AP1903 cell death induction. B. Batch or individual sorting of TRA181 and GFP-positive hiPSCs in 96-well plates. C. Batch-sorted GFP-positive hiPSCs were maintained in puromycin-free medium for extended periods to determine population distribution over time. D. Bright-field images of hiPSC clones derived from TRA181 and GFP cells sorted from single cells. E. Amplification of cloned hiPSCs, with varying degrees of loss of GFP expression after amplification.

[0052] Figure 4 The diagram illustrates the targeted insertion of CAG-promoted iCasp9 into the AAVS1 safe harbor of hiPSCs. A. hiPSCs were transfected with ZFNs specific to the AAVS1 locus and donor constructs containing CAG-promoted iCasp9 (see [link to diagram]). Figure 2A. Puromycin-resistant cells were sorted in batches or individually in 96-well plates. B. Batch-sorted GFP+ iPSCs were maintained in puromycin-free medium for 21 days, followed by flow cytometry analysis of GFP expression. C. GFP-positive cells showed positive staining for 7AAD when treated with AP1903. D. Bright-field images of typical colonies after individual sorting. E. Amplified colonies derived from single cells, with flow cytometry assessment showing high GFP and TRA181 expression. F. Mean fluorescence intensity of selected clonal lines. G. Ligation PCR of genomic DNA detected homologous recombination between parental hiPSCs and donor constructs and the AAVS1 locus in the CAG hiPSC population pool.

[0053] Figure 5 The pluripotency characterization of the CAG-hiPSC clonal line is illustrated graphically. A. Representative image of the CAG clonal line C38 maintained in a feeder-free culture. B. Immunofluorescence staining for the pluripotency markers NANOG and OCT4. C. Quantitative RT-PCR targeting the expression of multiple endogenous pluripotency markers. D. Inducing directed lineage-specific differentiation of selected CAG clonal lines and evaluating lineage markers: nestin (ectoderm); αSMA (mesoderm); SOX17 (endoderm). E. Assessing the potential of CAG-C38 to generate teratomas composed of multiple lineages. Teratomas were collected 6 weeks post-injection.

[0054] Figure 6 The diagram illustrates the induced suicide gene-mediated killing of hiPSC clones in pluripotent and differentiated states, where CAG-driven iCasp9 targets the AAVS1 locus. A. Twenty CAG-iCasp9-targeted hiPSC clones were treated with AP1903 (or DMSO control) for 24 hours, followed by flow cytometry analysis of GFP and 7AAD staining. Flow cytometry measurements of clones CAG-C5 and CAG-C38 are also shown in this figure. B. One of fifteen CAG-iCasp9-targeted iPSC clones survived after AP1903 (10 nM) treatment. C. Viable cell coverage kinetics of CAG-C5 on a 10 cm culture dish after AP1903 (10 nM) treatment. D. CAG-iCasp9-targeted hiPSC clones were induced to differentiate into endoderm, mesoderm, or ectoderm cells, each treated with AP1903, and subsequently allowed to recover and differentiate. Repeat wells before and after treatment were stained with crystal violet and imaged. Images of the CAG-C5 clone are shown in the figure. E. CAG-iCasp9-targeted hiPSC clones were induced to differentiate into CD34+ cells, treated with AP1903 for 48 hours, and cell death was analyzed by flow cytometry.

[0055] Figure 7 The regression of teratomas derived from the iCasp9-iPSC clone line induced by AP1903 in vivo is illustrated graphically. A. Illustration of subcutaneous injection localization in NSG mouse studies. B. Teratomas collected on day 34 after treatment with parental hiPSC and CAG-C38 clone lines with AP1903 (intraperitoneal, total 200 µg) on ​​days 7–11. C. Measurement of the volume of collected teratomas. D. Collected teratomas derived from parental hiPSC or CAG-C38 clone lines stained with hematoxylin and eosin. While most parental hiPSC lines showed a trilineage differentiation cell type, CAG-C38 appeared to be primarily composed of highly histified adipoid cells. E. Parental hiPSC and CAG clone lines injected at 4E6 cell per cell line and treated with AP1903 (intraperitoneal, total 200 µg) on ​​days 40–46. The volume of all tumors was measured using standard methods.

[0056] Figure 8 The characterization of escaped clonal lines is illustrated graphically. A. The method for selecting clonal lines that survived AP1903 treatment is illustrated. B. PCR amplification of the iCasp9 transgene, followed by purification and genome sequencing to determine if any sequence changes were present. C. Flow cytometry evaluation of the escaped clonal lines showed high GFP and TRA181 expression. D. The single-point mutation K189E was identified in all sequenced clonal lines. E. To determine whether K189E was the direct cause of refractory clonal lines, the iCasp9 mutant form was generated and tested. F. Demonstration that the iCasp9 mutant transgene was the cause of survival after AP1903 treatment.

[0057] Figure 9 This diagram illustrates genome editing of the human ROSA26 locus using CRISPR / Cas9 to target or nuclease-independently insert iCasp9. A. Diagram illustrating the design of a CRISPR / Cas9-targeted ROSA26 locus construct. B. hiPSCs were transfected with ROSA26 locus-specific gRNA / Cas9-RFP plastids and donor constructs containing CAG-driven iCasp9, as shown in A. Two days after transfection, GFP expression in the cell population was analyzed by flow cytometry to determine transfection efficiency (approximately 89%). GFP expression in puromycin-resistant cells was analyzed by flow cytometry 20 days after transfection. C. AP1903-induced cell death analysis of 293 T cells transfected with the construct. D. Targeted insertion of the construct into 293 T cells. E. hiPSCs transfected with the construct and selected for puromycin to enrich target cells.

[0058] Figure 10The generation of iPSC clonal lines with targeted integration of monoallelic and biallelic iCasp9 is illustrated graphically. A. The copy number of the transgene (iCasp9-GFP) in the designated CAG promoter-driven iCasp9 clonal lines was assessed using quantitative PCR. B. The mean fluorescence intensity (MFI) of iCasp9-GFP in sorted mixed cultures (black) or clonal lines with monoallelic (light gray) or biallelic (dark gray) iCasp9-targeted insertions, as determined by flow cytometry. C. PCR analysis of transgene integration was performed using primers that overlapped with the AAVS1-transgene junction in the targeted mixed cells and clonal lines. D. The number of targeted integrations within the AAVS1 allele was assessed using PCR-based methods and primers specific to the AAVS1 integration site. E. Immediately after adding 10 nM AP1903, live-cell imaging was used to assess the coverage of unsorted target pool cells, iCasp9-GFP-sorted target pool cells, and cloned CAG-C38 and CAG-C5 cell lines on the plate surface.

[0059] Figure 11 The following diagram illustrates how the exogenous CAG promoter allows iCasp9-mediated exclusion of established iPSC-derived teratomas. A. Schematic description of the study design. B. Bioluminescent images of the three specified NSG mouse groups (n=2) before AP1903 administration (top panel) and on day 3 after administration (125 µg, intraperitoneal, days 19–25, bottom panel). C. Total flux (photons / sec) from teratomas of the specified cell types, expressed as mean and SD. D. Ratio of total flux of the specified cell types after treatment to before treatment (mean - / +SD). E. Results of injection of cloned CAG-C38 and CAG mixed cells into mice. F. Terminal volume measurement of teratomas derived from the specified cell types (day 28). G. Teratoma images on day 28. No teratomas were detected from the lateral aspect of the injected CAG-C38 clone or CAG-iCasp9 mixed cells. p-value < 0.05, measured by unpaired t-test. ns: not significant.

[0060] Figure 12The diagram illustrates the induced killing effect of transplanted biallelic iCasp9 clones compared to monollelic iCasp9 clones. A. Schematic description of the study design. B. Bioluminescent images of NSG mice transplanted with specified cell types (biallelic CAG-C5 clone, monollelic CAG-C38 clone, and parental iPSCs) on days 7, 14, and 42 post-iPSC transplantation. Day 7 imaging was performed just before administration of AP1903 (125 µg, intraperitoneal, days 7–13). C. The image below shows images of teratomas collected (if found) on day 42. D. Total flux (photons / sec) of teratomas from specified groups is shown as mean and SD. p-value < 0.05; p-value < 0.01; p-value < 0.001, as measured by an unpaired t-test. ns: not significant.

[0061] Figure 13 The generation of a safety protection system in which dual suicide genes target individual safe harbor loci is illustrated. A. PCR analysis was performed on the specific genomic integration of sr39TK into the H11 safe harbor locus using primers specific to sr39TK, sr39TK-blastactin (Bsd) junctions, or endogenous H11 sequence-transgenic junctions. GAPDH was used as an internal reference. B. CAG-C38 iCasp9 iPSC clones in which sr39TK targets insertion into the H11 locus grew (i) without GCV or AP1903 treatment; (ii) treated with AP1903 only for 2 days; (iii) treated with GCV only for 9 days; and (iv) treated with AP1903 and GCV in parallel for 2 days, then AP1903 was washed off, and GCF treatment was continued for more than 7 days.

[0062] Figure 14 The generation of B2M- / -, HLA-I-deficient iPSCs is illustrated graphically. A. Flow cytometry analysis of GFP, B2M, and HLA-I expression before transfection (left panel) and after transfection (middle panel). Cells negative for both B2M and HLA-I are sorted in batches (right panel). B. Flow cytometry analysis of B2M and HLA-I negative cells sorted clonally in a 96-well plate.

[0063] Figure 15 illustrates the improved survival of B2M- / -, HLA I-deficient iPSCs. A. B2M- / - hiPSCs were not killed by pre-sensitized T cells. B. B2M- / - hiPSCs co-cultured in a single well were not recognized by NK cells. C. B2M- / - hiPSCs co-cultured in individual wells were not recognized by NK cells. D. B2M- / - hiPSCs have been preserved in immune-competent mice.

[0064] Figure 16 illustrates how HLA class I regulation of iPSCs enhances iPSC retention in immune-competent recipients. A. Genetically engineered HLA-modified iPSCs express HLA-E on their cell surface and maintain a pluripotent phenotype. B. In vivo fluorescein imaging of teratomas 72 hours after injection of B2M- / -HLAE iPSCs shows enhanced retention compared to wild-type iPSCs.

[0065] Figure 17 This indicates that iPSCs genetically engineered to express the non-cleavable, high-affinity CD16 receptor and the 41BBL co-stimulatory molecule maintained expression throughout differentiation into iCD34 cells. A. Undifferentiated cells on day 0; B. Differentiated cells on day 10.

[0066] Figure 18 The reprogramming of CAR-T cells into iPSCs (TiPSCs) was described based on PCR analysis of CAR (FTV106) integration into iPSCs derived from genetically engineered T cells with FTV106, which retain the same genetic imprint as the source T cells.

[0067] Figure 19 shows that genetically engineered iPSCs expressing CD19 chimeric antigen receptor (CAR) and truncated LNGFR cell surface markers as co-recognition markers of CAR maintained expression during differentiation into iCD34 cells. A. Undifferentiated cells on day 0; B. Differentiated cells on day 10.

[0068] Figure 20 We depicted cell type-specific expression of CAR driven by endogenous TCR promoters, and TCR expression and functional gene knockout caused by locus-specific insertion of CAR.

[0069] Figure 21 The results showed that engineered hiPSCs expressing immunosuppressive proteins could generate CD34 cells: A. Expression of HLA E, HLA G, and PDL1 on the cell surface of HLA I-deficient engineered iPSCs; B. All HLA I-deficient engineered iPSCs differentiated into CD34+ HE cells after 10 days of differentiation culture.

[0070] Figure 22 This indicates that hiPSCs engineered to express immunosuppressive proteins can generate hematopoietic lineage cells.

[0071] Figure 23 This indicates that during the hematopoietic differentiation of hiPSCs, the expression of engineered non-classical HLA immunosuppressive proteins is downregulated: A. HLA-E and HLA-G proteins are absent, while PDL1 expression is maintained; B. HLA-E and HLA-G proteins are present in the supernatant fraction, indicating protein shedding.

[0072] Figure 24 The design and expression of non-cleavable HLAG fusion proteins in hiPSCs were described. A. Design of cleavage-resistant forms of HLA-E / B2M and HLA-G / B2M fusion proteins; B. Expression of non-cleavable HLA-G / HLA-A3 proteins on the cell surface without affecting iPSC quality.

[0073] Figure 25 The generation of TAP2- / - iPSCs exhibiting reduced HLA class I expression was described: A. Flow cytometry analysis showed that the expression of HLA class I in the two selected clonal lines was significantly reduced compared to the parental FTi121; B. After treatment with IFNγ, the expression of HLA class I in the TAP2- / - clonal lines was reduced compared to wild-type FTi121.

[0074] Figure 26 The results indicate that host NK cells induce hiPSC teratoma rejection in immune-competent recipients: A. Three days after antibody injection, CD4+ T cells, CD8+ T cells, and NK cells were absent; B. NK-depleted mice exhibited the highest resistance to tumor rejection 120 hours after iPSC injection compared to animals treated with IgG.

[0075] Figure 27 shows that HLA I-deficient modified iPSCs achieved enhanced survival and resistance in vitro. Expression of immunosuppressive proteins on hiPSC-derived cells prevented the recognition and proliferation of the following cells: A. purified allogeneic human T cells; B. PBMC allogeneic human T cells; C. PBMC allogeneic human NK cells.

[0076] Figure 28 shows that the expression of hnCD16 protein does not affect the hematopoietic differentiation potential of engineered iPSCs. A. Engineered iNK cells with hnCD16 surface expression; B. hnCD16 is constitutively expressed and continuously maintained in iNKs derived from genetically engineered iPSCs.

[0077] Figure 29 illustrates the enhanced function of iNK cells expressing hnCD16: A. CD16 stimulation induces enhanced cytokine production in iNK cells expressing hnCD16; B. hnCD16 enhances antibody-dependent cell-mediated cytotoxicity (ADCC) in iNK cells expressing hnCD16.

[0078] Figure 30 The quality of iPSC-derived iNK cells was demonstrated by expanding iNK cells.

[0079] Figure 31 This indicates that iNK cells have longer telomeres compared to adult NK cells.

[0080] Figure 32 illustrates the Tet-induced CAR-2A-LNGFR expression in a genetically engineered iPSC line with doxycycline-inducible CAR: A. Inducible CD19CAR expression in TetCAR iPSCs; B. iNK-mediated cytotoxicity assessed by flow cytometry based on caspase-3 expression on target cells, with iNK cytotoxicity enhanced by inducible CD19CAR expression.

[0081] Figure 33 This indicates that Cre or doxycycline in iPSCs can induce expression: A. Removal of the stop codon and CAR expression is induced by: A. a single Cre recombinase induction; B. activation after doxycycline induction.

[0082] Figure 34 This section describes the insertion of the gene of interest into the selected locus at the interruption site, and the hiPSC clonal selection after gene editing: A. Architectural design for the target vector and donor vector to insert the gene of interest into the B2M locus; B. hiPSC selection of the clonal population containing the selected gene at the selected locus for insertion after gene editing.

[0083] Figure 35 The following data were depicted: iProT transformation, cryopreservation, and expression of CD34 and CD7 in proT cells (based on flow cytometry): A. Before proT cell isolation; B. Day 3 after thawing; C. Cryopreserved on day 10+13; D. Not cryopreserved; E. proT cells survived after thawing and exhibited enhanced cell proliferation when cultured in T cell differentiation medium.

[0084] Figure 36Functional characterization of iPSC-derived and CB-derived ProT cells was described: cytokine release and chemotaxis: A. iPSC-derived ProT cells were able to successfully migrate to CCL21 and SDF; B. iPSC-derived ProT cells were able to respond by producing and secreting IFNγ and TNFα cytokines.

[0085] Figure 37 This indicates a decrease in the percentage of germline cells from the TCR γ locus retained by iPSC-derived ProT cells, thereby triggering VDJ recombination at the TCR locus. Detailed Implementation

[0086] Genome modifications of stem cells (e.g., hESCs (embryonic stem cells) or iPSCs (induced pluripotent stem cells)) include polynucleotide insertions, deletions, and substitutions. Exogenous gene expression in genome-engineered iPSCs often encounters problems, such as gene silencing or reduced gene expression after long-term clonal expansion or cell differentiation in genome-engineered iPSCs, and dedifferentiated cell types derived from genome-engineered iPSCs. This invention provides an efficient and reliable targeting method for the stable integration of one or more exogenous genes (including suicide genes and other functional modalities) into iPSCs and maintaining the functional response of said genes in expanded iPSCs and differentiated cells derived from genome-engineered iPSCs. In one embodiment, the iPSC is a single-cell-source clone. In another embodiment, this invention provides a gene-encoded induced "suicide" system suitable for targeted integration at one or more selected sites, the system responding to specific, non-toxic chemical inducing factors in iPSCs, expanded iPSCs, and differentiated cells derived therefrom. This invention therefore represents a highly efficient and reliable targeting method for eliminating delivered cells without damaging surrounding cells and tissues, applicable to a variety of cell therapies. Additionally, this invention provides a method and system for obtaining cloned iPSCs integrated with various gene patterns related to: reprogramming and dedifferentiation, iPSC differentiation, promoting iPSC or derivative cell transplantation, transport, homing, migration, cytotoxicity, viability, maintenance, expansion, long lifespan, self-renewal, retention, and / or survival, including (but not limited to) HSCs (hematopoietic stem cells and progenitor cells), T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, and NK cells. Furthermore, this invention describes the identification of induced caspase 9 gene mutations that make the cell difficult to treat with chemical induction and allow escape from induced cell death; and methods to overcome or prevent such escape, such as a dual suicide gene safety protection system or biallelic suicide gene insertion.

[0087] definition

[0088] 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 pertains. For the purposes of this invention, the following terms are defined as follows. The article “a / an / the” is used herein to refer to one or more (i.e., at least one) grammatical object of the article. For example, “a component” means one component or more components.

[0089] The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination of alternatives.

[0090] The term “and / or” should be understood to mean one or both of the alternatives.

[0091] As used herein, the term "about" or "approximately" means that a quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" means a range of a quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length that is ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a reference quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length.

[0092] As used herein, the terms “substantially” or “basically” mean that the quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length is approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher of a reference quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length. In one embodiment, the terms “substantially identical” or “substantially identical” mean that the range of the quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length is approximately the same as that of a reference quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length.

[0093] As used herein, the terms “substantially free” and “substantially free” are used interchangeably and, when used to describe a composition (e.g., a cell population or culture medium), mean a composition that is free of the specified substance or its source, for example, 95%, 96%, 97%, 98%, or 99% free of the specified substance or its source, or undetectable, as measured by conventional methods. The terms “free” or “substantially free” of a component or substance in a composition also mean (1) that the composition does not contain any concentration of such component or substance, or (2) that the composition includes such component or substance that is functionally inert but at low concentrations. A similar meaning can be applied to the term “lacking”, which means that a particular substance or its source is absent in the composition.

[0094] Throughout this specification, unless the context otherwise requires, the term "comprise / comprises / comprising" should be understood to imply that it includes the stated steps or components or a group of steps or components, but does not exclude any other steps or components or a group of steps or components. In certain embodiments, the terms "comprising," "having," "containing," and "including" are used synonymously.

[0095] The phrase “composed of” means that it includes and is limited to anything that follows the phrase “composed of.” Therefore, the phrase “composed of” indicates that the listed components are necessary or required, and that no other components can exist.

[0096] "consistently composed of..." means including any of the components listed after the phrase, and is limited to other components that do not interfere with or affect the activity or effect specified for the listed components in this disclosure. Thus, the phrase "consistently composed of..." indicates that the listed components are required or necessary, but other components are optional and may or may not be present depending on whether they affect the activity or effect of the listed components.

[0097] Throughout this specification, references to "an embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "another embodiment," or combinations thereof, refer to specific features, structures, or characteristics described in connection with the embodiment, which are included in at least one embodiment of the invention. Therefore, the aforementioned phrases appearing throughout this specification do not necessarily refer to the same embodiment. Furthermore, in one or more embodiments, specific features, structures, or characteristics can be combined in any suitable manner.

[0098] The term "in vitro" generally refers to activities occurring outside an organism, such as experiments or measurements performed in or on living tissue in an artificial environment outside the organism, preferably with minimal alteration to natural conditions. In certain embodiments, an "in vitro" procedure involves obtaining living cells or tissues from an organism and culturing them in a laboratory apparatus, typically for several hours or up to about 24 hours under sterile conditions, but including up to 48 or 72 hours or longer, as applicable. In some embodiments, such tissues or cells may be collected and frozen, and later thawed for in vitro processing. Tissue culture experiments or procedures using living cells or tissues lasting longer than several days are typically considered "in vitro," but in some embodiments, this term may be used interchangeably with "in vitro."

[0099] The term "living body" generally refers to activities that take place inside an organism.

[0100] As used herein, the terms "reprogramming," "dedifferentiation," "enhanced cell efficacy," or "enhanced developmental efficacy" refer to a method of improving cell efficacy or causing cells to dedifferentiate into a less differentiated state. For example, cells with increased cell efficacy have greater developmental plasticity (i.e., they can differentiate into more cell types) compared to the same cells in a non-reprogrammed state. In other words, reprogrammed cells are cells in a less differentiated state than the same cells in a non-reprogrammed state.

[0101] As used herein, the term “differentiation” is the process by which undifferentiated (“non-specialized”) or weakly specialized cells acquire the characteristics of specialized cells (such as blood cells or muscle cells). Differentiated cells, or differentiation-inducing cells, are cells that are already in a more specialized (“specialized”) position within a cell lineage. The term “specialization,” when applied to the differentiation process, refers to a point in the differentiation pathway where a cell, under normal circumstances, would continue to differentiate into a specific cell type or a subpopulation of that cell type, and under normal circumstances, it cannot differentiate into different cell types or revert to a weaker differentiated cell type. As used herein, the term “pluripotent” refers to the ability of a cell to form all lineages of the body or somatic tissue (i.e., the embryo itself). For example, embryonic stem cells are a type of pluripotent stem cell capable of forming cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuous developmental potency, ranging from incomplete or partially pluripotent cells that cannot produce a complete organism (such as ectodermal stem cells or EpiSCs) to more primitive, multipotent cells that can produce a complete organism (such as embryonic stem cells).

[0102] As used herein, the term "induced pluripotent stem cells" or iPSCs refers to stem cells derived from differentiated adult, neonatal, or fetal cells that have been induced or altered, i.e., reprogrammed, to differentiate into cells capable of differentiating into tissues of all three germ layers or dermis: mesoderm, endoderm, and ectoderm. The resulting iPSCs do not refer to cells as they are found in nature.

[0103] As used herein, the term "embryonic stem cell" refers to naturally occurring pluripotent stem cells within the internal cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and generate all derived cells from the three primary germ layers during development: the ectoderm, endoderm, and mesoderm. They do not contribute to the outer membranes of the embryo or the placenta; that is, they are not totipotent.

[0104] As used herein, the term "pluripotent stem cell" refers to a cell that has the developmental potential to differentiate into cells of one or more germ layers (ectoderm, mesoderm, and endoderm), but not all three. Therefore, pluripotent cells can also be called "partially differentiated cells." Pluripotent cells are well-known in the field, and examples of pluripotent cells include adult stem cells, such as hematopoietic stem cells and neural stem cells. "Pluripotent" means that the cell can form many types of cells within a specified lineage, rather than cells from other lineages. For example, pluripotent hematopoietic cells can form many different types of blood cells (red blood cells, white blood cells, platelets, etc.), but they cannot form neurons. Therefore, the term "pluripotency" refers to a cell state whose developmental potential is less than that of totipotency and pluripotency.

