Modified hematopoietic stem cells and their progeny

Abstract

The present disclosure in various aspects and embodiments relates to cell compositions (and methods for making or using same) comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem progenitor cells (HSPCs), where the HSCs and / or HSPCs have one or more endogenous genes modified in their expression, thereby avoiding or reducing targeting of these cells by targeted therapies. In certain aspects, the present disclosure provides such HSCs and / or HSPCs from gene-edited human induced pluripotent stem cells (iPSCs).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 413,550, filed October 5, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted via EFS-Web in XML format and is incorporated herein by reference in its entirety. The XML copy, created on September 26, 2023, is named GRU-0010PC_Sequence_Listing.xml and is 30,045 bytes in size. [Background technology]

[0003] The ideal antigen for targeted therapy is a disease-specific antigen, e.g., a cancer-specific antigen. Cancer-specific antigens are antigens that are exclusively expressed in malignant cells, thereby providing a unique target leading to maximal disease elimination with minimal off-target toxicity. However, such disease antigens are rare because most antigens expressed by pathological or malignant cells are also found in normal tissues. For example, targeting the myeloid marker CD33 in acute myeloid leukemia (AML) (e.g., using CAR-T cells) results in toxicity from the destruction of normal bone marrow cells. To avoid the loss or destruction of normal cells, hematopoietic stem cells (HSCs) genetically engineered to disrupt the expression of the target antigen offer a promising therapeutic approach for replenishing normal cells lost or damaged due to the destruction of normal cells.

[0004] However, methods for generating clinically relevant numbers of genetically modified HSCs and / or cell lines therefrom that have clinically advantageous phenotypes remain a significant obstacle. There is a need for reliable, commercially available, expandable HSCs (and their progeny) with genetic modification of one or more cancer-associated antigens to treat diseases, including, but not limited to, hematological cancers.

[0005] In various aspects and embodiments, the present invention meets these objectives. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure in various aspects and embodiments relates to cell compositions (and methods for making or using the same) comprising hematopoietic stem cells (HSCs) that have one or more endogenous genes modified in their expression, thereby avoiding or reducing targeting of these cells by targeted therapies. In one aspect, the present disclosure provides such HSCs from gene-edited human induced pluripotent stem cells (iPSCs).

[0007] In one aspect, the present disclosure provides a method for preparing a population of HSCs and / or hematopoietic stem progenitor cells (HSPCs), which is useful for replenishing hematopoietic cells in a subject undergoing a therapy that targets one or more hematopoietic lineages for destruction. The method includes preparing a human iPSC population and modifying one or more endogenous genes within the iPSC population. Generally, the one or more endogenous genes include one or more cancer-associated antigens, and thus the present disclosure involves disrupting expression of the cancer-associated antigen(s) in iPSC-derived HSCs or HSPCs. The method further includes differentiating the iPSC population into a CD34+ population (e.g., recovered from dissociated embryoid bodies) and inducing endothelial-to-hematopoietic transition (EHT) of the CD34+ population to prepare a population comprising HSCs and / or HSPCs with reduced expression of the cancer-associated antigen(s). When these HSCs and / or HSPCs (or cell populations derived therefrom) are administered in conjunction with cancer therapies that target cancer-associated antigens, targeting of the HSCs and / or HSPCs or their progeny can be reduced or avoided entirely.

[0008] In embodiments, the one or more endogenous genes are selected from CD33, CD19, CD7, CD123, and CD371, among others.

[0009] HSCs and / or HSPCs can be derived from gene-edited iPSCs. In various embodiments, iPSCs are prepared by reprogramming somatic cells, such as (but not limited to) CD34+ cells isolated from peripheral blood. In embodiments, iPSCs can be further gene-edited to delete one or more of HLA-A, HLA-B, and HLA-C, and to delete one or more of HLA-DP, HLA-DQ, and HLA-DR. In certain embodiments, iPSCs retain expression of at least one HLA class I (e.g., HLA-A, HLA-B, HLA-C, and HLA-E) and at least one HLA class II complex. In certain embodiments, iPSCs are homozygous for at least one retained class I and class II locus. In some embodiments, iPSCs lack HLA-A or HLA-B or HLA-C or HLA-DQ or HLA-DQ.neg , homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg In some embodiments, the iPSCs are further homozygous for HLA-DRB1.

[0010] In some embodiments, the process for producing a population containing HSCs and / or HSPCs can include generating CD34+ enriched cells from pluripotent stem cells (e.g., EBs) and inducing endothelial and hematopoietic differentiation. HSCs containing a relatively high frequency of LT-HSCs can be generated from the cell population using a variety of stimuli or factors, including mechanical, biochemical, metabolic, and / or local stimulation.