[0105] Pluripotency can be determined in part by assessing the pluripotency characteristics of cells. Pluripotency characteristics include (but are not limited to): (i) pluripotent stem cell morphology; (ii) 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 composed of the three somatic cell lineages; and (vi) embryomorph formation composed of cells from the three somatic cell lineages.

[0106] Two types of pluripotency have been previously described: the “excited” or “metastable” pluripotent state is equivalent to ectodermal stem cells (EpiSCs) of late blastocysts, and the “initial” or “basal” pluripotent state is equivalent to the internal cell mass of early / preimplantation blastocysts. While both pluripotent states exhibit the characteristics described above, the initial or basal state further exhibits: (i) pre-activation or reactivation of the X chromosome in female cells; (ii) improved clonality and viability during single-cell culture; (iii) a general reduction in DNA methylation; (iv) reduced deposition of the H3K27me3 repressive chromatin marker on promoters of developmental regulatory genes; and (v) reduced expression of differentiation markers relative to excited-state pluripotent cells. Standard cell reprogramming methods (whereby exogenous pluripotent genes are introduced into somatic cells, expressed, and then silenced or removed from all pluripotent cells) typically exhibit the characteristics of an excited-state pluripotency. Under standard pluripotent cell culture conditions, such cells remain in an excited state unless exogenous transgene expression is maintained (where the characteristics of the basal state are observed).

[0107] As used in this article, the term "pluripotent stem cell morphology" refers to the classic morphological characteristics of embryonic stem cells. Normal embryonic stem cells are characterized by a small, round shape, a high nucleus-to-cytoplasm ratio, a prominent nucleolus, and typical intercellular spacing.

[0108] As used herein, the term “subject” refers to any animal, preferably a human patient, livestock or other domesticated animal.

[0109] "Multipotent factors" or "reprogramming factors" refer to agents that, alone or in combination with other agents, enhance cellular developmental efficacy. Multipotent factors include (but are not limited to) polynucleotides, peptides, and small molecules that can improve cellular developmental efficacy. Exemplary multipotent factors include, for example, transcription factors and small molecule reprogramming agents.

[0110] "Cultivation" or "cell culture" refers to the maintenance, growth, and / or differentiation of cells in a test tube environment. "Cell culture medium," "culture medium" (in all cases, the singular form "medium"), "supplement," and "culture medium supplement" refer to a nutrient composition for cultivating cell cultures.

[0111] "Cultivation" or "maintenance" refers to the maintenance, proliferation (growth), and / or differentiation of cells outside a tissue or body (e.g., in sterile plastic (or coated plastic) cell culture dishes or flasks). "Cultivation" or "maintenance" can utilize culture media as a source of nutrients, hormones, and / or other factors that contribute to cell proliferation and / or maintenance.

[0112] As used in this article, the term "mesoderm" refers to one of the three germ layers that appears during early embryonic development and produces various specialized cell types, including blood cells of the circulatory system, muscles, heart, dermis, bones, and other supporting and connective tissues.

[0113] As used herein, the terms "permanent hematopoietic endothelial cells" (HE) or "multipotent stem cell-derived permanent hematopoietic endothelial cells" (iHE) refer to a subset of endothelial cells that generate hematopoietic stem cells and progenitor cells during the transformation from endothelial cells to hematopoietic cells. Hematopoietic cell development in the embryo proceeds sequentially: from the lateral plate mesoderm to angiogenic cells to permanent hematopoietic endothelial cells and hematopoietic progenitor cells.

[0114] The terms "hematopoietic stem cells and progenitor cells," "hematopoietic stem cells," "hematopoietic progenitor cells," or "hematopoietic precursor cells" refer to cells that have reached a hematopoietic lineage but are capable of further differentiation into hematopoiesis. These include pluripotent hematopoietic stem cells (blood embryonic cells), bone marrow progenitor cells, megakaryocyte progenitor cells, erythrocyte progenitor cells, and lymphocyte progenitor cells. Hematopoietic stem cells and progenitor cells (HSCs) are pluripotent stem cells that generate all types of blood cells, including bone marrow (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphocyte lineages (T cells, B cells, NK cells). As used herein, the term "permanent hematopoietic stem cells" refers to CD34+ hematopoietic cells capable of generating mature bone marrow cell types and lymphocyte types, including T cells, NK cells, and B cells. Hematopoietic cells also include various subsets of primitive hematopoietic cells, which generate primitive erythrocytes, megakaryocytes, and macrophages.

[0115] As used herein, the terms “T lymphocyte” and “T cell” are used interchangeably and refer to the primary type of leukocyte that matures in the thymus and plays a variety of roles in the immune system, including identifying specific foreign antigens in the body and activating and deactivating other immune cells. T cells can be any type of T cell, such as cultured T cells, primary T cells, or T cells derived from cultured T cell lines, such as Jurkat, SupT1, etc., or T cells derived from mammals. T cells can be CD3+ cells. T cells can be any type of T cell and can be at any developmental stage, 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 monocytes (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, untreated T cells, regulatory T cells, gamma delta T cells (γδ T cells), and so on. Other types of helper T cells include, for example, Th3 (Treg), Th17, Th9, or Tfh cells. Other types of memory T cells include, for example, central memory T cells (Tcm cells) and effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically engineered T cells, such as T cells modified to express the T cell receptor (TCR) or chimeric antigen receptor (CAR). T cells can also differentiate from stem cells or progenitor cells.

[0116] "CD4+ T cells" refer to a subset of T cells that express CD4 on their surface and are associated with cell-mediated immune responses. They are characterized by a secretion profile following stimulation, which may include the 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 also found on other cells including monocytes / macrophages. CD4 antigens are members of the immunoglobulin family and are indicated as relevant recognition components in MHC (major histocompatibility complex) class II restricted immune responses. On T lymphocytes, they define a subset of helper / inducer factors.

[0117] "CD8+ T cells" refer to a subset of T cells that express CD8 on their surface, are MHC class I restricted, and act as cytotoxic T cells. The CD8 molecule is a differentiation antigen found on thymocytes and on cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin superfamily and a key recognition component in interactions restricted by the major histocompatibility complex class I.

[0118] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). As used herein, the terms "adaptive NK cells" and "memory NK cells" are interchangeable and refer to a subset of NK cells that are CD3- and CD56+ phenotyped, expressing NKG2C and CD57 and optionally present CD16, but lacking expression of one or more of the following: PLZF, SYK, FceR. And EAT-2. In some embodiments, the isolated CD56+ NK cell subset includes expression of CD16, NKG2C, CD57, NKG2D, NCR ligand, NKp30, NKp40, NKp46, activating and repressive KIRs, NKG2A, and DNAM-1. CD56+ expression may be weak or strong.

[0119] As used herein, the term "NKT cells" or "natural killer T cells" refers to CD1d-bound 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 have been identified. Constant or type I NKT cells express a very limited TCR lineage: a typical α chain (Vα24-Jα18 in humans) with a limited range of β chains (Vβ11 in humans). A second NKT cell population, called non-classical or non-constant type II NKT cells, exhibits a more uneven utilization of TCR αβ. Type I NKT cells are currently considered suitable for immunotherapy. Adaptive or constant (type I) NKT cells can be identified by the expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, αGalCer, CD161, and CD56.

[0120] As used herein, the term "isolated," etc., refers to a cell or cell population that has been separated from its original environment, i.e., the environment in which the isolated cells are isolated is substantially free of at least one component found in the environment where "unisolated" reference cells are present. The term includes cells stripped of some or all of the components found in their natural environment (e.g., tissue, biopsy). The term also includes cells stripped of at least one, some, or all of the components as if the cells were found in a non-natural environment, such as a culture or cell suspension. Thus, isolated cells are partially or completely separated from at least one component (including other substances, cells, or cell populations) as if they were found in nature or as if they were grown, stored, or survived in a non-natural environment. Specific examples of isolated cells include partially pure cells, substantially pure cells, and cells cultured in non-natural media. Isolated cells can be obtained by separating the desired cells or populations from other substances or cells in the environment or by removing one or more other cell populations or subpopulations from the environment. As used herein, the term "purified," etc., refers to increasing purity. For example, the purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%.

[0121] As used herein, the term "coding" refers to the inherent characteristics of a specific sequence of nucleotides in a polynucleotide (e.g., gene, cDNA, or mRNA) that serves as a template for the synthesis of other polymers and macromolecules in biological processes, which have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the biological characteristics derived therefrom. Thus, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, then the gene encodes that protein. Both coding strands (whose nucleotide sequences are identical to the mRNA sequence and are typically provided in sequence listings) and non-coding strands (which serve as templates for gene or cDNA transcription) can be referred to as proteins or other products encoding said gene or cDNA.

[0122] "Construction" refers to a macromolecule or molecular complex containing a polynucleotide to be delivered to a host cell in vitro or in vivo. As used herein, "vector" refers to any nucleic acid construct capable of guiding the delivery or transfer of foreign genetic material to a target cell, in which the nucleic acid construct is capable of replication and / or expression. As used herein, the term "vector" includes the construct to be delivered. Vectors can be linear or circular molecules. Vectors can be integrated or non-integrated vectors. Major vector types include (but are not limited to) plastids, free vectors, viral vectors, myxosomes, and artificial chromosomes. Viral vectors include (but are not limited to) adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, Sendai virus vectors, etc.

[0123] "Integration" refers to the stable insertion of one or more nucleotides of a construct into the cellular genome, i.e., covalently linked to a nucleic acid sequence within the cellular chromosomal DNA. "Targeted integration" refers to the insertion of a nucleotide of a construct into a pre-selected site or "integration site" in the cellular chromosomal or mitochondrial DNA. As used herein, the term "integration" also refers to a process involving the insertion of one or more exogenous sequences or nucleotides of a construct into an integration site, with or without the deletion of an endogenous sequence or nucleotide. In the case of a deletion at the insertion site, "integration" may further include replacing the deleted endogenous sequence or nucleotide with one or more inserted nucleotides.

[0124] As used herein, the term "exogenous" means the introduction of a reference molecule or reference activity into a host cell. This molecule can be introduced, for example, by introducing a nucleic acid encoding a reference molecule into the host genetic material, such as by integration into the host chromosome or into non-chromosomal genetic material (e.g., plastids). Therefore, when used with respect to the expression of a nucleic acid encoding a reference molecule, it means that the nucleic acid encoding a reference molecule is introduced into the cell in an expressible form. The term "endogenous" means a reference molecule or activity present in the host cell. Similarly, when used with respect to the expression of a nucleic acid encoding a reference molecule, it means the expression of a nucleic acid encoding a reference molecule that is present within the cell but not introduced exogenously.

[0125] As used herein, a “gene of interest” or “polynucleotide sequence of interest” is a DNA sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into RNA in vivo and, in some cases, translated into a polypeptide. Genes of interest or polynucleotide sequences may include (but are not limited to) prokaryotic sequences, cDNA derived from eukaryotic mRNA, genomic DNA sequences derived from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, a gene of interest may encode miRNA, shRNA, native polypeptides (i.e., polypeptides found in nature) or fragments thereof; variant polypeptides (i.e., mutants of native polypeptides with less than 100% sequence identity to native polypeptides) or fragments thereof; engineered polypeptides or peptide fragments, therapeutic peptides or polypeptides, imaging markers, optional markers, and so on.

[0126] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides) of any length, or similar molecules. A polynucleotide sequence consists of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) (uracil replaces thymine when the polynucleotide is RNA). Polynucleotides can include genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonucleases, cDNA, recombinant polynucleotides, branched-chain polynucleotides, plastids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides also refer to double-stranded and single-stranded molecules.

[0127] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to molecules in which amino acid residues are covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, the terms refer to short chains (often also referred to in the field as, for example, peptides, oligopeptides, and oligomers) and longer chains (often referred to in the field as polypeptides or proteins). “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and others. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0128] "Operationally ligated" refers to the binding of nucleic acid sequences on a single nucleic acid fragment such that the function of one is influenced by the other. For example, a promoter is operatively ligated to a coding sequence or functional RNA when it can influence the expression of that sequence or RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). The coding sequence can be operatively ligated to the regulatory sequence in a sense or antisense orientation.

[0129] As used herein, the term “genetic imprinting” refers to genetic or epigenetic information that contributes to preferred therapeutic properties of a source cell or iPSC and is retained in source cell-derived iPSCs and / or iPSC-derived hematopoietic lineage cells. As used herein, a “source cell” is a non-pluripotent cell that can be used to generate iPSCs through reprogramming, and source cell-derived iPSCs can further differentiate into specific cell types, including any hematopoietic lineage cell. Depending on the context, source cell-derived iPSCs and their differentiated cells are sometimes collectively referred to as “derived cells.” As used herein, genetic imprinting that confers preferred therapeutic properties is incorporated into iPSCs by reprogramming selected source cells that are specific to donor, disease, or treatment response, or by introducing genetic modification patterns into iPSCs through genome editing. In the aspects of source cells obtained from specially selected donors, disease, or treatment contexts, genetic imprints contributing to preferred therapeutic properties may include any background-specific genetic or epigenetic modifications exhibiting a retainable phenotype, i.e., preferred therapeutic property, which is transferred to derived cells of the selected source cells, regardless of whether the underlying molecular events are identified or not. Source cells specific to donor, disease, or treatment responses may contain genetic imprints that are retainable in iPSCs and derived hematopoietic lineage cells, including (but not limited to) pre-arranged single-specific TCRs, such as those from virus-specific T cells or constant-type natural killer T (iNKT) cells; traceable and desirable genetic polymorphisms, such as isotype for point mutations encoding high-affinity CD16 receptors in the selected donor; and predetermined HLA requirements, i.e., the selected HLA-matched donor cells exhibiting haplotypes as the population grows. As used herein, preferred therapeutic properties include transplantation, transport, homing, viability, self-renewal, survival, regulation and modulation of immune responses, survival, and improved cytotoxicity of derived cells. Preferred therapeutic properties may also involve receptor expression of the target antigen; HLA presentation or its absence; resistance to the tumor microenvironment; induction and immunomodulation of bystander immune cells; improved target specificity while reducing extratumor effects; and resistance to therapies such as chemotherapy.

[0130] As used herein, the term "enhanced therapeutic properties" refers to cells whose therapeutic properties are enhanced compared to typical immune cells of the same general cell type. For example, NK cells with "enhanced therapeutic properties" will exhibit enhanced, improved, and / or strengthened therapeutic properties compared to typical, unmodified, and / or naturally occurring NK cells. Therapeutic properties of immune cells can include (but are not limited to) cell transplantation, transport, homing, viability, self-renewal, survival, regulation and modulation of immune responses, survival, and cytotoxicity. Therapeutic properties of immune cells are also manifested through: receptor expression targeting antigens; HLA presentation or its absence; resistance to the tumor microenvironment; induction and immunomodulation of bystander immune cells; improved target specificity while reducing extratumor effects; and resistance to therapies such as chemotherapy.

[0131] As used herein, the term "conjugate" refers to a molecule, such as a fusion polypeptide, that enables the formation of a connection between immune cells (e.g., T cells, NK cells, NKT cells, B cells, macrophages, neutrophils) and tumor cells, and activates said immune cells. Examples of conjugates include (but are not limited to) bispecific T cell conjugates (BiTEs), bispecific killer cell conjugates (BiKEs), trispecific killer cell conjugates, or multispecific killer cell conjugates, or universal conjugates compatible with multiple immune cell types.

[0132] As used herein, the term "surface triggering receptor" refers to a receptor capable of triggering or initiating an immune response (e.g., a cytotoxic response). Surface triggering receptors can be engineered and expressed on effector cells, such as T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils. In some embodiments, surface triggering receptors facilitate bispecific or multispecific antibody conjugation between effector cells and specific target cells (e.g., tumor cells), independent of the effector cell's native receptor and cell type. Using this approach, iPSCs containing a universal surface triggering receptor can be generated, and such iPSCs can then be differentiated into populations of various effector cell types expressing the universal surface triggering receptor. "Universal" means that the surface triggering receptor can be expressed on any effector cell and activate any effector cell (regardless of cell type), and that all effector cells expressing the universal receptor can be coupled or linked to conjugates with the same antigenic determinants that are recognizable by the surface triggering receptor (regardless of the conjugate's tumor-binding specificity). In some embodiments, conjugates with the same tumor-targeting specificity are used for coupling with the universal surface triggering receptor. In some embodiments, conjugates with different tumor-targeting specificities are used for coupling with the universal surface triggering receptor. Therefore, it is possible to conjugate one or more effector cell types, thereby killing a specific type of tumor cell in some cases and two or more types of tumor cells in others. Surface trigger receptors typically contain a co-stimulatory domain for effector cell activation and an anti-antigen determinant specific to the antigenic determinant of the conjugate. Bispecific conjugates are specific to the anti-antigen determinant of the surface trigger receptor at one end and to the tumor antigen at the other end.

[0133] As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent potential toxicity of cell therapy or otherwise prevent adverse effects. In some cases, the expression of a safety switch protein is conditionally controlled to address safety concerns of transplanted engineered cells that have permanently incorporated the gene encoding the safety switch protein into their genome. This conditional regulation can be variable and may include control via small molecule-mediated post-translational activation and tissue-specific and / or timed transcriptional regulation. The safety switch can mediate the induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene regulation, and / or antibody-mediated depletion. In some cases, the safety switch protein is activated by exogenous molecules (e.g., prodrugs), which, upon activation, trigger apoptosis and / or cell death in the treated cells. Examples of safety switch proteins include, but are not limited to, suicide genes such as caspase 9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase, B-cell CD20, modified EGFR, and any combination thereof. In this strategy, prodrugs administered in adverse event conditions are activated by suicide gene products and kill transduced cells.

[0134] As used herein, the term "medicatologically active protein or peptide" refers to a protein or peptide capable of exerting biological and / or pharmaceutical effects on an organism. Medicinally active proteins possess curative or palliative properties against a disease and can be administered to improve, alleviate, relieve, reverse, or reduce the severity of a disease. Medicinally active proteins also possess preventative properties and are used to prevent the onset of a disease or to reduce the severity of such a disease or pathological symptom when it manifests. Medicinally active proteins include intact proteins or peptides or pharmaceutically active fragments thereof. They also include pharmaceutically active analogs of said proteins or peptides or analogs of fragments of said proteins or peptides. The term "medicinally active protein" also refers to a variety of proteins or peptides that act in a cooperative or synergistic manner to provide therapeutic benefits. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth inhibitory proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0135] As used herein, the term "signaling molecule" refers to any molecule that regulates, participates in, inhibits, activates, reduces, or increases cellular signal transduction. Signal transduction refers to the transmission of molecular signals in chemically modified forms, which is achieved by recruiting protein complexes along pathways that ultimately trigger biochemical events in the cell. Signal transduction pathways are well-known in the field and include, but are not limited to, G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, TG point signaling, ligand-gated ion channel signaling, ERK / MAPK signaling pathway, Wnt signaling pathway, cAMP-dependent pathway, and IP3 / DAG signaling pathway.

[0136] As used herein, the term “targeting modality” refers to the genetic incorporation of molecules (e.g., peptides) into cells to promote antigen and / or antigenic determinant specificity, including (but not limited to) i) antigen specificity (when it involves a unique chimeric antigen receptor (CAR) or T-cell receptor (TCR); ii) conjugate specificity (when it involves a monoclonal antibody or a bispecific conjugate); iii) targeting transformed cells; iv) targeting cancer stem cells; and v) other targeting strategies in the absence of specific antigens or surface molecules.

[0137] As used herein, the term “specific” can be used to refer to the ability of a molecule (e.g., a receptor or conjugate) to selectively bind to a target molecule, in contrast to nonspecific or nonselective binding.

[0138] As used herein, the term “adoptive cell therapy” refers to a cell-based immunotherapy, which, as used herein, refers to the infusion of autologous or allogeneic lymphocytes identified as genetically modified or unmodified T or B cells that have been expanded in vitro prior to the infusion.

[0139] As used herein, "therapeuticly adequate" includes, within its meaning, a non-toxic but sufficient and / or effective amount of the specific therapeutic and / or pharmaceutical composition mentioned herein to provide the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient's overall health status, age, and stage and severity of the condition. In certain embodiments, a therapeutically adequate amount is sufficient and / or effective in reducing, decreasing, and / or improving at least one symptom associated with the disease or condition of the subject being treated.

[0140] Differentiation of pluripotent stem cells requires alterations to the culture system, such as changes to the stimulants in the culture medium or the physical state of the cells. Most traditional strategies utilize the formation of embryonic bodies (EBs) as a common and crucial intermediate step in initiating lineage-specific differentiation. An "embryonic body" is a three-dimensional cluster that has been shown to mimic embryonic development because it generates multiple lineages within its three-dimensional region. Through the differentiation process, typically several hours to several days, simple EBs (such as aggregated pluripotent stem cells induced to differentiate) continue to mature and develop into cystic EBs, at which point they are typically further treated for several days to several weeks to continue differentiating. EB formation is initiated by bringing pluripotent stem cells close together to form three-dimensional, multi-layered cell clusters, typically achieved through one of several methods, including allowing pluripotent cells to settle in droplets, allowing cells to settle in a U-shaped bottom plate, or by mechanical agitation. Further differentiation cues are needed to promote EB development because aggregates maintained in pluripotent culture maintenance media do not form suitable EBs. Therefore, pluripotent stem cell aggregates need to be transferred to a differentiation medium that provides inducing cues to the selected lineage. EB-based pluripotent stem cell culture typically induces the generation of differentiated cell populations (ectoderm, mesoderm, and endoderm germ layers) through moderate proliferation within EB cell clusters. While EBs have been shown to promote cell differentiation, they produce heterogeneous cells with variable differentiation states due to inconsistent exposure of cells in a three-dimensional structure to differentiation cues from the environment. Furthermore, the generation and maintenance of EBs are cumbersome. Additionally, cell differentiation via EBs is accompanied by moderate cell proliferation, which also leads to reduced differentiation efficiency.

[0141] In contrast, "aggregate formation," distinct from "EB formation," can be used to expand pluripotent stem cell populations. For example, during aggregate-based pluripotent stem cell expansion, a culture medium that maintains proliferation and pluripotency is selected. Cell proliferation typically increases aggregate size, forming larger aggregates that can dissociate into smaller aggregates using conventional mechanical or enzymatic methods, thereby maintaining cell proliferation and increasing cell number within the culture. Unlike EB culture, cells cultured within aggregates maintain pluripotency markers. Pluripotent stem cell aggregates require further differentiation cues to induce differentiation.

[0142] As used herein, “monolayer differentiation” is a term referring to a differentiation method that differs from differentiation through three-dimensional, multi-layered cell clusters, i.e., “EB formation.” Monolayer differentiation, along with other advantages disclosed herein, avoids the need for EB formation to initiate differentiation. Because monolayer culture does not mimic embryonic development, such as EB formation, differentiation toward a specific lineage is considered minimal compared to all three germ layer differentiations within EB.

[0143] As used herein, "dissociated" cells refer to cells that have been substantially separated from or purified from other cells, or cells that have been substantially separated from a surface (e.g., a culture plate surface). For example, cells can be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells aggregated in a test tube can dissociate from each other, for example, by enzymatic or mechanical dissociation into clusters, single cells, or a suspension of a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells dissociate from a culture plate or other surface. Thus, dissociation can involve interrupting the interaction between cells and the extracellular matrix (ECM) and substrate (e.g., a culture surface), or interrupting the ECM between cells.

[0144] As used herein, "feeder cells" or "feeder layers" are terms used to describe a 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, as the feeder cells provide growth factors and nutrients that support the second cell type. Feeder cells optionally originate 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 and immortalized mouse embryonic fibroblasts. When co-cultured with other cells, feeder cells can typically be inactivated by irradiation or treatment with an antimitotic agent (such as mitomycin) to prevent them from growing beyond their supporting cells. Feeder cells can include endothelial cells, stromal cells (such as epithelial cells or fibroblasts), and leukemia cells. Not limited to the foregoing, a particular type of feeder cell can be a human feeder layer, such as human skin fibroblasts. Another type of feeder cell can be mouse embryonic fibroblasts (MEF). Generally, various feeder cells are capable of being used in part to maintain pluripotency, directly differentiate into a certain lineage, and promote maturation into specialized cell types, such as effector cells.

[0145] As used herein, a "feeder-free" (FF) environment refers to an environment, such as culture conditions, cell cultures, or culture media, that substantially does not contain a feeder layer or matrix cells, and / or has not been preconditioned by culturing feeder cells. A "preconditioned" medium refers to a culture medium collected after feeder cells have been cultured in it for a period of time (e.g., at least one day). Preconditioned media contain various mediators, including growth factors and cytokines secreted by feeder cells cultured in the medium.

[0146] In the context of genome editing or modification of iPSCs and their derivative non-pluripotent cells or in the context of genome editing or modification of non-pluripotent cells and their reprogrammed derivative iPSCs, "function" refers to (1) successful knock-in, knockout, reduction of gene expression, transgenesis, or controlled gene expression at the gene level, such as inducible or timely expression at a desired stage of cell development, achieved through direct genome editing or modification or through "transmission," through differentiation or reprogramming of the initial cell for which genome engineering was initially performed; or (2) successful removal, addition, or alteration of cell function / characteristics at the cellular level, achieved by: (i) through direct... (ii) gene expression changes obtained through direct genome editing in the cells; (iii) gene expression changes maintained in the cells by "transmission," by differentiation or reprogramming from the initial cells that were originally genome-engineered; (iv) downstream gene regulation in the cells as a result of gene expression changes that occur only in the earlier developmental stages of the cells or only in the initial cells that generated the cells through differentiation or reprogramming; or (iv) enhanced or newly acquired cell functions or properties presented in mature cell products that were originally obtained by genome editing or modification of iPSCs derived from progenitor or dedifferentiated cells.

[0147] "HLA deficiency," including deficiency of HLA class I, or deficiency of HLA class II, or both, refers to the absence or cessation of surface expression of the complete MHC complex containing HLA class I protein heterodimers and / or HLA class II heterodimers, or a reduction in the level of said surface expression such that the weakened or reduced level is below levels that are naturally detectable by other cells or detectable by synthetic methods. HLA class I deficiency can be achieved by loss of function in any region of the HLA class I locus (chromosome 6p21) or by loss of function or reduced expression of HLA class I-related genes (including, but not limited to, the β-2 microglobulin (B2M) gene, the TAP 1 gene, the TAP 2 gene, and TAP-related glycoproteins). HLA class II deficiency can be achieved by loss of function or reduced expression of HLA-II-related genes (including, but not limited to, RFXANK, CIITA, RFX5, and RFXAP). Prior to this invention, it was unclear whether iPSCs lacking or altered HLA complexes were capable of entering developing, maturing, and generating functionally differentiated cells while maintaining regulatory activity. Furthermore, prior to this invention, it was unclear whether differentiated cells lacking the HLA complex could be reprogrammed into iPSCs and maintained as pluripotent stem cells simultaneously lacking the HLA complex. Unexpected failures occurring during cell reprogramming, pluripotency maintenance, and differentiation may involve, but are not limited to, aspects including (but not limited to): developmental stage-specific gene expression or its absence, the need for HLA complex presentation, protein shedding from introduced surface expression patterns, the need for appropriate and efficient clonal reprogramming, and the need for reconfiguration of differentiation protocols.

[0148] As used herein, “HLA-deficient modified iPSCs” refers to HLA-deficient iPSCs that are further modified by introducing gene-expressed proteins associated with (but not limited to) improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, survival, immune evasion, inhibitory resistance, proliferation, co-stimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cell-mediated cytotoxicity, such as 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. “HLA-deficient modified” cells also include cells other than iPSCs.

[0149] "Fc receptors" (abbreviated as FcRs) are classified based on the types of antibodies they recognize. For example, those receptors that bind to the most common class of antibodies (IgG) are called Fc-γ receptors (FcγRs), those that bind to IgA are called Fc-α receptors (FcαRs), and those that bind to IgE are called Fc-ε receptors (FcεRs). FcRs are also classified by the cells that express them (macrophages, granulocytes, natural killer cells, T and B cells) and the signal transduction characteristics of each receptor. Fc-γ receptors (FcγRs) include several members: FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b), which have different affinities for antibodies due to their different molecular structures.

[0150] Two isoforms of CD16 have been identified: Fc receptor FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG on target cells to activate NK cells and promote antibody-dependent cell-mediated cytotoxicity (ADCC). As used herein, “high-affinity CD16,” “non-cleavable CD16,” or “non-cleavable high-affinity CD16” refers to CD16 variants. Wild-type CD16 has low affinity and undergoes extracellular domain shedding, a protein cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes after NK cell activation. F176V and F158V are exemplary high-affinity CD16 variants; while the S197P variant is an example of a non-cleavable form of CD16.

[0151] I. Methods for targeted genome editing at selected loci in iPSC cells

[0152] As used interchangeably in this article, genome editing or gene editing is a type of genetic engineering in which DNA insertion, deletion, and / or substitution are performed in the genome of a target cell. Targeted genome editing (which is interchangeable with “targeted genome editing” or “targeted gene editing”) enables insertion, deletion, and / or substitution at pre-selected sites in the genome. When an endogenous sequence is deleted at an insertion site during targeted editing, the endogenous gene containing the affected sequence can be knocked out or its expression can be reduced due to the sequence deletion. Therefore, targeted editing can also be used to disrupt the expression of endogenous genes. Similarly, the term “targeted integration” is used in this article, which refers to a method involving the insertion of one or more exogenous sequences with or without the endogenous sequence being deleted at an insertion site. In contrast, randomly integrated genes undergo positional effects and silencing, resulting in unreliable and unpredictable expression. For example, centromeres and subtelomere regions are particularly susceptible to transgene silencing. Conversely, newly integrated genes can influence surrounding endogenous genes and chromatin, potentially altering cellular properties or favoring cell transformation. Therefore, inserting exogenous DNA into preselected loci (such as safe harbor loci or genomic safe harbor (GSH)) is of great importance for safety, efficiency, copy number control, and reliable control of gene responses.

[0153] Targeted editing can be achieved through nuclease-independent or nuclease-dependent methods. In nuclease-independent targeted editing, homologous recombination is guided by the host cell's enzymatic mechanisms, through the reception of exogenous polynucleotides on the homologous sequence side.

[0154] Alternatively, targeted editing can be achieved by specifically introducing double-strand breaks (DSBs) using rare, specific-cutting endonucleases. This type of 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, NHEJ typically results in a small number of random insertions or deletions of endogenous nucleotides (insertions / deletions). In contrast, when a donor vector containing exogenous genetic material flanked by a pair of homologous arms is present, exogenous genetic material can be introduced into the genome via homologous recombination during homology-directed repair (HDR), resulting in “targeted integration.”

[0155] Available endonucleases capable of introducing specific and targeted DSBs include (but are not limited to) zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided CRISPR-Cas9 nucleases (CRISPR / Cas9; clustered regularly spaced short palindromic repeats associated with Cas9). Additionally, the DICE (dual integrase box exchange) system utilizing phiC31 and Bxb1 integrases is also a promising tool for targeted integration.

[0156] ZFNs are targeted nucleases that contain nucleases fused to zinc finger DNA-binding domains. A “zinc finger DNA-binding domain” or “ZFBD” refers to a polypeptide domain that binds to DNA via one or more zinc fingers in a sequence-specific manner. A zinc finger is a domain of approximately 30 amino acids within the zinc finger-binding domain, its structure stabilized by coordination with zinc ions. Examples of zinc fingers include (but are not limited to) C2H2, C3H, and C4 zinc fingers. A “designed” zinc finger domain is a domain that does not exist in nature; its design / composition is primarily derived from rational guidelines, such as the application of substitution rules and computerized algorithms to process information in databases storing existing ZFP designs and binding data. See, for example, U.S. Patent Nos. 6,140,081, 6,453,242, and 6,534,261; also see WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496. A “selected” zinc finger domain is a domain not found in nature, which is generated primarily by empirical methods such as phage presentation, interaction interception, or hybridization selection. ZFNs are described in more detail in U.S. Patent Nos. 7,888,121 and 7,972,854, the full disclosure of which is incorporated herein by reference. The most well-recognized example of a ZFN in the art is a fusion of the FokI nuclease with a zinc finger DNA-binding domain.

[0157] TALENs are targeted nucleases comprising a nuclease fused to a TAL effector DNA-binding domain. The terms "transcription activator-like effector DNA-binding domain," "TAL effector DNA-binding domain," or "TALE DNA-binding domain" refer to the polypeptide domain of a TAL effector protein responsible for binding TAL effector proteins 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 effector-specific DNA sequences via their DNA-binding domain, and activate gene transcription of these sequences via their transactivation domain. The specificity of the TAL effector DNA-binding domain depends on the variable number of imperfect 34-amino acid repeats of the effector, which includes polymorphisms at selected repeat positions, termed variable double residue repeats (RVDs). TALENs are described in more detail in U.S. Patent Application No. 2011 / 0145940, which is incorporated herein by reference. The most widely accepted example of a TALEN in the art is a fusion polypeptide of the FokI nuclease and a TAL effector DNA-binding domain.

[0158] Another example of a targeted nuclease used in the method of the present invention is a Spo11-targeting nuclease, a polypeptide comprising a Spo11 polypeptide having nuclease activity fused to a DNA-binding domain (e.g., a zinc finger DNA-binding domain, a TAL effector DNA-binding domain, etc., specific to the DNA sequence of interest). See, for example, U.S. Application No. 61 / 555,857, the disclosure of which is incorporated herein by reference.

[0159] Other examples of the target nucleases suitable for use in this invention include (but are not limited to) Bxb1, phiC31, R4, PhiBT1 and Wβ / SPBc / TP901-1, whether used individually or in combination.

[0160] Other non-limiting examples of targeted nucleases include naturally occurring recombinant nucleases such as CRISPR / cysteine ​​9, restriction endonucleases, large nucleases, homing endonucleases, and so on.

[0161] CRISPR / Caspase-9 requires two main components: (1) the caspase-9 endonuclease (Casp9) and (2) a crRNA-tracrRNA complex. When co-expressed, these two components form a complex recruited to the target DNA sequence, containing a PAM and a seeding region adjacent to the PAM. The crRNA and tracrRNA can combine to form a chimeric guide RNA (gRNA) to direct Casp9 to target the selected sequence. These two components can then be delivered to mammalian cells via transfection or transduction.

[0162] DICE-mediated insertion utilizes a pair of recombinases (e.g., phiC31 and Bxb1) to provide unidirectional integration of exogenous DNA, strictly limited to the small attB and attP recognition sites of each enzyme. Since these att targets are not naturally present in the mammalian genome, they must first be introduced into the desired integration site within the genome. See, for example, U.S. Patent Application Publication No. 2015 / 0140665, the disclosure of which is incorporated herein by reference.

[0163] One aspect of the invention provides a construct comprising one or more exogenous polynucleotides for targeted genome integration. In one embodiment, the construct further comprises a pair of homologous arms specific to the desired integration site, and the targeted integration method comprises introducing the construct into a cell to allow site-directed homologous recombination by a cell-host enzyme mechanism. In another embodiment, the method of achieving targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell and introducing a ZFN expression cassette comprising a DNA-binding domain specific to the desired integration site into the cell to achieve ZFN-mediated insertion. In yet another embodiment, the method of achieving targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell and introducing a TALEN expression cassette comprising a DNA-binding domain specific to the desired integration site into the cell to achieve TALEN-mediated insertion. In yet another embodiment, the method of achieving targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell, introducing a Cas9 expression cassette and a gRNA comprising a guide sequence specific to the desired integration site into the cell to achieve Cas9-mediated insertion. In yet another embodiment, the method for achieving targeted integration in a cell includes introducing a construct containing one or more att sites of a pair of DICE recombinases into a desired integration site in the cell, introducing a construct containing one or more exogenous polynucleotides into the cell, and introducing an expression cassette of the DICE recombinases to achieve DICE-mediated targeted integration.

[0164] Potential sites for targeted integration include (but are not limited to) safe harbor loci or genomic safe harbors (GSHs), which are intragenic or extragenic regions of the human genome that, theoretically, can accommodate the predictable expression of newly integrated DNA without adverse effects on the host cell or organism. A suitable safe harbor must allow transgene expression sufficient to produce the desired amount of the vector-encoded protein or non-coding RNA. The safe harbor must also not make the cell susceptible to malignant transformation or alter cellular function. If the integration site is a potential safe harbor locus, ideally it should meet criteria including (but not limited to) the following: no disruption of regulatory elements or genes, as determined by sequence annotation; it is an intergenic region within a densely gene-rich area, or a convergence point between two genes transcribed in opposite directions; it maintains distance to minimize the possibility of long-range interactions between the vector-encoded transcriptional activator and the promoters of adjacent genes (particularly cancer-related and microRNA genes); and it exhibits clearly pervasive transcriptional activity, as reflected by sequence tag (EST) expression profiles over a wide spatial and temporal range, indicating pervasive transcriptional activity. This latter characteristic is particularly important in stem cells, where chromatin remodeling during differentiation typically leads to the silencing of some loci and the potential activation of others. Within a suitable region for exogenous insertion, the exact locus selected for insertion should be free of repetitive elements and conserved sequences, and primers for amplifying homologous arms should be readily designed for it.

[0165] Sites suitable for human genome editing, or specifically for targeted integration, include (but are not limited to) adeno-associated virus site 1 (AAVS1), the chemokine (CC motif) receptor 5 (CCR5) locus, and human orthologs of the mouse ROSA26 locus. Additionally, human orthologs of the mouse H11 locus may also be suitable sites for insertion using the targeted integration compositions and methods disclosed herein. Furthermore, collagen and HTRP loci may also be used as safe havens for targeted integration. However, validation of each selected site has proven essential, particularly in stem cells with specific integration events, and often requires optimization of insertion strategies, including promoter selection, exogenous gene sequence and configuration, and construct design.

[0166] Editing sites for targeted insertions / deletions are typically contained in endogenous genes whose expression and / or function are intended to be disrupted. In one embodiment, endogenous genes containing targeted insertions / deletions are associated with the regulation and control of immune responses. In some other embodiments, endogenous genes containing targeted insertions / deletions are associated with: targeting patterns, receptors, signaling molecules, transcription factors, drug target candidates, immune response regulation and control, or proteins that inhibit the transplantation, transport, homing, viability, self-renewal, retention, and / or survival of stem cells and / or progenitor cells and their derived cells.

[0167] Therefore, one aspect of the present invention provides a method for targeted integration at selected loci, the selected loci including genomic safe harbors or known or proven safe pre-selected loci adequately regulated to achieve continuous or timed gene expression, such as B2M, TAP1, TAP2, or TAP-associated glycoprotein loci as provided herein. In one embodiment, the genomic safe harbor used in the targeted integration method comprises one or more desired integration sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the genomic safe harbor criteria. In one embodiment, a method for targeted integration in a cell includes: introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing a construct comprising a pair of homologous arms specific to the desired integration site and one or more exogenous sequences to achieve site-directed homologous recombination via a cell-host enzyme mechanism, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the genomic safe harbor criteria.

[0168] In another embodiment, the method of targeted integration in cells includes: introducing a construct containing one or more exogenous polynucleotides into the cell, and introducing a ZFN expression cassette containing a DNA-binding domain specific to the desired integration site into the cell to achieve ZFN-mediated insertion, wherein the desired integration site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the genomic safe harbor criteria. In yet another embodiment, the method of targeted integration in cells includes: introducing a construct containing one or more exogenous polynucleotides into the cell, and introducing a TALEN expression cassette containing a DNA-binding domain specific to the desired integration site into the cell to achieve TALEN-mediated insertion, wherein the desired integration site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the genomic safe harbor criteria. In another embodiment, the method of targeted integration in cells includes: introducing a construct containing one or more exogenous polynucleotides into the cell, introducing a Cas9 expression cassette and a gRNA containing a guide sequence specific to the desired integration site into the cell to achieve Cas9-mediated insertion, wherein the desired integration site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR or RUNX1, or other loci that meet the genomic safe harbor criteria. In yet another embodiment, the method for targeted integration in a cell comprises: introducing a construct containing one or more att sites of a pair of DICE recombinases into a desired integration site in a cell; introducing a construct containing one or more exogenous polynucleotides into the cell; and introducing an expression cassette of the DICE recombinases to achieve DICE-mediated targeted integration, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the genomic safe harbor criteria.

[0169] Additionally, as provided herein, the methods described above for targeted integration into safe harbors are for inserting any polynucleotide of interest, such as those encoding: safety switch proteins, targeting patterns, receptors, signal transduction molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, and proteins that promote stem cell and / or progenitor cell transplantation, transport, homing, viability, self-renewal, retention, and / or survival. In some other embodiments, the construct containing one or more exogenous polynucleotides further includes one or more marker genes. In one embodiment, the exogenous polynucleotide in the construct of the present invention is a suicide gene encoding a safety switch protein. Suicide gene systems suitable for inducing cell death include (but are not limited to) caspase 9 (or caspase 3 or 7) and AP1903; thymidine kinase (TK) and ganciclovir (GCV); cytosine deaminase (CD) and 5-fluorocytosine (5-FC). Furthermore, some suicide gene systems are cell type specific; for example, genetic modification of T lymphocytes using the B-cell molecule CD20 allows them to be eliminated after administration of the mAb rituximab. Additionally, when genetically engineered cells are exposed to cetuximab, modified EGFR containing antigenic determinants recognized by cetuximab can be used to deplete said cells. Therefore, one aspect of the present invention provides a method for targeting and integrating one or more suicide genes encoding safety switch proteins selected from caspase 9 (caspase 3 or 7), thymidine kinase, cytosine deaminase, modified EGFR, and B-cell CD20.

[0170] In some embodiments, one or more exogenous polynucleotides integrated by the methods herein are driven by an operatively linked exogenous promoter contained in a construct for targeted integration. The promoter may be inducible or structural, and may be time-specific, tissue-specific, or cell-type-specific. Structural promoters suitable for the methods of the present invention include, but are not limited to, cellular giant virus (CMV), elongation factor 1α (EF1α), phosphoglycerate kinase (PGK), hybrid CMV enhancer / chicken β-actin (CAG), and ubiquitin C (UBC) promoters. In one embodiment, the exogenous promoter is CAG.

[0171] Exogenous polynucleotides integrated using the methods described herein can be driven at the integration site by endogenous promoters in the host genome. In one embodiment, the method of the present invention is used to target the integration of one or more exogenous polynucleotides into the AAVS1 locus in the cellular genome. 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 to target the integration of the ROSA26 locus in the cellular genome. In one embodiment, at least one integrated polynucleotide is driven by the endogenous ROSA26 promoter. In yet another embodiment, the method of the present invention is used to target the integration of the H11 locus in the cellular genome. 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 to target the integration of the collagen locus in the cellular genome. In one embodiment, at least one integrated polynucleotide is driven by the endogenous collagen promoter. In yet another embodiment, the method of the present invention is used to target the integration of the HTRP locus in the cellular genome. In one embodiment, at least one integrated polynucleotide is driven by the endogenous HTRP promoter. Theoretically, gene expression of exogenous genes driven by endogenous promoters can only be achieved if the desired insertion is correct.

[0172] In some embodiments, one or more exogenous polynucleotides contained in the construct used in the targeted integration method are driven by a single promoter. In some embodiments, the construct includes one or more linker sequences located between two adjacent polynucleotides driven by the same promoter, such that the physical spacing between the parts is greater and the accessibility of the enzyme mechanism is maximized. The linker peptide of the linker sequence may consist of amino acids selected to create physical spacing between the parts (exogenous polynucleotides, and / or proteins or peptides encoded by them), and may be flexible or rigid depending on the relevant function. The linker sequence may be cleaved by a protease or chemically to produce individual parts. Examples of enzyme cleavage sites in the linker include cleavage sites of proteases (e.g., enterokinase, factor Xa, trypsin, collagenase, and thrombin). In some embodiments, the protease is a naturally occurring host protease or is introduced exogenously. Alternatively, the cleavage site in the linker may be a site capable of cleavage upon exposure to a selected chemical substance (e.g., cyanogen bromide, hydroxylamine, or low pH). The optionally present linker sequence may serve purposes other than providing a cleavage site. The linker sequence should allow the part to be efficiently positioned relative to another adjacent part so that the part functions correctly. Linkers can also be simple amino acid sequences of sufficient length to prevent any steric hindrance between the portions. Additionally, the linker sequence can be post-translational modified, including (but not limited to) phosphorylation sites, biotinylation sites, sulfation sites, γ-carboxylation sites, etc. In some embodiments, the linker sequence is flexible so that the bioactive peptide cannot maintain a single undesirable conformation. The linker may primarily contain amino acids with small side chains, such as glycine, alanine, and serine, to provide flexibility. In some embodiments, about 80 or 90% or more of the linker sequence contains glycine, alanine, or serine residues, especially glycine and serine residues. In several embodiments, the G4S linker peptide separates the terminal processing domain and the endonuclease domain of the fusion protein. In other embodiments, the 2A linker sequence allows for the production of two separate proteins in a single translation. Suitable linker sequences can be readily identified empirically. Furthermore, suitable size and sequence of the linker sequence can also be determined using 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, any two adjacent connection sequences are different.

[0173] Methods for introducing a construct containing a foreign polynucleotide to be integrated into cells can be implemented using conventionally known methods of gene transfer into cells. In one embodiment, the construct comprises the backbone of a viral vector, such as an adenovirus vector, an adeno-associated virus vector, a retroviral vector, a lentiviral vector, or a Sendai virus vector. In some embodiments, plasmid vectors are used to deliver foreign polynucleotides into target cells and / or to express foreign polynucleotides in target cells (e.g., pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc.). In some other embodiments, free vectors are used to deliver foreign polynucleotides into target cells. In some embodiments, recombinant adeno-associated virus (rAAV) can be used for genetic engineering to introduce insertions, deletions, or substitutions via homologous recombination. Unlike lentiviruses, rAAVs cannot integrate into the host genome. Furthermore, free rAAV vectors mediate homology-directed gene targeting at a much higher rate than transfection with conventional targeting plasmids. In some embodiments, AAV6 or AAV2 vectors are used to introduce insertions, deletions, or substitutions into targets within the iPSC genome.

[0174] II. Methods for obtaining and maintaining genome-engineered iPSCs

[0175] This invention provides a method for obtaining genome-engineered iPSCs that maintain their full function at the selected editing sites, wherein the iPSCs comprise one or more targeted edits performed at one or more desired sites. The targeted edits preserve the full function of the genome-engineered amplified iPSCs or iPSC-derived non-pluripotent cells at the corresponding selected editing sites. The targeted edits introduce iPSC insertions, deletions, and / or substitutions into the genome; that is, targeted integration and / or insertions / deletions are introduced at the selected sites.

[0176] In a particular embodiment, genome-engineered iPSCs containing one or more targeted edits at one or more selected sites are maintained, passaged, and expanded as single cells in a fate maintenance medium (FMM) as shown in Table 1, wherein the iPSCs maintain targeted editing and functional modification at the selected sites. The components of the medium may be present in the medium in amounts within the optimal range shown in Table 1. iPSCs cultured in the FMM have been shown to continue maintaining their undifferentiated and basal or untreated profile; genomic stability without the need for culture washing or selection; and readily generate all three somatic lineages through in vitro embryoid or monolayer differentiation (without embryoid formation) and in vivo teratoma formation. See, for example, U.S. Application No. 61 / 947,979, the disclosure of which is incorporated herein by reference.

[0177]

[0178] In some embodiments, genome-engineered iPSCs containing one or more targeted integrations and / or insertions / deletions are maintained, passaged, and expanded in loci containing MEKi, GSKi, and ROCKi and containing little or no TGFβ receptor / ALK5 inhibitors, wherein the iPSCs maintain full functional targeted editing at selected sites. In some embodiments, the targeted integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet genomic safe harbor criteria. In some embodiments, the targeted insertion / deletion site is selected from endogenous genes associated with: targeting patterns, receptors, signal transduction molecules, transcription factors, drug target candidates; immune response regulation and modulation; or proteins that inhibit iPSCs or their derived cell transplantation, transport, homing, viability, self-renewal, retention, and / or survival. In some embodiments, genome-engineered, maintained, passaged, and amplified iPSCs contain one or more suicide-inducible genes integrated into one or more desired integration sites, said integration sites including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet genomic safe harbor criteria. In some other embodiments, genome-engineered, maintained, passaged, and amplified iPSCs contain polynucleotides located at the same or different desired integration sites, said polynucleotides encoding: safety switch proteins, targeting mechanisms, receptors, signal transduction molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote stem cell and / or progenitor cell transplantation, transport, homing, viability, self-renewal, retention, and / or survival.

[0179] In some embodiments, the genome-engineered iPSC containing one or more exogenous polynucleotides further includes insertions / deletions in one or more endogenous genes. In some embodiments, the insertions / deletions in the endogenous genes cause gene expression to be interrupted. In some embodiments, the insertions / deletions in the endogenous genes cause the edited gene to be knocked out. In some embodiments, the insertions / deletions in the endogenous genes cause the expression of the edited gene to be reduced. In some embodiments, the genome-engineered iPSC containing one or more exogenous polynucleotides at selected sites may further include one or more targeted edits, including insertions / deletions at selected sites. In some embodiments, the insertions / deletions are included in one or more endogenous genes related to the regulation and mediation of immune responses. In some embodiments, the insertions / deletions are included in one or more endogenous checkpoint genes. In some embodiments, the insertions / deletions are included in one or more endogenous T cell receptor genes. In some embodiments, the insertions / deletions are included in one or more endogenous MHC class I repressor genes. In some embodiments, the insertions / deletions are included in one or more endogenous genes associated with the major histocompatibility complex. In some embodiments, the insertion / deletion is contained in one or more endogenous genes, including (but not limited to) B2M, PD1, TAP1, TAP2, TAP-associated glycoproteins, and TCR genes. In one embodiment, the genome-engineered iPSC containing one or more exogenous polynucleotides located at selected sites further includes targeted editing in the B2M (β-2 microglobulin) encoding gene.

[0180] Another aspect of the invention provides a method for generating genome-engineered iPSCs via targeted editing of the iPSCs; or, firstly, generating genome-engineered non-pluripotent cells via targeted editing, and then reprogramming selected / isolated genome-engineered non-pluripotent cells to obtain iPSCs containing the same targeted editing as the non-pluripotent cells. Another aspect of the invention provides genome-engineered non-pluripotent cells that undergo reprogramming simultaneously by introducing targeted integration and / or targeted insertion / deletion into the cells, wherein the contacted non-pluripotent cells are under conditions sufficient for reprogramming, and wherein the reprogramming conditions include contacting the non-pluripotent cells with one or more reprogramming factors and small molecules. In various embodiments of the method of simultaneously performing genome engineering and reprogramming, targeted integration and / or targeted insertion / deletion can be introduced into the non-pluripotent cells by contacting them with one or more reprogramming factors and small molecules before or substantially simultaneously with initiating reprogramming.

[0181] In some embodiments, in order to simultaneously perform genome engineering and reprogramming on non-pluripotent cells, targeted integration and / or insertion / deletion may be introduced into the 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, wherein the vector carrying the construct is introduced before the reprogrammed cells exhibit stable expression of one or more endogenous pluripotent genes (including, but not limited to, SSEA4, Tra181, and CD30).

[0182] In some embodiments, reprogramming is initiated by contacting 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, genomically engineered iPSCs achieved by any of the above methods are further maintained and expanded using a mixture comprising a combination of MEK inhibitors, GSK3 inhibitors, and ROCK inhibitors (FMM; Table 1).

[0183] In some embodiments of a method for generating genome-engineered iPSCs, the method comprises: genome-engineering iPSCs by introducing one or more targeted integrations and / or insertions / deletions into selected sites in the iPSCs to obtain genome-engineered iPSCs. Alternatively, a method for generating genome-engineered iPSCs comprises: (a) introducing one or more targeted edits into non-pluripotent cells to obtain genome-engineered non-pluripotent cells containing targeted integrations and / or insertions / deletions at selected sites, and (b) contacting the genome-engineered non-pluripotent cells with one or more reprogramming factors and optionally a small molecule composition to obtain genome-engineered iPSCs containing targeted integrations and / or insertions / deletions at selected sites, said small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor. Alternatively, a method for generating genome-engineered iPSCs comprises: (a) contacting a non-pluripotent cell with one or more reprogramming factors and optionally a small molecule composition to initiate reprogramming of the non-pluripotent cell, said small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor; (b) introducing one or more targeted integration and / or insertion / deletion sites into the genome-engineered reprogrammed non-pluripotent cell; and (c) obtaining genome-engineered cloned iPSCs containing targeted integration and / or insertion / deletion sites located at selected sites.

[0184] 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. One or more reprogramming factors may be in peptide form. The reprogramming factor may also be in polynucleotide form and thus introduced into non-pluripotent cells via a vector (e.g., retrovirus, Sendai virus, adenovirus, cell-free genome, and small loop). In certain embodiments, one or more polynucleotides encoding at least one reprogramming factor are introduced via a lentiviral vector. In some embodiments, one or more polynucleotides are introduced via a cell-free vector. In several other embodiments, one or more polynucleotides are introduced via a Sendai virus vector.

[0185] In some embodiments, non-pluripotent cells are transferred using multiple constructs comprising different exogenous polynucleotides and / or different promoters via multiple vectors for targeted integration at the same or different selected sites. These exogenous polynucleotides may comprise suicide genes, or genes encoding targeting patterns, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or genes encoding proteins that promote iPSC or cell-derived cells to transplant, transport, homing, viability, self-renewal, retention, and / or survival. In some embodiments, the exogenous polynucleotides encode RNA, including (but not limited to) siRNA, shRNA, miRNA, 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 of activated promoters (e.g., in the presence of an inducer or in a specific differentiated cell type). In some embodiments, the polynucleotides are expressed in iPSCs and / or in cells differentiated from iPSCs. In one embodiment, one or more suicide genes are driven by a constitutive promoter, such as CAG-driven caspase-9. These constructs, containing different exogenous polynucleotides and / or different promoters, can be transferred simultaneously or sequentially to non-pluripotent cells. Non-pluripotent cells undergoing targeted integration of multiple constructs can be simultaneously contacted with one or more reprogramming factors to initiate reprogramming and genome engineering concurrently, thereby obtaining genome-engineered iPSCs containing multiple targeted integrations within the same cell pool. Thus, this stable approach enables reprogramming and engineering strategies to be performed simultaneously to obtain genome-engineered clone hiPSCs with multiple patterns integrated at one or more selected target sites.

[0186] In some embodiments, one or more insertions / deletions are introduced into selected sites in non-pluripotent cells, including one or more endogenous genes. In some embodiments, non-pluripotent cells containing one or more insertions / deletions further include one or more of the aforementioned targeted integrations. In some embodiments, the insertions / deletions included in the endogenous genes cause gene expression to be interrupted. In some embodiments, the insertions / deletions included in the endogenous genes cause the edited gene to be knocked out. In some embodiments, the insertions / deletions included in the endogenous genes cause the expression of the edited gene to be reduced. In some embodiments, genome-engineered iPSCs containing one or more exogenous polynucleotides located at selected sites may further include one or more targeted edits located at selected sites, including insertions / deletions. In some embodiments, the insertions / deletions are included in one or more endogenous genes related to the regulation and mediation of immune responses. In some embodiments, the insertions / deletions are included in one or more endogenous checkpoint genes. In some embodiments, the insertions / deletions are included in one or more endogenous T cell receptor genes. In some embodiments, the insertions / deletions are included in one or more endogenous MHC class I repressor genes. In some embodiments, the insertions / deletions are included in one or more endogenous genes associated with the major histocompatibility complex. In some embodiments, the insertion / deletion is contained in one or more endogenous genes, including (but not limited to) B2M, PD1, TAP1, TAP2, TAP-associated glycoproteins, and TCR genes. In one embodiment, the genome-engineered iPSC containing one or more exogenous polynucleotides located at selected sites further includes targeted editing in the B2M (β-2-microglobulin) encoding gene.

[0187] III. Methods for obtaining genetically engineered non-pluripotent cells by differentiating genome-engineered iPSCs.

[0188] Another aspect of the present invention provides a method for in vivo differentiation of genome-engineered iPSCs through teratoma formation, wherein the differentiated cells derived from genome-engineered iPSCs in vivo maintain complete functional targeted editing, including targeted integration and / or insertion / deletion at desired sites. In some embodiments, the differentiated cells derived from genome-engineered iPSCs in vivo through teratoma formation contain one or more suicide-inducible genes integrated at one or more desired sites, the desired sites including AAVS1, CCR5, ROSA26, collagen, HTRPH11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet genomic safe harbor criteria. In some other embodiments, the differentiated cells derived from genome-engineered iPSCs in vivo through teratoma formation contain polynucleotides encoding targeted patterns, or polynucleotides encoding proteins that promote stem cell and / or progenitor cell transport, homing, viability, self-renewal, retention, and / or survival. In some embodiments, differentiated cells containing one or more suicide-inducible genes from teratoma-derived, genome-engineered iPSCs further include one or more insertions / deletions in endogenous genes associated with the regulation and mediation of 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 repressor genes. In some embodiments, the insertions / deletions are in one or more endogenous genes associated with the major histocompatibility complex. In some embodiments, the insertions / deletions are in one or more endogenous genes, including (but not limited to) B2M, PD1, TAP1, TAP2, TAP-associated glycoproteins, and TCR genes. In one embodiment, genome-engineered iPSCs containing one or more exogenous polynucleotides at selected sites further include targeted editing in the B2M (β-2-microglobulin) coding gene.

[0189] In certain embodiments, genome-engineered iPSCs containing one or more targeted edits at selected sites, as provided herein, are used to derive hematopoietic cell lineages or any other specific cell type in vitro, wherein the derived non-pluripotent cells maintain functional targeted editing at the selected sites. In one embodiment, the genome-engineered iPSC source cells include (but are not limited to) mesodermal cells with permanent hematopoietic endothelial (HE) potential, permanent HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells (MPP), T cell progenitor cells, NK cell progenitor cells, bone marrow cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, and B cells, wherein these cells derived from the genome-engineered iPSCs maintain functional targeted editing at the desired sites.

[0190] Differentiation methods and compositions applicable to obtaining iPSC-derived hematopoietic cell lineages include, for example, those described in International Application No. PCT / US2016 / 044122, the disclosure of which is incorporated herein by way of introduction. As provided, the methods and compositions for generating hematopoietic cell lineages are achieved by using permanently derived hematopoietic endothelial cells (HE) (including hiPSCs) from pluripotent stem cells under serum-free, feeder-free, and / or matrix-free conditions in a scalable monolayer culture platform without the formation of EB. Cells that can be differentiated according to the provided methods range from pluripotent stem cells to progenitors that are typecast into specific terminally differentiated and transdifferentiated cells, directly converting to hematopoietic fate without pluripotent intermediates, across a variety of cell lineages. Similarly, the cells generated from stem cell differentiation range from pluripotent stem cells or progenitors to terminally differentiated stem cells, and all intermediate hematopoietic cell lineages.

[0191] A method for differentiating and expanding pluripotent stem cells into hematopoietic lineage cells in a monolayer culture comprises contacting the pluripotent stem cells with a BMP pathway activator and optionally present bFGF. As provided, pluripotent stem cell-derived mesodermal cells are obtained and expanded without the formation of embryonic 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 permanent hematopoietic endothelial (HE) potential without the formation of embryonic bodies from the pluripotent stem cells. The mesodermal cells with permanent HE potential are differentiated into permanent HE cells by subsequent contact with bFGF and optionally present ROCK inhibitors and / or WNT pathway activators, which also expand during differentiation.

[0192] The method presented in this paper for obtaining hematopoietic lineage cells is superior to EB-mediated pluripotent stem cell differentiation because: EB formation produces moderate to minimal cell expansion; it does not allow monolayer culture, which is important for many applications that require uniform expansion and differentiation of said cells in a population; and it is laborious and inefficient.

[0193] The provided monolayer differentiation platform promotes differentiation into permanent hematopoietic endothelial cells, thereby generating hematopoietic stem cells and differentiated progeny such as T, B, NKT, and NK cells. The monolayer differentiation strategy achieves a combination of enhanced differentiation efficiency and large-scale expansion, enabling the delivery of a therapeutically relevant number of pluripotent stem cell-derived hematopoietic cells in various therapeutic applications. Furthermore, monolayer cultures using the methods provided herein generate functional hematopoietic lineage cells that achieve a full range of in vitro differentiation, in vitro regulation, and long-term in vivo hematopoietic self-renewal, remodeling, and transplantation. As provided, iPSC-derived hematopoietic lineage cells include (but are not limited to) permanent hematopoietic endothelial cells, hematopoietic pluripotent progenitor cells, hematopoietic stem cells and progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0194] A method for guiding pluripotent stem cells to differentiate into permanent hematopoietic lineage cells, wherein the method comprises: (i) contacting the pluripotent stem cells with a composition comprising a BMP activator and optionally bFGF to initiate differentiation of the pluripotent stem cells into and expansion of mesodermal cells; (ii) contacting the mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor to initiate differentiation of the mesodermal cells into and expansion of mesodermal cells with permanent hematopoietic endothelial cell potential, wherein the composition optionally does not contain a TGFβ receptor / ALK inhibitor; and (iii) contacting the mesodermal cells with permanent hematopoietic endothelial cell potential with a composition to initiate differentiation of pluripotent stem cell-derived mesodermal cells with permanent hematopoietic endothelial cell potential into and expansion of permanent hematopoietic endothelial cells, wherein the composition comprises a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, wherein the composition optionally does not contain a TGFβ receptor / ALK inhibitor.

[0195] In some embodiments, the method further comprises contacting pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor to seed and expand the pluripotent stem cells, wherein the composition does not contain a TGFβ receptor / ALK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs, or untreated iPSCs, or iPSCs containing one or more genetic imprints; and the one or more genetic imprints contained in the iPSCs are retained in the hematopoietic cells differentiated from them. In some embodiments of the method for guiding the differentiation of pluripotent stem cells into hematopoietic lineage cells, the differentiation of pluripotent stem cells into hematopoietic lineage cells does not involve the production of embryonic bodies and is carried out in a monolayer culture format.

[0196] In some embodiments of the above method, the obtained pluripotent stem cell-derived permanent hematopoietic endothelial cells are CD34+. In some embodiments, the obtained permanent hematopoietic endothelial cells are CD34+CD43-. In some embodiments, the permanent hematopoietic endothelial cells are CD34+CD43-CXCR4-CD73-. In some embodiments, the permanent hematopoietic endothelial cells are CD34+CXCR4-CD73-. In some embodiments, the permanent hematopoietic endothelial cells are CD34+CD43-CD93-. In some embodiments, the permanent hematopoietic endothelial cells are CD34+CD93-.

[0197] In some embodiments of the above method, the method further comprises (i) contacting pluripotent stem cell-derived perpetual hematopoietic endothelial cells with a composition to initiate the differentiation of the perpetual hematopoietic endothelial cells into pre-T cell progenitor cells, the composition comprising a ROCK inhibitor, one or more growth factors and cytokines, and optionally a BMP activator, the cytokines being selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally, (ii) contacting the pre-T cell progenitor cells with the composition to initiate the differentiation of the pre-T cell progenitor cells into T cell progenitor cells or T cells, the composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but excluding one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor. In some embodiments of the method, the pluripotent stem cell-derived T cell progenitor cells are CD34+CD45+CD7+. In some embodiments of the method, the pluripotent stem cell-derived T cell progenitor cells are CD45+CD7+.

[0198] In further embodiments of the above-described method for guiding pluripotent stem cells to differentiate into hematopoietic lineage cells, the method further comprises: (i) contacting pluripotent stem cell-derived permatrophic hematopoietic endothelial cells with a composition to initiate the differentiation of the permatrophic hematopoietic endothelial cells into pre-NK cell progenitor cells, the composition comprising a ROCK inhibitor, one or more growth factors and cytokines, and optionally a BMP activator, the cytokines being selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15; and optionally, (ii) contacting pluripotent stem cell-derived pre-NK cell progenitor cells with a composition comprising one or more growth factors and cytokines to initiate the differentiation of the pre-NK progenitor cells into NK cell progenitor cells or NK cells, the cytokines being selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, wherein the culture medium is free of one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor. 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 defined by NKp46+, CD57+, and CD16+.

[0199] Therefore, the above differentiation methods can be used to obtain one or more iPSC-derived hematopoietic cell populations: (i) CD34+ HE cells (iCD34) using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; (ii) permanent hematopoietic endothelial cells (iHE) using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; and (iii) permanent HSCs using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2. NK-B2 culture medium; (iv) multipotent progenitor cells (iMPP) using iMPP-A; (v) T cell progenitor cells (ipro-T) using one or more culture media selected from iTC-A2 and iTC-B2; (vi) T cells (iTC) using iTC-B2; (vii) NK cell progenitor cells (ipro-NK) using one or more culture media selected from iNK-A2 and iNK-B2; and / or (viii) NK cells (iNK) and iNK-B2. In some embodiments, the culture medium:

[0200] a.iCD34-C contains a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, IL11, IGF and EPO, and optionally present Wnt pathway activators; and does not contain TGFβ receptor / ALK inhibitors;

[0201] b.iMPP-A contains 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.

[0202] c.iTC-A2 contains 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;

[0203] d.iTC-B2 contains one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7; wherein the composition does not contain one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors;

[0204] e.iNK-A2 comprises a ROCK inhibitor and one or more growth factors and cytokines, as well as an optionally present BMP activator, wherein the cytokines are selected from the group consisting of: SCF, Flt3L, TPO, IL3, IL7, and IL15, and

[0205] f.iNK-B2 contains one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7 and IL15.

[0206] In some embodiments, the genome-engineered iPSC source cells obtained by the above methods contain one or more suicide-inducible genes integrated into one or more desired integration sites, said integration sites including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the genomic safe harbor criteria. In some other embodiments, the genome-engineered iPSC source cells contain polynucleotides encoding safety switch proteins, targeting patterns, receptors, signal transduction molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote stem cell and / or progenitor cell transport, homing, viability, self-renewal, retention, and / or survival. In some embodiments, the genome-engineered iPSC source cells containing one or more suicide genes further contain one or more insertions / deletions contained in one or more endogenous genes related to the regulation and mediation of immune responses, said endogenous genes including (but not limited to) checkpoint genes, endogenous T cell receptor genes, and MHC class I repressor genes. In one embodiment, the genome-engineered iPSC source cell containing one or more suicide genes further includes an insertion / deletion located in the B2M gene, wherein the B2M is knocked out.

[0207] Additionally, methods and compositions applicable to the dedifferentiation of genome-engineered hematopoietic cells with a first fate to genome-engineered hematopoietic cells with a second fate include, for example, those methods and compositions described in International Publication No. WO2011 / 159726, the disclosure of which is incorporated herein by way of introduction. The methods and compositions provided therein allow for the partial reprogramming of initial non-pluripotent cells into non-pluripotent intermediate cells by restricting endogenous Nanog gene expression during reprogramming and subjecting the non-pluripotent intermediate cells to conditions for differentiating the intermediate cells into the desired cell type.

[0208] IV. Compositions that allow targeted integration at selected loci to incorporate genes of interest into iPSC cells.

[0209] In view of the foregoing, the present invention provides a construct that enables the targeted integration of one or more exogenous polynucleotides into a selected site in the genome of induced pluripotent stem cells (iPSCs), with or without deletion of any endogenous sequence at the integration site. In one embodiment, the construct comprises at least one exogenous promoter operatively linked to one or more exogenous polynucleotides expressing one or more proteins of interest. In one embodiment, the construct is transferred into iPSCs via a vector for targeted integration. In another embodiment, the construct is transferred into non-pluripotent cells via a vector for targeted integration, and the non-pluripotent cells are then reprogrammed to obtain genome-engineered iPSCs. By either method, the construct remains integrated and the exogenous polynucleotide functions in the reprogrammed or vector-transferred iPSCs, in subsequently expanded iPSCs, in differentiated cells derived from iPSCs, and in dedifferentiated cells derived therefrom.

[0210] In some embodiments, a construct comprising at least one exogenous promoter operatively linked to one or more exogenous polynucleotides further comprises a homologous arm specific to a selected site, wherein the homologous arm side-connects the promoter and the exogenous polynucleotide of interest within the construct. This embodiment of the construct achieves a nuclease-independent targeted integration strategy. In some embodiments, the exogenous promoter is a CAG.

[0211] 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 some 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.

[0212] Some embodiments of the construct comprising one or more exogenous promoters and one or more exogenous polynucleotides operatively linked 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, a polynucleotide encodes a fluorescent protein. In another embodiment, a polynucleotide is a puromycin resistance gene.

[0213] In some embodiments, the construct comprising operatively linked polynucleotides further includes components that allow targeted integration at a safe harbor locus, said safe harbor locus comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the genomic safe harbor criteria. In some embodiments, the safe harbor locus is selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, and RUNX1.

[0214] An architecture comprising operably linked polynucleotides may contain one or more polynucleotides encoding safety switch proteins, targeting mechanisms, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote the transplantation, transport, homing, viability, self-renewal, retention, and / or survival of genome-engineered iPSCs or their derivatives. 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 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 a particular embodiment, one or more exogenous polynucleotides operably linked to an exogenous or endogenous promoter encode one or more safety switch proteins. The safety switch protein may be selected from the group consisting of: caspase-9 (or caspase-3 or 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 the loci: AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, and other loci that meet the genomic safe harbor criteria. In some embodiments, the construct is for targeted integration at AAVS1. In some embodiments, the construct is for targeted integration at ROSA26. In some embodiments, the construct is for targeted integration at H11.

[0215] This invention also provides a genome-engineered iPSC comprising one or more exogenous polynucleotides and / or insertions / deletions at selected sites. In some embodiments, the genome-engineered iPSC is obtained by transferring constructs to iPSCs via vectors and / or by using NHEJ editing. In some other embodiments, the genome-engineered iPSC is obtained from reprogrammed non-pluripotent cells using targeted editing (including targeted integration and / or insertions / deletions). In still other embodiments, the genome-engineered iPSC is obtained by simultaneously reprogramming and genome-engineering non-pluripotent cells using reprogramming factors, reprogramming small molecules, and / or vectors carrying constructs for targeted integration of the polynucleotide of interest.

[0216] In some embodiments, the genome-engineered iPSCs contain one or more exogenous polynucleotides encoding proteins selected from safety switch proteins, targeting mechanisms, receptors, signal transduction molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates; or proteins that promote the transplantation, transport, homing, viability, self-renewal, retention, and / or survival of iPSCs or their derived cells. In some embodiments, the genome-engineered iPSCs contain insertions / deletions in one or more endogenous genes encoding proteins associated with: targeting mechanisms, receptors, signal transduction molecules, transcription factors, drug target candidates, immune response regulation and control; or proteins that inhibit the transplantation, transport, homing, viability, self-renewal, retention, and / or survival of iPSCs or their derived cells. In some embodiments, the genome-engineered iPSCs containing one or more exogenous polynucleotides further contain insertions / deletions in one or more endogenous genes. In some embodiments, the insertions / deletions in the endogenous genes cause gene expression to be interrupted. In some embodiments, the insertions / deletions in the endogenous genes cause the edited gene to be knocked out. In some embodiments, insertions / deletions in endogenous genes cause a decrease in the expression of the edited gene. In some embodiments, the genome-engineered iPSC containing one or more exogenous polynucleotides at selected sites may further contain one or more targeted edits at selected sites, including insertions / deletions. In some embodiments, the insertions / deletions are contained in one or more endogenous genes associated with the regulation and mediation of immune responses. In some embodiments, the insertions / deletions are contained in one or more endogenous checkpoint genes. In some embodiments, the insertions / deletions are contained in one or more endogenous T cell receptor genes. In some embodiments, the insertions / deletions are contained in one or more endogenous MHC class I repressor genes. In some embodiments, the insertions / deletions are contained in one or more endogenous genes associated with the major histocompatibility complex. In some embodiments, the insertions / deletions are contained in one or more endogenous genes, including (but not limited to) B2M, PD1, TAP1, TAP2, TAP-associated glycoproteins, and TCR genes. In one embodiment, the genome-engineered iPSC containing one or more exogenous polynucleotides at selected sites further contains targeted edits in the B2M (β-2-microglobulin) encoding gene. In one embodiment, the genome-engineered iPSC containing targeted editing of the B2M gene is a B2M knockout or low-content type and lacks HLA-I.

[0217] In some embodiments, the genome-engineered iPSC contains at least one suicide gene integrated at a selected site. In some embodiments, the genome-engineered iPSC contains 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 constitutive promoters, inducible promoters, time-specific promoters, and tissue or cell type-specific promoters. In some embodiments, the genome-engineered iPSC containing one or more suicide genes at a selected integration site further contains insertions / deletions present in one or more selected endogenous genes. As disclosed herein, non-pluripotent cells with a variety of desired targeted editing patterns are suitable for reprogramming using the culture platforms in Table 1 to obtain derived iPSCs of genome-engineered iPSCs. In some embodiments, genome engineering of non-pluripotent cells for targeted integration or insertion / deletion occurs simultaneously with the reprogramming of non-pluripotent cells and produces genome-engineered iPSCs in a stable, efficient, accurate, and reliable manner.

[0218] This invention further provides non-pluripotent cells derived from genome-engineered iPSCs, wherein the genome-engineered iPSCs are obtained by targeted editing of iPSCs or by reprogramming genome-engineered non-pluripotent cells with site-specific integration or insertion / deletion, wherein the targeted editing and reprogramming are performed sequentially (first obtaining reprogrammed iPSCs, then performing genome engineering; or first obtaining genome-engineered non-pluripotent cells, then performing reprogramming), or simultaneously (simultaneously performing reprogramming and genome engineering on the same cell pool). In some embodiments, the genome-engineered iPSC-derived non-pluripotent cells are progenitor cells or fully differentiated cells. In some embodiments, the genome-engineered iPSC-derived cells are mesodermal cells, CD34 cells, hematopoietic endothelial cells, hematopoietic stem cells or progenitor cells, hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells, or B cells.

[0219] This invention also provides a composition for obtaining genome-engineered iPSCs. In one embodiment, the composition comprises a genome-engineered 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 genome-engineered non-pluripotent cell into genome-engineered iPSCs containing the same targeted editing. In some embodiments of the above composition, the genome-engineered non-pluripotent cell for reprogramming comprises one or more polynucleotides encoding safety switch proteins, targeting modes, receptors, signal transduction molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote the transplantation, transport, homing, viability, self-renewal, retention, and / or survival of iPSCs or their 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 contained in different constructs capable of targeted integration at the same or different selected sites. In some embodiments, the one or more selected sites comprise AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the genomic safe harbor criteria.

[0220] In some other embodiments, the composition for obtaining genome-engineered iPSCs comprises non-pluripotent cells, one or more site-directed 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 operatively linked polynucleotides with homologous arms for targeted integration. The composition is suitable for simultaneously genome-engineering and reprogramming non-pluripotent cells to obtain genome-engineered iPSCs containing targeted integration and / or insertions / deletions at selected sites. In some embodiments, different constructs and / or insertions / deletions are introduced simultaneously or sequentially with the reprogramming factors into the non-pluripotent cells. In some embodiments, different constructs and / or insertions / deletions are introduced into the non-pluripotent cells during reprogramming to allow for simultaneous or parallel genome engineering and reprogramming.

[0221] In some other embodiments, the composition for obtaining genome-engineered iPSCs comprises non-pluripotent cells, one or more reprogramming factors; and a combination of TGFβ receptor / ALK inhibitors, MEK inhibitors, GSK3 inhibitors, and ROCK inhibitors; and one or more constructs comprising operatively linked polynucleotides with homologous arms for targeted integration. The composition is suitable for simultaneously genome-engineering and reprogramming non-pluripotent cells to obtain genome-engineered iPSCs containing targeted integration and / or insertions / deletions at selected sites. In some embodiments, different constructs and / or insertions / deletions are introduced simultaneously or sequentially with the reprogramming factors into the non-pluripotent cells. In some embodiments, different constructs and / or insertions / deletions are introduced into the non-pluripotent cells during reprogramming to allow for simultaneous or parallel genome engineering and reprogramming.

[0222] Additionally, this document provides a composition for maintaining genome-engineered iPSCs obtained by reprogramming genome-engineered non-pluripotent cells. In one embodiment, the composition comprises genome-engineered iPSCs obtained by reprogramming genome-engineered non-pluripotent cells; and a combination of a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor. In one embodiment, the reprogrammed iPSCs maintain pluripotency and targeted editing during long-term passage and expansion.

[0223] A composition is also provided comprising: genome-engineered iPSCs and a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, wherein the genome-engineered iPSCs are obtained as follows: (a) reprogramming genome-engineered non-pluripotent cells, wherein the resulting iPSCs comprise the same targeted edits contained in genome-engineered non-pluripotent cells; or (b) genome-engineering a cloned iPSC or iPSC pool by introducing one or more targeted edits to selected sites; or (c) genome-engineering a reprogrammed non-pluripotent cell pool by introducing one or more targeted edits to selected sites, wherein the reprogrammed non-pluripotent cells are 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, genome-engineered iPSCs comprise one or more polynucleotides encoding safety switch proteins, targeting mechanisms, receptors, signal transduction molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote the transplantation, transport, homing, viability, self-renewal, survival, and / or survival of iPSCs or their derivatives after reprogramming into non-pluripotent cells. In some embodiments, genome-engineered iPSCs comprise insertions / deletions present in one or more endogenous genes encoding proteins associated with: targeting mechanisms, receptors, signal transduction molecules, transcription factors, drug target candidates, immune response regulation and control; or proteins that inhibit the transplantation, transport, homing, viability, self-renewal, survival, and / or survival of iPSCs or their derivatives. In some embodiments, genome-engineered iPSCs comprising one or more exogenous polynucleotides further comprise insertions / deletions present in one or more endogenous genes.

[0224] V. Therapeutic applications of genetically engineered iPSCs and functionally modulated immune cells derived from them.

[0225] This invention provides a composition comprising a segregation or subset of genetically engineered iPSCs and / or immune cells derived from said iPSCs using the disclosed methods and compositions, wherein said immune cells are genetically engineered and suitable for cell-based adoptive therapy. In one embodiment, the segregation or subset of genetically engineered immune cells comprises iPSC-derived HSC cells. In one embodiment, the segregation or subset of genetically engineered immune cells comprises iPSC-derived HSC cells. In one embodiment, the segregation or subset of genetically engineered immune cells comprises iPSC-derived proT or T cells. In one embodiment, the segregation or subset of genetically engineered immune cells comprises iPSC-derived proNK or NK cells. In some embodiments, the genetically engineered iPSC-derived immune cells are further modulated in vitro to improve therapeutic potential. In one embodiment, the segregation or subset of genetically engineered immune cells derived from iPSCs comprises an increased number or ratio of untreated T cells, stem cell memory T cells, and / or central memory T cells. In one embodiment, the isolated population or subset of genetically engineered immune cells derived from iPSCs includes an increased number or ratio of type I NKT cells. In another embodiment, the isolated population or subset of genetically engineered immune cells derived from iPSCs includes an increased number or ratio of adaptive NK cells. In some embodiments, the isolated population or subset of genetically engineered CD34 cells, HSC cells, T cells, or NK cells derived from iPSCs is allogeneic. In some other embodiments, the isolated population or subset of genetically engineered CD34 cells, HSC cells, T cells, or NK cells derived from iPSCs is autogenous.

[0226] In some embodiments, the iPSCs used for differentiation contain a genetic imprint of the desired therapeutic properties of the effector cells, which is retained and function in the differentiated hematopoietic cells derived from the iPSCs.

[0227] In some embodiments, the genetic imprinting in pluripotent stem cells includes (i) one or more genetic modification patterns obtained during or after reprogramming non-pluripotent cells into iPSCs by genomic insertion, deletion, or substitution in the pluripotent cell genome; or (ii) one or more source-specific immune cells that retain therapeutic properties and are specific to donor, disease, or treatment response, wherein the pluripotent cells are reprogrammed from source-specific immune cells, wherein the iPSCs retain source therapeutic properties, and the source therapeutic properties are also contained in the iPSC-derived hematopoietic lineage cells.

[0228] In some embodiments, the genetic modification pattern includes one or more of the following: safety switch proteins, targeting patterns, receptors, signal transduction molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates; or proteins that promote the transplantation, transport, homing, viability, self-renewal, survival, immune response regulation and / or survival of iPSCs or their derivatives. In some other embodiments, the gene modification pattern includes one or more of the following: (i) deletion or reduced expression of any gene of B2M, TAP1, TAP2, TAP-associated glycoprotein, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5 or RFXAP, and the 6p21 region of chromosome; (ii) introduction or increase of expression of HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, CAR, TCR, Fc receptor, or surface trigger receptor for coupling with bispecific or multispecific conjugates or universal conjugates.

[0229] In some other embodiments, hematopoietic lineage cells include therapeutic properties of source-specific immune cells associated with one or more of the following: (i) receptor expression targeting antigens; (ii) HLA presentation or its absence; (iii) resistance to the tumor microenvironment; (iv) induction of bystander immune cells and immune modulation; (iv) improved target specificity while reducing extratumor effects; (v) resistance to therapies such as chemotherapy; and (vi) improved homing, survival, and cytotoxicity.

[0230] In some embodiments, iPSCs and their derived hematopoietic cells comprise one or more of the following: B2M knockout or low-content, HLA-E / G, PDL1, A2AR, CD47, LAG3 knockout or low-content, TIM3 knockout or low-content, TAP1 knockout or low-content, TAP2 knockout or low-content, TAP-associated glycoprotein knockout or low-content, NLRC5 knockout or low-content, PD1 knockout or low-content, RFKANK knockout or low-content, CIITA knockout or low-content, RFX5 knockout or low-content, and RFXAP knockout or low-content. These cells, modified with HLA class I and / or II, exhibit enhanced resistance to immune detection and thus improved in vivo retention. Furthermore, such cells avoid the need for HLA matching in adoptive cell therapy and thus provide a universal, readily available source of treatment options.

[0231] In some embodiments, iPSCs and their derived hematopoietic cells comprise hnCD16 (non-cleavable high-affinity CD16), HLA-E, HLA-G, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A One or more of R, CAR, or TCR. These cells have improved immune effector capacity.

[0232] In some embodiments, iPSCs and their derived hematopoietic cells are specific to antigens.

[0233] Many diseases can be improved by introducing the immune cells of this invention into subjects suitable for adoptive cell therapy. Examples of diseases include a variety of autoimmune disorders, including (but not limited to) alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, some forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Hugh Grant's syndrome, systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, granulomatous polyangiitis (Wegener's disease); hematologic malignancies, including This includes (but is not limited to) acute and chronic 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 infections, including (but not limited to) HIV (human immunodeficiency virus), RSV (respiratory syncytial virus), EBV (Epstein-Barr virus), CMV (cell cytomegalovirus), adenovirus-related conditions, and BK polyomavirus-related conditions.

[0234] Specific embodiments of the invention relate to a method of treating a subject by administering to a subject in need a composition comprising any of the cells described herein. In specific embodiments, the terms “treatment” and the like are generally used herein to refer to achieving a desired pharmacological and / or physiological effect. The effect may be preventative in terms of completely or partially preventing the disease, and / or therapeutic in terms of partially or completely curing it, for the sake of disease and / or adverse effects attributable to said disease. As used herein, “treatment” encompasses any treatment of a mammalian disease and includes: preventing the development of said disease in a subject who may be susceptible to it but has not yet been diagnosed with it; inhibiting said disease, i.e., halting its development; or alleviating said disease, i.e., causing said disease to regress. The therapeutic agent or composition may be administered before, during, or after the onset of a disease or injury. Treatment of an ongoing disease is of particular interest, wherein the treatment stabilizes or reduces adverse clinical symptoms in the patient.

[0235] In certain embodiments, the subject suffers from a disease, symptom, and / or lesion that can be treated, alleviated, and / or improved by cell therapy. Some embodiments anticipate subjects requiring cell therapy to be those suffering from an lesion, disease, or symptom, wherein the cell therapy (e.g., a therapy that delivers cellular material to the subject) is capable of treating, alleviating, improving, and / or reducing the severity of at least one symptom associated with said lesion, disease, or symptom. Some embodiments anticipate subjects requiring cell therapy to include (but are not limited to) bone marrow or stem cell transplant candidates, subjects who have received chemotherapy or radiation therapy, subjects who have or are at risk of developing a hyperplastic condition or cancer (e.g., hyperplastic syndrome or hematopoietic cancer), subjects who have a tumor or are at risk of developing a tumor (e.g., a solid tumor), and subjects who have a viral infection or a disease associated with a viral infection or are at risk of developing a viral infection or a disease associated with a viral infection.

[0236] Accordingly, the present invention further provides a pharmaceutical composition comprising pluripotent hematopoietic lineage cells prepared by the methods and compositions disclosed herein, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable medium. In one embodiment, the pharmaceutical composition comprises pluripotent T cells prepared by the methods and compositions disclosed herein. In one embodiment, the pharmaceutical composition comprises pluripotent NK cells prepared by the methods and compositions disclosed herein. In one embodiment, the pharmaceutical composition comprises pluripotent CD34+ HE cells prepared by the methods and compositions disclosed herein. In one embodiment, the pharmaceutical composition comprises pluripotent HSCs prepared by the methods and compositions disclosed herein.

[0237] Additionally, the above-mentioned pharmaceutical composition is provided for therapeutic use, which is achieved by introducing the composition into a subject suitable for adoptive cell therapy, wherein the subject suffers from an autoimmune disease; a hematologic malignancy; a solid tumor; or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0238] The isolated pluripotent stem cell-derived hematopoietic lineage cells may have at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% T cells, NK cells, NKT cells, proT cells, proNK cells, CD34+ HE cells, or HSCs. In some embodiments, the isolated pluripotent stem cell-derived hematopoietic lineage cells have about 95% to about 100% T cells, NK cells, NKT cells, proT cells, proNK cells, CD34+ HE cells, or HSCs. In some embodiments, the present invention provides subjects requiring cell therapy with pharmaceutical compositions having purified T cells, NK cells, NKT cells, CD34+ HE cells, proT cells, proNK cells, or HSCs, for example, compositions having an isolated population of about 95% T cells, NK cells, NKT cells, proT cells, proNK cells, CD34+ HE cells, or HSCs.

[0239] In some embodiments, the pharmaceutical composition comprises a segregated population of pluripotent stem cell-derived hematopoietic lineage cells, wherein the population has less than about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, or 30% of iPSC-derived T cells, NK cells, NKT cells, proT cells, proNK cells, CD34+ HE cells, or HSCs. In some embodiments, the segregated population of derived hematopoietic lineage cells may have more than about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, or 30% of T cells, NK cells, NKT cells, proT cells, proNK cells, CD34+ HE cells, or HSCs. In other embodiments, the isolated population of derived hematopoietic lineage cells may have about 0.1% to about 1%, about 1% to about 3%, about 3% to about 5%, about 10% to about 15%, about 15% to 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% of T cells, NK cells, NKT cells, proT cells, proNK cells, CD34+ HE cells, or HSCs.

[0240] In a particular embodiment, the derived hematopoietic lineage cells may have 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% of T cells, NK cells, NKT cells, proT cells, proNK cells, CD34+ HE cells, or HSCs.

[0241] As those skilled in the art will understand, both autologous and allogeneic immune cells can be used in cell therapy. Autologous cell therapy can reduce infection, lower the probability of GvHD, and accelerate immune remodeling. Allogeneic cell therapy can have immune-mediated graft-versus-malignant disease (GVM) effects and a lower relapse rate. Based on the specific conditions of the patient or subject requiring cell therapy, those skilled in the art can determine which specific type of therapy to administer.

[0242] In certain embodiments, the derived hematopoietic lineage cells in the pharmaceutical composition of the present invention are allogeneic relative to the subject. In certain embodiments, the derived hematopoietic lineage cells in the pharmaceutical formulation of the present invention are autologous relative to the subject. For autologous transplantation, the isolated population of derived hematopoietic lineage cells is fully or partially HLA matched relative to the patient. In another embodiment, the derived hematopoietic lineage cells are not HLA matched relative to the subject.

[0243] In some embodiments, the number of derived hematopoietic lineage cells in the pharmaceutical composition is at least 0.1 × 10⁻⁶. 5 1 cell, at least 0.5 × 10 5 10 cells, at least 1×10 5 1 cell, at least 5 × 10 5 10 × 10 cells, at least 10 × 10 5 1 cell, at least 0.5 × 10 6 1 cell, at least 0.75 × 10 6 10 cells, at least 1×10 6 1.25 × 10⁶ cells 6 1.5 × 10⁶ cells 6 1.75 × 10⁶ cells, at least 1.75 × 10⁶ 6 1 cell, at least 2 × 10 6 1 cell, at least 2.5 × 10 6 1 cell, at least 3 × 10 6 1 cell, at least 4 × 10 6 1 cell, at least 5 × 10 6 10 × 10 cells, at least 10 × 10 6 15 × 10 cells, at least 15 × 106 1 cell, at least 20 × 10 6 1 cell, at least 25 × 10 6 One cell or at least 30 × 10 6 Each cell.

[0244] In some embodiments, the number of derived hematopoietic lineage cells in the pharmaceutical composition is about 0.1 × 10⁻⁶. 5 From one cell to approximately 10 × 10 5 10 cells; approximately 0.5 × 10 6 From one cell to approximately 5 × 10 6 1 × 10⁻⁶ cells; approximately 1 × 10⁻⁶ 6 From one cell to approximately 3 × 10 6 1.5 × 10⁻⁶ cells; approximately 1.5 × 10⁻⁶ 6 From one cell to approximately 2.5 × 10⁻⁶ 6 10 cells; or approximately 2 × 10 6 From one cell to approximately 2.5 × 10⁻⁶ 6 Each cell.

[0245] In some embodiments, the number of derived hematopoietic lineage cells in the pharmaceutical composition is about 1 × 10⁻⁶. 6 From one cell to approximately 3 × 10 6 1.0 × 10⁶ cells; approximately 1.0 × 10⁶ cells 6 From one cell to approximately 5 × 10 6 1.0 × 10⁶ cells; approximately 1.0 × 10⁶ cells 6 From one cell to approximately 10 × 10 6 10 × 10⁶ cells; approximately 10 × 10⁶ 6 From one cell to approximately 20 × 10 6 10 × 10⁶ cells; approximately 10 × 10⁶ 6 From one cell to approximately 30 × 10 6 10 cells; or approximately 20 × 10 6 From one cell to approximately 30 × 10 6 Each cell.

[0246] In some other embodiments, the number of derived hematopoietic lineage cells in the pharmaceutical composition is from about 1 × 10⁻⁶ cells to about 30 × 10⁻⁶ cells. 6 1.0 × 10⁶ cells; approximately 1.0 × 10⁶ cells 6 From one cell to approximately 20 × 10 6 1.0 × 10⁶ cells; approximately 1.0 × 10⁶ cells 6 From one cell to approximately 10 × 10 6 10 cells; approximately 2.0 × 10 6 From one cell to approximately 30 × 10 6 10 cells; approximately 2.0 × 10 6 From one cell to approximately 20 × 10 6 10 cells; or approximately 2.0 × 10⁻⁶ cells. 6From one cell to approximately 10 × 10 6 Each cell.

[0247] In other embodiments, the number of derived hematopoietic lineage cells in the pharmaceutical composition is approximately 1 × 10⁻⁶. 6 1 cell, approximately 2 × 10 6 1 cell, approximately 5 × 10 6 1 cell, approximately 7 × 10 6 One cell, approximately 10 × 10 6 One cell, approximately 15 × 10 6 17 × 10⁶ cells 6 One cell, approximately 20 × 10 6 1 cell, approximately 25 × 10 6 One cell or approximately 30 × 10 6 Each cell.

[0248] In one embodiment, the number of derived hematopoietic lineage cells in the pharmaceutical composition is the number of immune cells in a portion or a single umbilical cord blood sample, or at least 0.1 × 10⁻⁶ cells. 5 Cells / kg body weight, at least 0.5 × 10⁻⁶ 5 Cells / kg body weight, at least 1×10 5 Cells / kg body weight, at least 5×10 5 Cells / kg body weight, at least 10×10 5 Cells / kg body weight, at least 0.5 × 10⁻⁶ 6 Cells / kg body weight, at least 0.75 × 10⁻⁶ 6 Cells / kg body weight, at least 1×10 6 Cells / kg body weight, at least 1.25 × 10⁻⁶ 6 Cells / kg body weight, at least 1.5 × 10 6 Cells / kg body weight, at least 1.75 × 10⁻⁶ 6 Cells / kg body weight, at least 2×10 6 Cells / kg body weight, at least 2.5 × 10⁻⁶ 6 Cells / kg body weight, at least 3×10 6 Cells / kg body weight, at least 4×10 6 Cells / kg body weight, at least 5×10 6 Cells / kg body weight, at least 10×10 6 Cells / kg body weight, at least 15 × 10 6 Cells / kg body weight, at least 20 × 10 6 Cells / kg body weight, at least 25 × 10⁻⁶ 6 Cells / kg body weight or at least 30 × 10⁻⁶ 6 Cells per kg of body weight.

[0249] The genetically engineered hematopoietic lineage cells provided by this invention can be administered to subjects without prior in vitro or in vitro expansion. In a particular embodiment, the isolated population of genetically engineered hematopoietic lineage cells is regulated and treated in vitro using one or more reagents to obtain immune cells with improved therapeutic potential. The regulated population of genetically engineered hematopoietic lineage cells can be washed to remove the treatment agents, and the improved population can be administered to the patient without further in vitro expansion of the population.

[0250] In other embodiments, the present invention provides isolated populations of genetically engineered derived hematopoietic lineage cells amplified prior to conditioning with one or more reagents. Isolated populations of derived hematopoietic lineage cells expressing TCR, CAR, or other proteins can be generated in a recombinant manner using the methods and compositions provided herein.

[0251] For genetically engineered hematopoietic lineage cells expressing recombinant TCRs or CARs, whether before or after gene modification, the cells can be activated and expanded using methods described in the following documents: for example, U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 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.

[0252] In some embodiments, different approaches can be used to provide the primary and co-stimulatory signals of genetically engineered hematopoietic lineage cells. For example, the reagent providing each signal can be present in solution or conjugated to a surface. When conjugated to a surface, the reagent can be conjugated to the same surface (i.e., the "cis" form) or individual surfaces (i.e., the "trans" form). Alternatively, one reagent can be conjugated to a surface and another reagent can be present in solution. In one embodiment, the reagent providing the co-stimulatory signal can bind to the cell surface and the reagent providing the primary activation signal can be present in solution or conjugated to the surface. In some embodiments, both reagents can be present in solution. In another embodiment, the reagent can be in a soluble form and then cross-linked to a surface, such as cells expressing Fc receptors or antibodies or other binding agents to which the reagent will bind, as disclosed in U.S. Patent Application Publications Nos. 20040101519 and 20060034810 concerning artificial antigen-presenting cells (aAPCs), which are intended to be used in this invention for activating and expanding T lymphocytes.

[0253] Compositions comprising the derived hematopoietic lineage cell populations of the present invention can be sterile and suitable for administration and preparation for administration (i.e., administration to human patients without any further processing). In some embodiments, the therapeutic composition is prepared for infusion into a patient. A cell-based composition prepared for administration means that the composition requires no further processing or manipulation prior to transplantation or administration to the recipient.

[0254] Suitable therapeutically acceptable sterile compositions for administration to patients may include one or more pharmaceutically acceptable carriers (additives) and / or diluents (e.g., pharmaceutically acceptable media, such as cell culture media), or other pharmaceutically acceptable components. The pharmaceutically acceptable carriers and / or diluents are determined in part by the specific composition being administered and the specific method of administration. Therefore, the therapeutic compositions of the present invention have a variety of suitable formulations (see, for example, Remington's Pharmaceutical Sciences, 17th edition, 1985, the disclosure of which is incorporated herein by reference in its entirety).

[0255] In certain embodiments, the therapeutic cell composition having a segregated population of genetically engineered derived hematopoietic lineage cells also has a pharmaceutically acceptable cell culture medium. The therapeutic composition comprising the genetically engineered derived hematopoietic lineage cell population as disclosed herein can be administered via enteral or parenteral administration methods, or in combination with other suitable compounds to achieve the desired therapeutic objective.

[0256] Pharmaceutically acceptable carriers and / or diluents must have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to the human subjects being treated. Additionally, the stability of the therapeutic composition should be maintained or enhanced.

[0257] Such carrier solutions may also contain buffers, diluents, and other suitable additives. A buffer is a solution or liquid whose chemical composition neutralizes an acid or base without causing a significant change in pH. Examples of buffers contemplated in this invention include (but are not limited to) Durbectos-buffered saline (PBS), Ringer's solution, 5% dextrose aqueous solution (D5W), and standard / physiological saline (0.9% NaCl).

[0258] These pharmaceutically acceptable carriers and / or diluents may be present in an amount sufficient to maintain the pH of the therapeutic composition between about 3 and about 10. Thus, the buffer may comprise up to about 5% (by weight) of the total composition. The therapeutic composition may also include electrolytes, such as (but not limited to) sodium chloride and potassium chloride. 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 solution with a pH in one of the said pH ranges. In another embodiment, the therapeutic composition has a pH of about 7. Alternatively, the therapeutic composition has a pH in the range of about 6.8 to about 7.4. In yet another embodiment, the therapeutic composition has a pH of about 7.4.

[0259] The sterile composition of the present invention can be a sterile solution or suspension present in a pharmaceutically acceptable, non-toxic medium. Suspension can refer to a non-adhesive condition, wherein cells do not adhere to a solid carrier. For example, cells maintained in a suspension can be agitated to prevent them from adhering to a carrier, such as a culture dish.

[0260] A suspension is a dispersion (mixture) in which finely divided substances combine with another substance, wherein the constituents are so finely divided and mixed that they cannot settle rapidly. Suspensions can be prepared using a mediator (e.g., a liquid medium, including 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, such as physiological saline or serum-free culture medium, and do not adhere to a solid carrier. In everyday life, the most common suspensions are those that are solid in liquid water. Acceptable diluents (e.g., mediators and solvents) that can be used are water, Ringer's solution, isotonic sodium chloride (physiological saline) solution, and serum-free cell culture medium. In some embodiments, a hypertonic solution is used to prepare the suspension. Additionally, sterile, non-volatile oils are conventionally used as solvents or suspension media. In parenteral administration, mediators are particularly suitable as solutions (preferably oils or aqueous solutions) as well as suspensions, emulsions, or implants. Aqueous suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and / or polydextrose. In some embodiments, the infusion solution is isotropic relative to the patient's tissue. In some embodiments, the infusion solution is hypertonic relative to the patient's tissue.

[0261] Pharmaceutically acceptable carriers, diluents, and other components constituting the ready-to-use pharmaceutical compositions of the present invention are derived from U.S. pharmaceutical-grade reagents permitted for use in clinical therapy. Typically, these finished reagents, including any media, solutions, or other pharmaceutically acceptable carriers and / or diluents, are sterilized according to conventional methods in the art, such as filtration sterilization, and tested for a variety of undesirable contaminants (e.g., mycoplasma, endotoxins, or viral contamination) prior to use. In one embodiment, the pharmaceutically acceptable carrier is substantially free of naturally occurring proteins of human or animal origin and is suitable for storing cell populations, including hematopoietic stem cells and progenitor cells, in the pharmaceutical composition. The pharmaceutical composition is intended for administration to human patients and is therefore substantially free of cell culture components, such as bovine serum albumin, horse serum, and fetal bovine serum.

[0262] This invention also provides in part the use of pharmaceutically acceptable cell culture media in certain compositions and / or cultures of the invention. Such compositions are suitable for administration to human subjects. Generally, any culture medium supporting the maintenance, growth, and / or health of the derived hematopoietic lineage cells of the invention is suitable as a pharmaceutical cell culture medium. In certain embodiments, a pharmaceutically acceptable cell culture medium is a serum-free and / or feeder-free medium.

[0263] The pharmaceutical composition may have a serum-free culture medium suitable for storing regulated and isolated derived hematopoietic lineage cell populations. In various embodiments, the serum-free culture medium is non-animal and may optionally be protein-free. Optionally, the culture medium may contain biomedically acceptable recombinant proteins. A non-animal culture medium is one in which the components are derived from non-animal sources. The recombinant protein replaces the protozoan protein in the non-animal culture medium, and the nutrients are derived from synthetic, plant, or microbial sources. In contrast, a protein-free culture medium is defined as substantially free of protein.

[0264] Those skilled in the art will understand that the above examples of culture media are illustrative and in no way limiting to the culture media formulations applicable to the present invention, and that there are many known and suitable culture media available to those skilled in the art.

[0265] The pharmaceutical composition is substantially free of mycoplasma, endotoxins, and microbial contamination. In certain embodiments, the therapeutic composition contains bovine serum albumin at concentrations below about 10, 5, 4, 3, 2, 1, 0.1, or 0.05 μg / ml.

[0266] Regarding mycoplasma and microbial contamination, as used herein, "substantially free" means a negative test reading known to those skilled in the art. For example, mycoplasma contamination is determined by passage of therapeutic composition samples in broth medium and partitioning them onto 37°C agar plates on days 1, 3, 7, and 14, using appropriate positive and negative controls. The morphology of the samples is compared to that of the positive and negative control groups under a 100x microscope. Additionally, inoculum of indicator cell cultures is incubated for 3 and 5 days and examined for the presence of mycoplasma using epifluorescence microscopy at 600x magnification with a DNA-binding fluorescent dye. If the agar and / or broth medium procedures and the indicator cell culture procedures indicate the absence of evidence of mycoplasma contamination, then the sample is considered satisfactory.

[0267] Organic solvents, or suitable organic solvents, generally refer to carbon-containing liquids or gases that dissolve solid, liquid, or gaseous solutes to produce solutions. Suitable organic solvents are those suitable for in vitro administration to mammalian cells or for co-cultivation with mammalian cells, and also suitable for in vivo administration to subjects, for example, exhibiting minimal toxicity or other inhibitory effects under in vitro (e.g., cell culture) or in vivo conditions, at selected concentrations, and during cultivation or administration. Suitable organic solvents should also be appropriate for the storage stability and handling of the reagents described herein.

[0268] Examples of suitable organic solvents include, but are not limited to, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME), and dimethylacetamide, including mixtures or combinations thereof. In some embodiments, the composition or organic solvent is substantially free of methyl acetate, which means that only trace amounts of methyl acetate are present in the composition or solvent, preferably undetectable amounts (e.g., as measured by high-performance liquid chromatography (HPLC), gas chromatography (GC), etc.).

[0269] Endotoxin-free containers or compositions mean that the container or composition contains at most trace amounts (i.e., amounts that have no adverse physiological effect on the subject) or undetectable amounts of endotoxin. Cells “substantially endotoxin-free” means that there are fewer endotoxins per dose of cells, which has been approved by the FDA for use in biological applications, with a total endotoxin level of 5 EU per kilogram of body weight per day, which translates to 350 EU per total cell dose for an average 70 kg person.

[0270] In one embodiment, the endotoxin-free container and / or composition is free of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% endotoxin. Endotoxins are toxins associated with certain bacteria (typically Gram-negative bacteria), but they can also be found in Gram-positive bacteria, such as Listeria monocytogenes. The most prevalent endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS) found in the outer membranes of many Gram-negative bacteria, representing important pathogenic characteristics of these bacteria's pathogenicity. Small amounts of endotoxins in humans can cause fever, decreased blood pressure, and activation of inflammation and coagulation, as well as other adverse physiological effects. Therefore, it is generally desirable to remove most or all trace amounts of endotoxins from pharmaceutical containers, as even small amounts can have adverse effects on the human body. Endotoxins can be removed from containers using methods known in the art, such as cleaning containers with endotoxin-free water in a HEPA filter washing device, removing pyrogens at 250°C, and cleaning the packaging in a HEPA filter workstation located inside a Class 100 / 10 cleanroom (e.g., a Class 100 cleanroom contains no more than 100 particles larger than half a micrometer in one cubic foot of air).

[0271] Example

[0272] The following examples are provided for illustrative purposes, not for limitation.

[0273] Example 1 - Materials and Methods

[0274] To efficiently select and test suicide systems under the control of different promoters combined with different safe harbor locus integration strategies, a proprietary hiPSC platform of the applicant is used, which is capable of single-cell passage and high-throughput 96-well plate-based flow cytometry sorting to derive cloned hiPSCs with single or multiple gene regulation.

[0275] Maintenance of hiPSCs in small molecule cultures: hiPSCs are passaged as single cells after reaching 75%-90% confluence, as is standard practice. For single-cell dissociation, hiPSCs are washed once with PBS (Mediatech) and treated with acutase (Millipore) at 37°C for 3-5 minutes, followed by pipetting to ensure single-cell dissociation. The single-cell suspension is then mixed with an equal volume of conventional culture medium, centrifuged at 225×g for 4 minutes, resuspended in FMM, and seeded onto a substrate-coated surface. The passage ratio is typically 1:6-1:8. Cells are transferred to pre-coated substrate-coated tissue culture plates and maintained at 37°C for 2-4 hours, then fed with FMM every 2-3 days. Cell cultures are maintained in a humidified incubator set at 37°C and 5% CO2.

[0276] Human iPSC genome editing using ZFN and CRISPR to target iCasp9 insertion into a genomic safe harbor: For ZFN-mediated genome editing, 2 million iPSCs were transfected with a mixture of 2.5 μg ZFN-L (FTV893; SEQ ID NO: 2 and 4), 2.5 μg ZFN-R (FTV894; SEQ ID NO: 3 and 5), and 5 μg AAVS1 iCasp9 donor constructs (FTV895, FTV930, FTV921, FTV931, FTV932, FTV952, or FTV953; inducing caspase 9 SEQ ID NO: 7 and 8) plastid DNA. For CRISPR-mediated genome editing, 2 million iPSCs were transfected with a mixture of 5 μg ROSA26-gRNA / Cas9 (FTV922; SEQ ID NO: 6) and 5 μg ROSA26 iCasp9 donor constructs (FTV955 or FTV956). Transfection was performed using a Neon transfection system (Life Technologies) with parameters of 1500 V, 10 ms, and 3 pulses. On day 2 or 3 post-transfection, transfection efficiency was measured by flow cytometry if the plasmids contained an artificial promoter driving GFP and / or RFP expression cassette. On day 4 post-transfection, puromycin was added to the culture medium at a concentration of 0.1 μg / ml for the first 7 days and 0.2 μg / ml for the next 7 days to select target cells. During puromycin selection, on day 10, cells were passaged in wells coated with fresh matrix gel. On day 16 or later, selected with puromycin, the percentage of GFP+ iPS cells in the surviving cells was analyzed by flow cytometry.

[0277] Batch sorting and clonal sorting of genome-edited iPSCs: Twenty days after puromycin selection, GFP+SSEA4+TRA181+ iPSCs were batch-sorted and cloned from iPSCs targeted by ZFN-mediated AAVS1 EF1α or CAG promoter-driven iCasp9-2A-GFP. Single-cell dissociated pools of targeted iPSCs were resuspended in cooled staining buffer containing Hanks' Balanced Salt Solution (MediaTech), 4% fetal bovine serum (Invitrogen), 1× penicillin / streptomycin (MediaTech), and 10 mM Hepes (MediaTech); freshly prepared for optimal performance. The bound primary antibodies, including SSEA4-PE and TRA181-AlexaFluor-647 (BD Biosciences), were added to the cell solution and incubated on ice for 15 minutes. All antibodies were used at a rate of 7 μL / 100 μL staining buffer per million cells. The solution was washed once with staining buffer, centrifuged at 225 g for 4 min, and resuspended in staining buffer containing 10 µM thiazolinone and maintained on ice for flow cytometry sorting. Flow cytometry sorting was performed using FACS Aria II (BDBiosciences). For batch sorting, GFP+SSEA4+TRA181+ cells were gate-sorted and sorted into 15 ml standard tubes containing 7 ml FMM. For clone sorting, sorted cells were directly sprayed into 96-well plates using a 100 µM nozzle at a concentration of 3 events per well. Each well was pre-filled with 200 µL of FMM supplemented with 5 μg / mL fibronectin and 1× penicillin / streptomycin (Mediatech) and pre-coated overnight with 5x matrix gel. 5x matrix gel pre-coating involved adding an aliquot of matrix gel to 5 mL of DMEM / F12, incubating overnight at 4°C to allow for proper resuspension, and finally adding 50 µL per well to a 96-well plate, followed by incubation overnight at 37°C. The 5x matrix gel was withdrawn just before adding the medium to each well. After sorting, the 96-well plate was centrifuged at 225 g for 1 to 2 minutes before incubation. The plates were kept undisturbed for seven days. On day 7, 150 µL of medium was removed from each well and replaced with 100 µL of FMM. On day 10 post-sorting, another 100 µL of FMM was fed into the wells. Colony formation was detected as early as day 2, and most colonies amplified between day 7 and day 10 post-sorting. In the first passaging, each well was washed with PBS and dissociated at 37°C with 30 µL of aculase for approximately 10 minutes. The need for prolonged aculase treatment reflects the compactness of the colonies that had been idled during long-term culture.After cell dissociation was observed, 200 µL of FMM was added to each well and aspirated several times to disrupt the colony. The dissociated colonies were transferred to another well in a 96-well plate pre-coated with 5x matrix gel and then centrifuged at 225 g for 2 min before incubation. This 1:1 passage was performed to expand the early colony before amplification. Subsequent passages were treated with aculase for 3–5 min as usual and amplified at 1:4–1:8 after reaching 75–90% confluence in FMM in larger wells pre-coated with 1x matrix gel. GFP fluorescence levels and TRA1-81 expression levels were analyzed for each clone cell line. Clones with approximately 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).

[0278] In vitro, iCasp9-integrated iPSCs were induced to die: a dimerization chemical inducer (CID; AP1903) was purchased from Medchemexpress (Monmouth Junction, NJ). Twenty-four hours after CID exposure, cells were collected 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 using Flowjo software.

[0279] To test whether AP1903 induces complete cell death in iCasp9-targeted iPSC clones, we treated three wells containing each targeted iPSC clone with AP1903 for 48 hours, washed the wells twice with PBS, and then added fresh iPSC medium. The wells were allowed to recover with fresh medium every two days for a total of five days. Each well was then stained with alkaline phosphatase staining reagent (Sigma).

[0280] In vitro tri-lineage directed differentiation and inducible killing of iCasp9 targeted clonal lines: For monolayer tri-lineage directed differentiation, hiPSCs were seeded in FMM into Matrigel-coated wells one day before differentiation began (e.g., 200K cells / well for endoderm, 50K cells / well for ectoderm, and 10K and 50K cells / well for mesoderm). Four replicate wells were configured for each lineage differentiation. For endoderm differentiation, the FMM medium was replaced with endoderm induction medium: RPMI-1640, ascorbic acid (50 µg / ml), monothioglycerol (450 µM), 1X glutamate, Knockout serum substitute (0.20%), activin A (100 ng / ml), CHIR99021 (Biovision) (3 µM), and 1X penicillin / streptomycin. Two days later, the medium was replaced with RPMI-1640, ascorbic acid (50 µg / ml), monothioglycerol (450 µM), 1X glutamate, Knockout serum substitute (0.50%), bFGF (5 ng / ml), activin A (100 ng / ml), and 1X 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. The other two wells were treated with AP1903 for 48 hours, then washed and replenished with fresh differentiation medium and cultured for another 5 days, followed by crystal violet staining. For mesodermal differentiation, the medium was replaced with DMEM / F12 supplemented with ITS, 10 ng / ml bFGF, 20 µM forskolin, and 3 µM CHIR99021 (Mediatech). 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. The other two wells were treated with AP1903 for 48 hours, then washed and replenished with fresh differentiation medium and cultured for another 5 days, followed by crystal violet staining. For ectoderm differentiation, FMM medium was replaced with neural induction medium: DMEM / F12 (Mediatech) supplemented with 1x B27 medium additive (Lifetech), 1x N2 medium additive (Lifetech), 10 μM SB431542 and 100 nM LDN-193189. The 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. The other two wells were treated with AP1903 for 48 hours, then washed and replenished with fresh differentiation medium and cultured for another 5 days, followed by crystal violet staining.

[0281] Crystal violet staining: Prepare a 0.5% crystal violet solution by dissolving 0.5 g of crystal violet dye (Sigma) in 100 mL of deionized water. Filter and store at room temperature. Before staining, wash cells twice with PBS. Replace with PBS containing 4% paraformaldehyde for 15 minutes at room temperature. Rinse twice with PBS. Stain with 0.1% crystal violet (prepared in 10% ethanol) for 15 minutes at room temperature. Discard the CV. Gently wash cells with deionized water from the wash bottle until the water no longer darkens. Remove the deionized water and allow the stained cells to dry, then photograph.

[0282] In vivo induction to kill teratomas derived from iCasp9-integrated iPSCs: All mice were housed in captivity at 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-ray dihydrogen glutathione). 剔除 Jackson Laboratory. In teratoma analysis, teratomas containing 1-2 x 10⁻⁶ cells will be analyzed. 6 200 μl of a mixture of 50% FMM medium and 50% Matrigel was subcutaneously introduced into the dorsal region of each mouse. Each mouse received two bilateral cell injections, with iCasp9-integrated iPSCs injected on the left and unmodified parental iPSCs injected on the right. To initiate in vivo kill at designated time points, mice were administered AP1903 (125 µg / mouse) daily via intraperitoneal (ip) injection for 5 or 7 days. Teratomas were monitored for formation and volume measurements were initiated at week 3 or as directed. Teratomas were collected at designated time points and processed for histological or molecular analysis. An AP1903 stock solution (25 µg / ml) was prepared in DMSO. For a single intraperitoneal injection, 5 µl of the stock solution was added to 200 µl of PBS, followed by sonication in a water bath for 10 minutes. For histological analysis, teratomas were collected and fixed overnight in 4% paraformaldehyde at room temperature, followed by maintenance in 70% ethanol at room temperature for further processing. Samples were submitted to the UCSD Histology Core Facility for sectioning and hematoxylin and eosin staining. Sections were examined, interpreted, and photographed using a Nikon Eclipse TS100 microscope equipped with a Nikon DS-Fi1 camera.

[0283] In vivo visualization of inducible killing of transplanted luciferase-expressing iPSCs: To visualize AP1903-induced in vivo killing, iCasp9-integrated iPSCs and unmodified parental iPSCs were infected with a lentivirus containing a polycistronic cassette composed of the firefly luciferase gene and RFP linked via IRES and expressed under the control of EF1α (Biosettia). RFP+ cells were sorted from the infected cells and injected as described above. 2 x 10 6 One iCasp9-integrated iPSC was subcutaneously injected into the left side of NSG mice, and the same number of parental iPSCs were injected into the right side. AP1903 was administered intraperitoneally on the designated day. Immediately after iPSC implantation (day 0), graft and teratoma volumes were monitored weekly using the Xenogen IVIS imaging system. For imaging, D-fluorescein (4 mg / mouse, ThermoFisher) was injected intraperitoneally into the mice, and bioluminescence was recorded 10 minutes later. Analysis was performed using Xenogen Living Image software.

[0284] Example 2 - The iCasp9 suicide gene targeting the AAVS1 safe harbor, regulated by the CMV promoter, elicits a variable response when activated by AP1903.

[0285] To enable high-throughput analysis of the correct integration and expression strategies of iPSCs, the iCasp9 suicide gene platform was chosen, where small molecule chemical dimerization inducers (e.g., AP1903) can induce caspase-9-mediated rapid cell death. Noting the efficacy of small molecule induction at various stages of development toward the final product, the iCasp9 gene needs to be continuously expressed at every stage of pluripotent stem cell maintenance and differentiation. Following a single-well hiPSC approach, various suicide gene expression cassettes were integrated and maintained in safe harbor loci, including (but not limited to) AAVS1, ROSA26, and H11 loci, for efficient and precise integration and maintenance. Several integrative vectors, each containing gene expression cassettes located downstream of various exogenous and endogenous promoters, including endogenous AAVS1 or ROSA26, cellular giant virus (CMV), elongation factor 1α (EF1α), phosphoglycerate kinase (PGK), hybrid CMV enhancer / chicken β-actin (CAG), and ubiquitin C (UBC) promoters, were tested to systematically analyze and compare the activity of different suicide systems in hiPSCs and hiPSC-derived differentiated cells.

[0286] Design targeting AAVS1 locus ( Figure 1A) A donor construct that integrates the Casp9 suicide gene into 293T cells, the donor construct containing CMV-driven iCasp9 (AAVS1-G1.0). The donor construct also contains GFP and puromycin genes, whose expression is driven by the AAVS1 endogenous promoter after targeted insertion. Figure 1 A). 293T cells were transfected with ZFNs specific to the AAVS1 locus and the donor construct AAVS1-G1.0. Three days after transfection, puromycin selection was initiated and continued for 12 days. GFP expression in the puromycin-selected population was analyzed by flow cytometry, and the range of GFP expression levels was observed. Approximately 82% of the puromycin-selected cell population expressed GFP (…). Figure 1 B). Next, 293T cells selected with puromycin were treated with 10 nM or 120 nM AP1903 (DMSO used as a control) for 24 hours. The treated cells were then collected and stained with 7AAD, which selectively stains membrane-damaged dead cells, and analyzed by flow cytometry. The percentage of 7AAD-negative cells (potentially viable cells) for each treatment was plotted (DMSO as control; AP1903 10 nM; and AP1903 120 nM) (n=2). Significant cell death was detected after AP1903 treatment at both concentrations. Figure 1 C). AP1903 dosage appeared to have no effect on cell death rate. This was confirmed by ligation PCR on genomic DNA from these 293T cells selected with puromycin. Figure 1 (D) Based on the presence of PCR amplification products that are specific to the junctions formed by the insertion of the donor vector, the targeted insertion of the donor vector into the AAVS1 locus of 293T cells selected by puromycin was further verified.

[0287] Similar to 293T cells, hiPSCs were transfected with ZFNs specific to the AAVS1 locus and a donor construct (AAVS1-G1.0) containing CMV-driven iCasp9. Puromycin selection was initiated 3 days after transfection and continued for 16 days. GFP expression in puromycin-resistant cells was analyzed by flow cytometry. Based on GFP intensity, three populations were selected by flow cytometry: GFP (negative), GFP (low), and GFP (high). Figure 1E). Separate GFP (negative), GFP (low), and GFP (high) populations were amplified and then treated with AP1903 (10 nM or 100 nM) or DMSO control for 24 hours. Treated cells were collected, stained with 7AAD, and analyzed by flow cytometry. The percentage of 7AAD-negative cells (potentially viable cells) was plotted for each treatment. However, compared to 293T cells with Casp9-targeted insertion into the AAVS1 locus, no AP1903-mediated cell death was detected in any treatment of iPSCs with Casp9-targeted insertion into the AAVS1 locus. Figure 1 F). Ligation PCR of genomic DNA from puromycin-selected and sorted hiPSCs showed that donor vector-targeted insertion into the AAVS1 locus occurred only in the GFP (low) population, and not in the GFP (negative) or GFP (high) populations. Figure 1 G). Therefore, it is believed that the absence of cell death in the GFP (high) population is related to incorrect (untargeted) genomic insertion and that iCasp9 expression did not occur under the control of the CMV promoter. Figure 1 G, lane 4; lane 1 - marker). However, it is believed that the absence of cell death in GFP (low) is related to reduced expression of the iCasp9 gene under the control of the CMV promoter, despite correct targeting insertion in iPSCs ( Figure 1 G, lane 5). Therefore, the iCasp9 suicide gene targeting the AAVS1 safe harbor, regulated by the CMV promoter, elicits a variable response when activated by AP1903, which may depend on the cell type used for integration and the integration site in the genome.

[0288] Example 3 - iCasp9 targeted insertion into the safe harbor locus of hiPSCs under the control of various endogenous and exogenous promoters

[0289] In previous studies, puromycin selection was stably expressed under the control of the AAVS1 promoter to maintain viability and growth during selection. Therefore, a next donor construct containing iCasp9 was designed to target the AAVS1 locus by driving iCasp9 gene expression (and GFP, and puromycin marker gene) via the endogenous AAVS1 promoter after insertion. Figure 2 A). hiPSCs were transfected with ZFNs specific to the AAVS1 locus and donor constructs containing iCasp9 under the control of the AAVS1 endogenous promoter. Three days after transfection, puromycin selection was initiated and continued for 20 days. Flow cytometry analysis of puromycin-resistant cells subsequently showed GFP expression (…). Figure 2B). The expanded puromycin-resistant iPSCs were then treated with AP1903 (or DMSO control) for 24 hours. Treated cells were collected, stained with 7AAD, and analyzed by flow cytometry. The percentage of 7AAD-negative cells (i.e., viable cells) was plotted for each treatment. However, AP1903-mediated cell death was not detected in any treatments of iPSCs with Casp9-targeted insertion into the AAVS1 locus and iPSCs whose Casp9 expression is controlled via the endogenous AAVS1 promoter. Figure 2 C). Ligation PCR of genomic DNA from the puromycin resistance pool showed that the donor vector was targeted and inserted into the AAVS1 locus in this population. Figure 2 (D) Therefore, the iCasp9 suicide gene targeting the AAVS1 safe harbor, regulated by the endogenous AAVS1 promoter, does not appear to induce iCasp9 expression, despite correct insertion at the AAVS1 locus. Similarly, based on GFP expression intensity, low levels of expression do not appear sufficient to achieve iCasp9-mediated cell death.

[0290] Unlike other gene delivery strategies, such as lentivirus-mediated transgene integration (where multiple copies of a gene are introduced into each individual cell), in locus-specific targeting approaches, the characteristics of different promoters appear to play a significant role in functional output. To investigate which promoter maintains stable expression at the desired level, constructs were then designed to drive iCasp9 expression via various exogenous promoters and to target the AAVS1 locus via the AAVS1 endogenous promoter using GFP and puromycin marker genes. Figure 2 E). The exogenous promoters tested included CMV, UBC, PGK, EF1α, and CAG. hiPSCs were transfected with ZFNs specific to the AAVS1 locus and donor constructs containing iCasp9 under the control of one of the selected exogenous promoters. Puromycin selection was initiated 3 days after transfection and continued for 20 days, followed by flow cytometry analysis of GFP expression in puromycin-resistant cells. hiPSCs transfected with constructs containing iCasp9 under the control of EF1α and CAG (see SEQ ID NO: 1) showed higher GFP expression after transfection with CAG alone, which exhibited stable expression. Figure 2 (F and Table 1). These two strategies were selected for further analysis.

[0291] Example 4 - The EF1α promoter-driven iCasp9 is targeted and inserted into the AAVS1 safe harbor of the iPSC.

[0292] according to Figure 2E. A donor construct containing EF1α-driven iCasp9 targeting the AAVS1 locus was designed. hiPSCs transfected with AAVS1 locus-specific ZFNs and the donor construct containing EF1α-driven iCasp9 were selected with puromycin for 20 days and then treated with AP1903. Figure 3 Flow cytometry analysis of the gated region in Figure A highlights GFP-positive iPSCs with targeted insertion, albeit few, that responded to AP1903-induced cell death. To determine whether these GFP-positive populations were enriched and maintained, TRA181 (a marker indicating pluripotency and undifferentiated state) and GFP-positive (indicating iCasp9 expression) hiPSCs were batch- or individually sorted in 96-well plates approximately day 16 post-selection. Figure 3 B). Based on the time-dependent maintenance of GFP-positive batch-sorted hiPSCs in puromycin-free medium, the population distribution curve over time was determined. Figure 3 C). The percentage of cells expressing high TRA181 and high GFP was observed to decrease over time (e.g., from day 2 to day 16 post-sorting). This data suggests that although EF1a showed high levels of expression, it could not maintain this expression, likely due to quiescence of the pluripotent state. To determine whether individual hiPSCs containing stable EF1a-mediated expression could be identified, individual hiPSC sorting cells were screened. Bright-field images of cloned hiPSCs derived from single-cell sorting of TRA181 and GFP cells show the morphology of hiPSC communities derived from single-cell sorting. Figure 3 D). Then, cloned hiPSCs with EF1α-driven iCasp9 targeting insertion into the AAVS1 locus were amplified and GFP expression was assessed. However, after amplification, GFP expression was lost to varying degrees in all communities, indicating that the EF1α promoter cannot maintain expression in hiPSCs after insertion and during amplification (see [link to article]). Figure 3 E, for example, EF1α clones C1 and C23). Overall, at both the batch and individual sorting levels, EF1a cannot stably maintain GFP and consequently cannot stably maintain iCasp9 expression.

[0293] Example 5 - iCasp9 targeted insertion of CAG bootloader driver into hiPSC's AAVS1 safe harbor

[0294] Several endogenous promoters were found to drive the surviving expression of iCasp9 during hiPSC clonal expansion, but the expression level was determined to be too low to effectively respond to AP1903 treatment. iCasp9 expression under various exogenous promoters also showed loss during prolonged hiPSC clonal expansion and failed to drive AP1903-induced cell death. Next, hiPSCs transfected with ZFNs specific to the AAVS1 locus and donor constructs containing CAG-driven iCasp9 were evaluated. Figure 2 E and Table 1).

[0295] Three days after transfection, puromycin selection was initiated and continued for 16 days. Subsequently, puromycin-resistant cells were sorted in batches or individually in 96-well plates based on high GFP expression. Following targeting and puromycin selection, iPSCs sorted in batches based on GFP positivity were maintained in puromycin-free medium for 21 days, followed by flow cytometry analysis to demonstrate whether high GFP expression could be maintained during iPSC amplification. Figure 4 B indicates that the percentage of GFP-positive cells was not only maintained during long-term amplification, but also remained above 99%. This is the first observation that a promoter targeting a specific locus can maintain stable expression, similar to a lentiviral approach that inserts multiple copies into the genome. Next, the iCasp9 gene response in batch-sorted cells maintaining 99% GFP expression was tested. Figure 4 C indicates that GFP-positive cells undergo induced cell death when treated with AP1903 and become 7AAD-positive after treatment. Following stable expression conversion to effective iCasp9-mediated cell death in bulk CAG populations, we then evaluated individual clonal lines. Next, we evaluated puromycin-resistant iPSCs clonally sorted in matrix gel-coated 96-well plates. Bright-field images of typical individual sorted populations showed high-quality pluripotent morphology. Figure 4 D). Individually amplified single-cell populations and their GFP and TRA181 expression were assessed by flow cytometry. All selected populations (e.g., CAG-C5, CAG-C8, CAG-C12, CAG-C38) showed high percentages of GFP and TRA181 expression maintained during amplification. Figure 4 E). The mean fluorescence intensity (MFI) of the selected clonal lines is plotted on Figure 4 In the F cloning series, CAG-C5 had the highest MFI of 1500, while CAG-C38 had the lowest MFI of 858 among the four cloning lines. Ligation-based PCR was performed on the genomic DNA of all selected GFP+ cloning lines to detect homologous recombination between the donor constructs and the AAVS1 locus. Parental hiPSCs represent the original hiPSC lines before transfection.

[0296] The CAG hiPSC population pool represents the population following targeted transfection and 16-day puromycin selection. GAPDH genomic sequences were amplified to ensure genomic DNA quality. All selected GFP-positive clones exhibited the correct insertion at the AAVS1 locus. Figure 4 (G). Therefore, the above data indicate that, after targeted insertion into the AAVS1 locus in hiPSC cells, CAG is the only promoter capable of stably maintaining high levels of expression of the gene under its control during iPSC amplification.

[0297] Similarly, the performance of other promoters was evaluated at transfection (days 2-3 post-transfection), maintenance after puromycin selection (day 20 post-puromycin selection), and functional response after AP1903 treatment. Table 1 below summarizes the performance of exogenous promoters CMV, UBC, EF1α, CAG, and PGK, as well as the endogenous promoter AAVS1. In Table 1, the suicide gene response was quantified based on the number of GFP-positive cells responding to AP1903 treatment.

[0298] Table 1: Promoter targeting AAVS1 mediates gene expression maintenance, intensity, and functional response.

[0299]

[0300] Therefore, among all the promoters tested, CAG exhibited the best performance in all counting aspects, and the CAG-iPSC clone line is further characterized as follows.

[0301] Example 6 - Pluripotency Characterization of CAG-hiPSC Cloning Line

[0302] Pluripotency was characterized by analyzing all selected CAG-hiPSC cloning lines. Figure 5 Image A is a representative image of the CAG clone line C38 maintained on a feeder-free culture, showing a homogeneous hiPSC culture in which individual cells have a high nucleus-to-cytoplasm ratio. Figure 5Immunofluorescence staining of the pluripotency markers NANOG and OCT4 in cG1 indicated that the CAG-hiPSC clones were pluripotent and undifferentiated. To characterize the pluripotency of the selected CAG-hiPSC clones (CAG-C5, CAG-C8, CAG-C12, CAG-C38, and CAG-C40), quantitative RT-PCR was performed to assess the expression of various endogenous pluripotency markers, including NANOG, OCT4, SOX2, REX1, DPPA2, and MYC. NANOG expression was shown to be lower in all CAG-hiPSC clones than in parental iPSCs. Flow cytometry analysis was also performed to analyze the expression of TRA181, SSEA3, and CD30 markers. Subsequently, the selected CAG clones were induced to undergo directed lineage-specific differentiation, and lineage markers were evaluated. Figure 5 D shows the expression of nestin (an ectoderm marker), αSMA (a mesodermal marker), and SOX17 (an endoderm marker) after directed differentiation of the selected CAG clonal line. Further evaluation was conducted on the potential of one of the CAG-iPSC clonal lines (CAG-C38) to generate teratomas composed of various lineages. Figure 5 In E, teratomas were collected at week 6 post-injection and exhibited three germ layers (endoderm, mesoderm, or ectoderm). These data suggest that the CAG-iPSC clonal lineage maintains pluripotency and the ability to differentiate into non-pluripotent cells of all lineages.

[0303] Example 7 - Induced suicide genes mediate the killing of hiPSC clones in pluripotent and differentiated states through CAG-driven iCasp9 targeting insertion into the AAVS1 locus.

[0304] Twenty hiPSC clones targeting CAG-iCasp9 were treated with AP1903 (or DMSO control) for 24 hours, followed by 7AAD staining, and GFP and 7AAD staining were analyzed by flow cytometry. All clones showed greater than 95% 7AAD positivity after AP1903 treatment. Figure 6 A). Flow cytometry plots from two representative clonal lines (CAG-C5 and CAG-C38) also illustrate the response of the Casp9 gene to the pluripotency of the CAG-iPSC clonal line. Figure 6A). Fifteen iPSC clones targeting CAG-iCasp9 were cultured in triple-duplicate wells of 12-well plates. 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 allow any residual viable cells to recover. Eight-day recovery wells were stained for alkaline phosphatase and photographed to record any viability of the iPSC clones. Of the fifteen clones, only clone CAG-C14 contained surviving clones, i.e., clones that had escaped induced cell death (…). Figure 6 B). The other fourteen clonal lines showed no staining, indicating that no cells survived after Casp9 induction. Figure 6 C illustrates the viable cell coverage kinetics of the CAG-C5 clonal line on a 10 cm culture dish after AP1903 (10 nM) treatment. Viable cell death began approximately 8 minutes after treatment, and almost all viable cells were killed within 12 minutes of AP1903 treatment. Subsequently, the hiPSC clonal line targeting CAG-iCasp9 was induced to differentiate into cells of three germ layers (endoderm, mesoderm, or ectoderm), each treated with AP1903 for 48 hours and allowed to recover in differentiation medium for 5 days. Repeat wells before and after treatment were stained with crystal violet and imaged, as shown. Figure 6 As shown in Figure D. Images from the CAG-C5 clone line are illustrated, showing that cells from all lineages were killed after AP1903 treatment. Finally, the hiPSC clone line targeting CAG-iCasp9 was induced to differentiate into CD34+ cells, and cell death was analyzed by flow cytometry after 48 hours of AP1903 treatment. Figure 6 Elevated 7AAD staining in E indicates CD34+ cell death following AP1903 treatment. The data suggest that regardless of the cell's epigenetic state (i.e., pluripotency relative to ectoderm, endoderm, or mesoderm) or cell type-specific state (e.g., CD34-positive cells), CAG targeting the AAVS1 locus stably maintains iCasp9 expression.

[0305] Example 8 - In vivo evidence of AP1903-induced regression of teratoma derived from the iCasp9-iPSC clone.

[0306] The subcutaneous injection site in the NSG mouse study is shown in... Figure 7 In Figure A, in vivo cell death mediated by iCasp9 expression and induction was demonstrated. Parental hiPSC and CAG-C38 clones were injected at 4E6 cells per cell line (day 0) and treated with AP1903 (intraperitoneal, total 200 µg) on ​​days 7–11. Teratomas were collected and imaged on day 34 to demonstrate size. Figure 7B). In the CAG-C38 collection, one injection site contained no tumor, while the other consisted primarily of fat-like cells. The volume of the collected teratomas was also measured. Figure 7 C). Among the collected tumors, teratomas derived from CAG-38 hiPSCs were either undetectable or significantly smaller than their parental controls. The collected teratomas were stained with hematoxylin and eosin, and the staining results demonstrated histological differences between the parental iPSC and CAG-hiPSC clones. Figure 7 (D) Although the parental hiPSC line exhibited most of the three lineages of differentiated cell types, CAG-C38 appeared to consist primarily of highly organized adipoid cells, likely derived from mice. Subsequently, the parental hiPSC and CAG clones were injected at a rate of 4E6 cells per cell line and treated with AP1903 (intraperitoneal, total 200 µg) on ​​days 40–46. Tumor volume was measured in all cases using standard procedures, and CAG-iPSC remained responsive to Casp9 gene expression and reduced tumor volume in mice injected with either selected CAG-iPSC clone. Overall, the data demonstrate that the CAG promoter targeting the AAVS1 locus is stably expressed both in vitro and in vivo.

[0307] Example 9 - Genome editing of the human ROSA26 locus using CRISPR / Cas9 for targeted or nuclease-independent insertion of iCasp9

[0308] To test the synergy of other targeting strategies with the safe harbor locus, this experiment examined the potential for nuclease-independent targeted insertion of exogenous genes driven by the CAG promoter at different endogenous loci in the iPSC genome. A construct targeting the ROSA26 locus via CRISPR / Cas9 was designed, as shown in Figure 9A. hiPSCs were transfected with gRNA / Cas9-RFP plastids specific to the ROSA26 locus and donor constructs containing CAG-driven iCasp9. Two days after transfection, GFP expression in the cell population was analyzed by flow cytometry to determine transfection efficiency, which was indicated to be between approximately 80% and 85%. Figure 9 B). Puromycin selection was initiated 3 days after transfection and continued for 20 days, followed by flow cytometry analysis of GFP expression in puromycin-resistant cells. GFP expression was maintained during iPSC amplification. Figure 9 B). Table 2 below summarizes those with Figure 9 The iPSC expression level and functional response of the structure shown in Figure A.

[0309] Table 2: Gene expression maintenance, intensity, and functional response mediated by the CAG promoter targeting ROSA26:

[0310]

[0311] Table 2 demonstrates that CAG-driven exogenous gene targeting insertion is suitable not only for the AAVS1 locus but also for the ROSA26 locus and can be mediated by ZFN and CRISPR strategies. To test the feasibility of the strategy, another construct was designed to target the ROSA26 locus using a nuclease-independent strategy by utilizing homologous arms. The construct used for the nuclease-independent ROSA26 locus consists of a CAG promoter-driven GFP and iCasp9, while RFP indicates off-target integration (…). Figure 9 C). Figure 9 In C, constructs transfected with puromycin and selected 293 T cells and hiPSCs were enriched against their respective target cells. Figure 9 D and Figure 9 E). Data demonstrate that nuclease-independent strategies can also be applied to the current platform to efficiently generate targeted hiPSCs.

[0312] In summary, during the long-term clonal expansion of hiPSCs, the CAG promoter maintained high levels of iCasp9 expression, regardless of targeting strategy, locus, or differentiation state. Furthermore, these iCasp9-integrated clonal lines experienced rapid cell death in the presence of AP1903, and no residual cell survival was observed when the cultures were allowed to recover in the absence of dimerizing molecules. Additionally, hiPSC clonal lines differentiated into three somatic cell lineages in vitro, and all were found to undergo AP1903-induced cell death. The clonal lines also specifically differentiated into hematopoietic cells, exhibiting complete AP1903-induced cell death. Similar cell death-mediated responses were observed in vivo when injected into NSG mice to form teratomas. CAG-driven nuclease-free targeting integration into iPSCs yielded similar results. Therefore, the CAG-driven targeted integration system presented herein is not only efficient, precise, and sustainable, but also reliable because it appears not to induce epigenetic profile changes typically found in other promoter and / or construct cases. These changes eliminate suicide gene-mediated iPSC responses, amplifying iPSCs or iPSC-derived differentiated cells both in vitro and in vivo. Other exogenous genes and / or different selected loci can also be applied to this platform. For example, in addition to generating constructs containing GFP and iCasp9, genes with different functions can be incorporated into the constructs. Targeting modalities (e.g., chimeric antigen receptors or engineered T-cell receptors) can also be introduced into specific loci. In another instance, various gene-targeted specific loci can be performed during reprogramming. As previously discussed, the hiPSC platform can select individual cells with multiple properties, including successfully reprogrammed hiPSCs that have access to the desired gene introduced into the specific locus.

[0313] Notably, a hiPSC clone line contains certain rare cells that evade induced cell death, and this clone line and these rare cells were characterized to assess the molecular mechanisms of escape.

[0314] Example 10 - Characterization of escape clones reveals single-point mutations in all expanded clones.

[0315] The procedure for identifying surviving clones after AP1903 treatment further facilitates the selection and amplification of clones that survived the AP1903 treatment. Figure 8 A). As demonstrated, all escaped clonal lines had the correct targeted insertion, as shown by ligation PCR. To determine whether sequence changes were found in the escaped clonal lines, the iCasp9 transgene was amplified by PCR, followed by purification and genome sequencing. Figure 8 B). Among all sequenced clonal lines, those escaped clonal lines showed a single-point mutation K189E in their iCasp9 transgenic sequences. Figure 8 C). To confirm whether K189E is the direct cause of the refractory clone line, according to Figure 2 E produced the iCasp9 mutant form and was used to transfect and test the resulting targeted iPSC cells. Figure 8 E depicts GFP-expressing CAS-iCasp9m iPSC cells as unresponsive to AP1903 treatment, and all cells surviving after treatment exhibit the same high level of GFP expression compared to CAS-iCasp9wtiPSC cells.

[0316] Example 11 - Methods for rescuing escaped clones containing a common single-point mutation

[0317] The examples above demonstrate that, for all tested clonal lines, those resistant to in vitro treatment derived from a single recalcitrant line contain a common inactivation point mutation in iCasp9. However, selecting hiPSC clonal lines with biallelic iCasp9 insertion resulted in improved in vitro and in vivo killing kinetics and reduced resistance rates. Therefore, to guard against the possibility of resistance to a single inducible safety system, the generation and selection of hiPSC clonal lines containing multiple conditional suicide genes, including iCasp9 and herpes simplex virus thymidine kinase, each incorporated into a unique safe harbor locus, was investigated. The dual safety protection system was compared with a single iCasp9 safety switch in vitro and in vivo to assess the safety and effective exclusion of transplanted cells.

[0318] Example 12 - Copy number of iCasp9 targeted insertion of CAG bootloader driver into hiPSC's AAVS1 safe harbor

[0319] Quantitative PCR assessment of transgene (iCasp9-GFP) copy number in CAG promoter-driven iCasp9 clonal lines revealed that a few clonal lines had more than one transgene copy. For example... Figure 10 As shown in Figure A, a copy number of 1 indicates monoallelic iCasp9 integration, a copy number of 2 indicates biallelic iCasp9 integration, and a copy number greater than 2 indicates potential random integration. This integration occurs in sorted mixed cultures or in clonal lines with targeted insertion of monoallelic or biallelic iCasp9, such as... Figure 10 The mean fluorescence intensity (MFI) of iCasp9-GFP shown in Figure B demonstrates that the transgene expression level corresponds to the copy number; the higher the copy number, the higher the MFI reading. PCR analysis using primers overlapping with the AAVS1-transgene junction in mixed target cells and clonal lines demonstrates transgene integration, confirming that the iCasp9-GFP transgene specifically integrates into the AAVS1 locus. Figure 10C). The number of targeted integration alleles was assessed using primers specific to the AAVS1 integration site. The absence of a band indicates that the integration sites of both AAVS1 alleles are disrupted, i.e., biallelic insertion, while the presence of a band indicates that one or both alleles are undisturbed. Figure 10 D). For example Figure 10 As shown in C and 10D, two transgenic copies in the CAG-C5 and CAG-C35 clonal lines underwent biallelic insertion, while one copy in the CAG-C38 clonal line inserted into the target site of one allele. Immediately after adding 10 nMAP1903, live-cell imaging was used to assess the surface coverage of the culture plate. Figure 10 As shown in Figure E, unsorted mixed target cells (integrated and non-integrated) exhibited minimal induced killing ability, while mixed target cells sorted against iCasp9-GFP (targeted and random integration, variable copy number) showed slightly better induced killing ability. Cloned CAG-C38 (monoallelic targeted insertion) and CAG-C5 (bialic targeted insertion) cell lines showed significantly higher killing rates and efficiencies than the mixed cells.

[0320] Example 13 - Comparison of transplanted biallelic and monollelic iCasp9 clones in terms of in vivo induced killing and teratoma regression

[0321] Figure 11 Figure A illustrates a study in NSG mice with subcutaneous injection, demonstrating in vivo cell death mediated by iCasp9 expression and induction in CAG-C38 clones, CAG pool, PGK pool (PGK-iCasp9 transgene), UBC pool (UBC-iCasp9 transgene), and CMV pool (CMV-iCasp9 transgene). On day 0, the CAG-C38 clone and pools were injected into three groups of NSG mice (n=2) at 4E6 cells per cell line. From days 19 to 25, the mice were treated with AP1903 (intraperitoneal, 125 µg). On day 28, teratomas were collected and imaged to show size. The three groups of NSG mice were before AP1903 administration (…). Figure 11 B, above image) and the 3rd day after the investment ends ( Figure 11 Bioluminescence imaging (see figure B below) shows that only the exogenous CAG promoter enabled the exclusion of established iPSC-derived teratomas mediated by iCasp9. This observation is further described in... Figure 11 C-11G. Figure 11 The total flux (photons / second) presented in C as average and SD indicates the number of tumor cells derived from teratomas of a specified cell type, confirming... Figure 11 Teratoma size results in B. The ratio of the specified cell type to the previous total throughput (mean ± SD) is shown in [data / image / image]. Figure 11 D. Results of mice injected with cloned CAG-C38 and CAG hybrid cells are plotted with high resolution in D. Figure 11 In section E, the final volume of teratomas derived from the specified cell type was measured on day 28, and the results are shown in [image / description]. Figure 11 F in. Figure 11 G shows images of teratomas derived from the specified cell type on day 28. No teratomas were detected on the lateral aspect of cells injected with either the CAG-C38 clone or the CAG-iCasp9 hybrid. Overall, these in vitro studies with confirmed data on iCasp9-GFP transgene expression driven by different promoters demonstrate that the CAG promoter is the most effective in both in vitro and in vivo assays. The data also indicate that the CAG-C38 clone, despite being a monoallelic type for iCasp9-GFP, is more effective than the CAG hybrid in teratoma exclusion after suicide gene induction.

[0322] In addition, the inducible killing of the transplanted biallelic iCasp9 clone was tested and compared with that of the monollelic iCasp9 clone. Figure 12 Figure A illustrates a study in NSG mice with subcutaneous injection sites to demonstrate in vivo cell death mediated by iCasp9 expression and induction in parental cells, the CAG-C5 clone, and the CAG-C38 clone. On day 0, the corresponding cell types were injected into four groups of NSG mice (n=2), and on days 7–13, the mice were treated with AP1903 (intraperitoneal, 125 µg). Teratomas were imaged on days 7, 14, and 42. On day 42, teratomas were collected and imaged to demonstrate size.

[0323] Bioluminescence imaging was performed on NSG mice transplanted with specified cell types (biallelic CAG-C5 clone, monollelic CAG-C38 clone, and parental iPSCs) on days 7, 14, and 42 post-iPSC transplantation. Figure 12 B). Imaging was performed on day 7 prior to administration of AP1903 (125 µg, intraperitoneal; days 7–13). The middle image shows images of teratomas (if found) collected on day 14, and the bottom image shows images of teratomas (if found) collected on day 42. As shown, both CAG-C5 and CAG-C38 were able to exclude teratomas in all test mice by day 14. However, teratomas in mice transplanted with CAG-C38 regressed no later than day 42, while teratomas excluded in mice transplanted with CAG-C5 did not recur. Figure 12 C shows an image of a teratoma of a specified cell type on day 42. Figure 12The total flux (photons / second) of teratomas in the specified group, shown in D as the mean and SD, confirms... Figure 12 Imaging data from B and 12C. Overall, these data demonstrate that the transplanted biallelic iCasp9 clones are more effective at inducible killing than the monollelic iCasp9 clones.

[0324] Example 14 - The Development of a Safety Protection System for Targeted Insertion of Dual Suicide Genes into Individual Safe Harbor Loci

[0325] To demonstrate the capabilities and efficacy of high-resolution precision engineering at the single-cell level, cloned iPSCs with a safety protection system were generated, in which dual suicide genes were targeted and inserted into individual safe harbor loci. In addition to the integration of CAG-iCasp9 into the AAVS1 locus, CAG-sr39TK was also integrated into the H11 safe harbor of the CAG-C38 cloned iPSC using the targeted insertion method described herein. Figure 13 As shown in Figure A, PCR analysis confirmed that specific genomic integration of sr39TK into the H11 safe harbor locus was achieved using primers specific to sr39TK, the sr39TK-blastactin (Bsd) junction, or the endogenous H11 sequence-transgenic junction, with GAPDH used as an internal reference. iCasp9 iPSC clones targeting the H11 locus with sr39TK (for non-clonal sr39TK clones) were treated with 25 µg / mL ganciclovir (GCV) for nine days, with the first two days treated with 10 nM AP1903 or untreated. Treatment of the CAG-C38 iCasp9 clone (for monoallelic iCasp9 clones) with AP1903 alone for two days induced the killing of most cells, but a small number of cells survived. Figure 13 B(ii)). Treatment of the CAG-C38 iCasp9 clone line with GCV alone for nine days induced persistent GCV cell proliferation. Figure 13 B(iii)). However, parallel treatment with AP1903 and GCV effectively eliminated all refractory cells, indicating that using a dual suicide safety system is more effective than using the iCasp9 suicide gene alone in killing residual cells that have escaped under induced suicide genes.

[0326] This article also provides the design of iPSCs containing inducible cassettes located at safe harbor loci for the controlled expression of genes of interest. Inducible cassette-specific insertion into the locus occurs at the iPSC level. Expression of the gene of interest (CAR, described as an example in this article) is achieved through inactivation by side-attached translation stop codons at two loxP sites. This is achieved using Cre recombinase (…). Figure 33 A) or doxycycline ( Figure 33B) After one induction, the stop codon is thus removed and CAR expression is activated. The inducer can be administered at any selected stage during in vitro iPSC differentiation, thereby facilitating the optimization of the differentiation procedure. The CAR is driven by a strong constitutive promoter to achieve high and sustained expression in the final product, such as iT or iNK.

[0327] A novel strategy was also developed that uniquely targets selected loci to achieve gene-specific disruption while inserting the novel gene of interest into the hiPSC population. Subsequently, single-cell sorting was performed in a high-throughput manner to select hiPSC clones containing all the required criteria, including unique inserted gene copy number, disruption of the target gene function, and lack of off-target effects.

[0328] Figure 34 A illustrates a construct design for a target vector and a donor vector, wherein the target vector contains a negative and a positive strand to guide nuclease binding, and the donor vector contains a B2M homologous arm, an exemplary gene for interruption, and a gene of interest (e.g., CD16, HLA-G, and PDL1) for inserting into the interrupted locus. Figure 34 B depicts a diagram illustrating hiPSC selection performed on a population of clones containing the selected gene at the selected locus after gene editing.

[0329] Example 15 - Generating B2M 剔除 (B2M- / -), iPSCs lacking HLA I and their modifications

[0330] To demonstrate the ability to perform single-cell gene editing to achieve HLA complex modification, iPSC lines were transfected with plastids expressing the Cas9 nickase using a pair of B2M-targeting gRNAs. These plastids were engineered to provide fewer off-target effects than wild-type Cas9. Figure 14 A shows flow cytometry analysis of GFP and B2M / HLA-I expression before (left) and after (middle) transfection. B2M and HLA-I were simultaneously detected using B2M-specific and HLA-I-specific antibodies bound to the same dyes. Cells negative for both B2M and HLA-I (closed population) were batch-sorted in 96-well plates. Figure 14 A) or by cloning to produce clones ( Figure 14 B). Further analysis using cloned genomic DNA sequencing of B2M- / - clones lacking HLA I, confirmed by small deletions or insertions, yielded a B2M gene knockout phenotype.

[0331] Then, HLA class I-deficient iPSCs (B2M- / - iPSCs, also known as HLA I knockout iPSCs) were genetically engineered, for example, by introducing HLA-E / B2M fusion proteins using lentiviruses to further engineer HLA I-deficient cells. The quality (i.e., pluripotency status) of the HLA I-deficient engineered iPSCs (B2M- / - HLA-E iPSCs) was assessed using flow cytometry based on the expression of pluripotency markers TRA-181 and SSEA4, as well as HLA-E. Figure 16A This indicates that the modified iPSCs lacking HLA I express HLA-E on the cell surface and maintain a pluripotent phenotype. When the HLA-E / B2M fusion protein expressing nucleotides is integrated into iPSCs lacking HLA I, similar results to those observed with lentiviral transduction were found.

[0332] Subsequently, B2M- / - HLA-E iPSCs were genetically engineered to contain the PDL1 protein, resulting in HLA I-deficient modified iPSCs: B2M- / - HLA-E PDL1 iPSCs. Additionally, B2M- / - iPSCs were genetically engineered to contain the HLA-G / B2M fusion protein, and then further genetically engineered to contain the PDL1 protein, resulting in another HLA I-modified B2M knockout iPSC: B2M- / - HLA-G PDL1 iPSCs. Figure 21 A shows the expression of HLA-E, HLA-G, and PDL1 on the cell surface of modified iPSCs lacking HLA I. Similar results were found when the HLA-G / B2M fusion protein expressing nucleotides was integrated into iPSCs lacking HLA I.

[0333] To determine whether HLA-I-deficient modified iPSCs maintain their hematopoietic differentiation capacity, the various HLA-I-deficient modified 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 in this paper. Figure 21 B indicates that all modified iPSCs lacking HLA I were able to differentiate into CD34+ HE, as observed by flow cytometry 10 days after differentiation. CD34+ cells were isolated and placed into iNK or iT lymphocyte differentiation cultures. Figure 22This indicates that, 10 days after redifferentiation, regulating HLA I-deficient iPSCs through genetically engineered immune resistance modalities (e.g., HLA-E, HLA-G, or PDL1) does not interfere with the ability of iCD34 cells to generate ProNK (NK progenitor cells; CD45+CD56) and ProT (T cell progenitor cells; CD45+CD34+CD7+) cells, as found by flow cytometry. Similar results were observed when the nucleotide-expressing PDL1 protein was integrated into HLA I-deficient iPSCs.

[0334] To determine whether the expression of genetically engineered immune resistance proteins was maintained during hematopoietic differentiation, the expression of HLA-E, HLA-G, and PDL1 on the cell surface of iNK cells, which were derived from HLA-I-deficient modified CD34+ cells differentiated for 20 days, was assessed by flow cytometry. Figure 23 A indicated the absence of HLA-E and HLA-G proteins, while PDL1 expression was maintained. To determine whether HLA-E and HLA-G proteins cleaved from the cell surface during hematopoietic differentiation, Western blot analysis was used to examine the expression of HLA-E and HLA-G proteins in protein lysates from day 20 iNK cells and day 20 culture supernatant. Figure 23 B indicates that HLA-E and HLA-G proteins were found in the supernatant fraction, thus concluding that HLA-E and HLA-G proteins detached from the cell surface of iPSC-derived NK cells.

[0335] To maintain the enhanced survival of HLA I-deficient modified iPSC-derived lymphocyte effector cells, anti-cleavage forms of the HLA-E / B2M and HLA-G / B2M fusion proteins were designed. Figure 24 A) Furthermore, B2M- / - iPSCs were genetically engineered to contain an cleavable HLA-G / HLA-A3 fusion protein. Figure 24 B indicates that, based on flow cytometry targeting HLA-G expression, the HLA-G / HLA-A3 non-cleavable proteins are expressed on the cell surface without affecting iPSC quality, as found based on the expression of pluripotent proteins (TRA-181, SSEA4, NANOG, and OCT3 / 4).

[0336] An alternative approach to generating iPSCs lacking HLA class I is to disrupt the peptide-carrying complex (PLC), which is responsible for loading peptides onto HLA class I molecules. The antigen-associated transporter (TAP) is an integral component of the PLC, and disruption of this gene results in a significant reduction in stable HLA class I molecules on the cell surface. However, the TAP2 mechanism is unknown. 剔除Does the absence of HLA I in (TAP2- / -) iPSCs have any impact on their differentiation potential or survival? 剔除 The iPSC line FTi121 was generated by transfecting iPSCs with plastids expressing Cas9 nickase using TAP2 gRNA pairs. HLA-I negative / low-positive cells were clonally sorted in 96-well plates to produce clonal lines. Figure 25 A indicates that flow cytometry analysis of the two selected clonal lines showed a significant reduction in HLA class I expression compared to the parental FTi121. Treatment with IFNγ induced upregulation of HLA class I molecules on the surface of iPSCs. Figure 25 B indicates that the TAP2- / - clones, after treatment with IFNγ, exhibited reduced expression of HLA class I compared to wild-type FTi121, implying a unique strategy for identifying optimal HLA complex expression levels to evade T-cell and NK-cell detection.

[0337] Example 16 - Retention Improvement of B2M- / - and HLA-I-Deficient iPSCs

[0338] To determine whether HLA-I deficiency enables B2M- / - hiPSCs to evade T lymphocyte responses, peripheral blood T cells were pre-sensitized by co-culturing with hiPSCs for 10 days. After 10 days, expanded and pre-sensitized T cells were collected and used as cytotoxic effector cells. Wild-type hiPSCs (WT) or B2M- / - (engineered) hiPSC target cells were seeded individually or co-cultured with pre-sensitized T cells at a 1:3 ratio. The number of target cells per well was measured using Incucyte Zoom over 5 days. Figure 15A As shown, pre-sensitized T cells recognize and kill WT hiPSC target cells, while engineered hiPSCs are unaffected.

[0339] While B2M- / - hiPSCs have been shown to evade T cell recognition and killing, the problem is that these cells can be killed more efficiently by NK cells. This is because NK cells are known to be activated by cells lacking MHC class I, and this is also true for cells lacking B2M; and if so, this would lead to in vivo rejection. To test whether B2M- / - hiPSCs are more readily recognized by NK cells, K562 cells, CD45+ cells differentiated from wild-type iPSCs (WT), or cells differentiated from B2M- / - iPSCs (B2M- / - hiPSCs) were individually labeled with CFSE, e670 proliferation dye (eBioscience), or e450 proliferation dye (eBioscience), respectively. - / -CD45+ cells differentiated from NK cells. All three types of labeled target cells were mixed in equal proportions and added to single-well NK cell effector cells at specified ratios. Figure 15B ), or three types of labeled target cells are each added to the corresponding individual wells of NK cell effector cells ( Figure 15C Cells were cultured for 4 hours, and then cytotoxicity was quantified by flow cytometry. Figure 15B and 15C As shown, in vitro, NK cell recognition did not appear to increase, with both B2M- / - and WThiPSC CD45+ cells being killed at the same rate and in relatively lower amounts than K562 cells (a known NK cell target, included as a positive control). Therefore, the data suggest that this B2M- / - iPSC line can avoid T cell-mediated killing without inducing NK cell recognition, indicating improved survival of universal donor / recipient compatible hiPSC clones.

[0340] Further evaluation was conducted on the improved in vivo survival of the universal B2M- / - hiPSC clone. To determine whether HLA-A deficiency resulted in increased in vivo survival in the presence of a competent immune system, fluoresceinized WT hiPSCs (WT) and engineered B2M- / - hiPSCs were subcutaneously and bilaterally injected into immunocompromised NSG (Nuclear-mediated NSG). Figure 15D (Left) or immune competence WTC57BL / 6 ( Figure 15D (Right) Teratomas were formed in recipient mice. After 96 hours, the mice were subjected to bioluminescent IVIS imaging to detect the teratomas. In NSG recipients, both WT and engineered teratomas were detected with equal intensity, while in WT C57BL / 6 mice, the retention of engineered B2M- / - hiPSCs was increased compared to WT hiPSCs.

[0341] Example 17 - HLA class I regulation of iPSCs increases iPSC retention in vivo.

[0342] To determine whether the in vivo retention of HLA I-deficient modified iPSCs was increased, fluoresceinized wild-type iPSCs and B2M- / - HLA-E iPSCs were subcutaneously injected into the opposite flank of fully immunocompetent C57BL / 6 recipients in teratoma analysis. Mice were analyzed daily by IVIS imaging in conjunction with fluorescein injections (to visualize the forming teratomas). Figure 16B The results showed that B2M- / - HLA-E iPSCs exhibited increased quantitative retention, approximately 6 times that of wild-type iPSCs, between 72 and 144 hours post-injection. Figure 16BThe study also presented three representative mice with enhanced fluorescein imaging of B2M- / - HLA-E iPSC teratomas. Similar improvements in survival were observed when HLA-G was used instead of HLA-E. Furthermore, as mentioned above, the application of modified HLA-E or HLA-G forms to avoid lysis further enhanced the in vivo survival of HLA class I modified iPSCs. Interestingly, we recognized that in some cases where NK cells derived from CMV+ donors expressed NKG2C and were thus activated, HLA-E surface expression actually activated NKG2C and therefore had adverse effects, including NK cell recognition and subsequent killing of HLA-E-expressing cells. In contrast, NK cells expressing NKG2A could not be activated by HLA-E-expressing iPSCs and their differentiated progeny, thus enhancing the survival of these HLA-E-expressing cells.

[0343] To determine which component of the host immune response is involved in the rejection of HLA I-deficient modified enhanced iPSCs in wild-type recipient mice, CD4+ T cells, CD8+ T cells, and NK cells were individually depleted by injection of anti-CD4, anti-CD8a, and anti-NK1.1 antibodies, respectively. Figure 26 Three days after antibody injection, CD4+ T cells, CD8+ T cells, and NK cells were absent. On the third day following antibody-mediated depletion, fluoresceinized B2M- / -HLA I-modified iPSCs were subcutaneously injected into the flanks of immunocompetent C57BL / 6 mice to induce teratoma formation. Mice were analyzed daily using IVIS imaging in conjunction with fluorescein injections (to visualize the forming teratomas). Figure 26 B indicates that, 120 hours after iPSC injection, NK cell-depleted mice exhibited the highest resistance to tumor rejection compared to animals treated with the IgG control.

[0344] To determine whether genetically engineered HLA-I modified iPSCs exhibit enhanced in vitro retention and resistance, 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. The treated iPSCs were then compared with purified allogeneic T cells pre-labeled with Cell Trace Violet (CTV) to monitor cell proliferation. Figure 27A ), all peripheral blood mononuclear cells (PBMCs, Figure 27B and 27CAllogeneic T cells and NK cells contained in the sample were co-cultured. Individual T cells / PBMCs (unstimulated) and T cells / PBMCs stimulated with anti-CD3 / CD28 beads to induce activation and proliferation (stimulated) served as controls. On day 12 after co-culture initiation, cells were assessed by flow cytometry to measure the proliferation of CD4+ T cells, CD8+ T cells, and CD56+ NK cells as a measure of their ability to recognize and respond to HLA I-modified iPSC-derived cells. Figure 27A and 27B This indicates that T cells co-cultured with B2M- / - and other B2M- / - HLA I-modified iPSC cells showed less proliferation compared to those co-cultured with wild-type iPSC-derived cells, as observed based on the dilution of CTV proliferation dye. Figure 27C The similar effects on NK cell proliferation suggest that HLA class I modifications can enhance immune resistance to T and NK cells.

[0345] Example 18 - Creating iPSCs with Enhanced Features Through Additional Editing

[0346] Molecules modified or regulated at the iPSC level can be used to enhance the desired properties of immunotherapies using derived lymphocytes obtained through the differentiation platform of this invention. These molecules may include safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates; or proteins that promote the transplantation, transport, homing, viability, self-renewal, survival, regulation and control of immune responses, and / or survival of iPSCs or their derived cells. In addition to B2M / HLA-I and HLA-E / G, targeting molecules contributing to the desired properties further include (but are not limited to) the CD16 receptor and 41BBL co-stimulatory molecule, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, and A. 2AR, CAR (chimeric antigen receptor), TCR (T cell receptor), TAP1, TAP2, TAP-associated glycoprotein, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, any gene in the 6p21 region of chromosome 6, conjugates, and surface trigger receptors for coupling with bispecific, multispecific, or universal conjugates. More specifically, gene modifications of the targeting molecules in iPSCs include one or more of the following:...

Claims

1. A cell or a population thereof, wherein (i) the cell is an induced pluripotent stem cell (iPSC); (ii) the cell contains one or more exogenous polynucleotides at a constant region of an endogenous T cell receptor (TCR); (iii) at least one of the one or more exogenous polynucleotides encodes a chimeric antigen receptor (CAR); (iv) the CAR is expressed under the control of an endogenous promoter of the TCR; and (v) the endogenous TCR is knocked out.

2. The cell or population thereof according to claim 1, wherein at least one of the one or more exogenous polynucleotides is present in the nucleic acid encoding the constant region of the TCRα locus.

3. The cell or population thereof according to claim 2, wherein the endogenous TCRα gene is knocked out.

4. The cell or population thereof according to claim 3, wherein the CAR is expressed at approximately the same level as endogenous TCRα in a reference cell in which the endogenous TCRα gene has not been knocked out.

5. The cell or population thereof according to claim 3, wherein the CAR is expressed at approximately the same developmental stage as endogenous TCRα in a reference cell in which the endogenous TCRα gene has not been knocked out.

6. The cell or population thereof according to claim 1, wherein the CAR comprises a CD19-related CAR.

7. The cells or populations thereof according to claim 1, further comprising the introduction or increase of CD16 expression, wherein the CD16 is a non-cleavable high-affinity CD16 (hnCD16).

8. The cells or populations thereof according to claim 1, further comprising the introduction or increase of CD3 expression.

9. The cells or populations thereof according to claim 1, further comprising the deletion or reduced expression of at least one of B2M and CIITA, and optionally the introduction or increase of HLA-G expression.

10. The cell or population thereof according to claim 1, wherein the iPSC is obtained by reprogramming T cells into a pluripotent state (TiPSC) and genome editing the TiPSC.

11. The cell or population thereof according to claim 10, wherein the iPSC is obtained by reprogramming T cells, the T cells comprising the one or more exogenous polynucleotides present in nucleic acids encoding constant regions in the TCR.

12. The cells or populations thereof according to claim 1, further comprising the deletion or reduced expression of at least one of the following: any gene of the following: TAP1, TAP2, TAP-associated glycoprotein, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and the 6p21 region of chromosome; and / or the introduction or increase of expression of at least one of the following: HLA-E, HLA-G, CD16, 41BBL, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, Fc receptor, conjugate, and surface trigger receptor for coupling with bispecific, multispecific, or universal conjugates.

13. A pharmaceutical composition comprising a pharmaceutically acceptable medium and cells or populations thereof as described in any one of claims 1-12.

14. Use of the pharmaceutical composition of claim 13 in the preparation of a medicament for treating autoimmune diseases; cancer; or infections associated with HIV, RSV, EBV, CMV, adenovirus or BK polyomavirus.

15. The application according to claim 14, wherein the cancer is a hematologic malignancy or a solid tumor.

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