[0011] Traditionally, hematopoietic stem cells or hematopoietic lineages are prepared by differentiating iPSCs into embryoid bodies up to day 8 and harvesting CD34+ cells. CD34 is commonly used as a marker for hemogenic endothelial cells, hematopoietic stem cells, and hematopoietic progenitor cells. According to aspects and embodiments of the present disclosure, it has been discovered that inducing endothelial-to-hematopoietic transition (EHT) (e.g., over at least 2 days but not more than 12 days) of a CD34+ cell population, which may be derived from iPSC embryoid bodies, can be used for the ex vivo generation of superior HSCs and hematopoietic lineages. Inducing endothelial-to-hematopoietic transition (EHT) can include increasing the expression or activity of dnmt3b, including pharmacologically increasing it using Piezo1 activation.

[0012] In various embodiments, CD34+ cells (e.g., suspension and / or adherent cells) are harvested from cultures undergoing endothelial-hematopoietic transition between days 10 and 20 of iPSC differentiation. In some embodiments, non-adherent cells are collected.

[0013] In embodiments, HSCs are further differentiated into hematopoietic lineages for therapy. In embodiments, the hematopoietic lineages are selected from progenitor T cells, T lymphocytes, and natural killer cells. Other lineages that can be produced and used for therapy include common myeloid progenitors (CMPs) or common lymphoid progenitors (CLPs). CMPs give rise to progeny such as red blood cells / erythrocytes, platelets, mast cells, osteoclasts, granulocytes, monocytes-macrophages, and dendritic cells. CLPs give rise to progeny such as T cells / T lymphocytes, B cells / B lymphocytes, NK cells / natural killer cells, and dendritic cells.

[0014] In some aspects and embodiments, modified HSCs can be administered to replace normal cells destroyed by antibodies, T cell therapy (e.g., CAR-T therapy), or NK cell therapy (e.g., CAR-NK). HSCs and their progenitor cells help the bone marrow recover and generate healthy cells, e.g., myeloid cells, in AML treatment. HSC rescue allows more targeted therapy to be given to patients so that more cancer cells are killed.

[0015] In other aspects, the present disclosure provides cell populations or pharmaceutically acceptable compositions thereof produced by the methods described herein. In various embodiments, the HSC and / or HSPC populations, as described herein, are derived from iPSCs, have genetically disrupted expression of one or more endogenous genes that are cancer-associated antigens, proliferate in vivo but do not exhibit uncontrolled proliferation or tumorigenesis in vivo, and differentiate in vivo to reconstitute hematopoietic lineages.

[0016] In certain aspects, the present disclosure provides a population of HSCs having genetically disrupted expression of one or more endogenous genes that are tumor-associated antigens, wherein the iPSCs are HLA-A. neg , homozygous for both HLA-B and HLA-C, HLA-DPB1 neg , HLA-DQB1 neg, as well as homozygous for HLA-DRB1. Such cells can be easily matched to the recipient for immunocompatibility.

[0017] In various aspects and embodiments, the HSCs / HSPCs of the present disclosure can be used to treat or ameliorate a disease or disorder (e.g., in methods of treatment), such as treating a hematopoietic malignancy. Non-limiting examples of hematopoietic malignancies include cytogenetically normal acute myeloid leukemia (CN-AML), acute myeloid leukemia (AML), acute lymphocytic leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia or chronic lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, or refractory or relapsed forms thereof. In some embodiments, the subject has received, is receiving, or will receive a targeted therapy, e.g., a therapy that targets hematopoietic cells (or their lineages).

[0018] In various embodiments, the modified HSCs and / or HSPCs (or cell lineages derived therefrom) of the present disclosure are administered in conjunction with the therapeutic use of CAR-T therapy. Generally, the CAR-T cells target a cancer antigen that has reduced or eliminated expression in the modified HSCs. Exemplary CAR-T cells include CD33-specific CAR-T cells, CD7-specific CAR-T cells, CD8-specific CAR-T cells, CD19-specific CAR-T cells, CD20-specific CAR-T cells, CD22-specific CAR-T cells, CD123-specific CAR-T cells, CD125-specific CAR-T cells, CD133-specific CAR-T cells, and CD371-specific CAR-T cells.

[0019] In various embodiments, HSCs and / or HSPCs or cell lines derived therefrom (e.g., T cells or NK cells, including CAR-T cells or CAR-NK cells) are administered in connection with the treatment of a non-hematologic malignancy, and the target antigen is expressed (even at a low level) in normal hematopoietic cells or lineages.

[0020] Other aspects and embodiments of the present disclosure will be apparent from the following detailed disclosure and examples. [Brief explanation of the drawings]

[0021] [Figure 1] Figure 1 shows that ETV2 overexpression (OE) does not affect pluripotency. Figure 1 shows a FACS plot showing the transduction efficiency of iPSCs using an adenoviral vector to overexpress ETV2 and GFP sequences. ETV2 overexpression does not affect iPSC stemness, as indicated by the expression of the TRA-1-60 stemness marker. [Figure 2] ETV2 overexpression (OE) increases the yield of hemogenic endothelial cells. Representative flow cytometry analysis and relative quantification of hemogenic endothelial cells (described as CD235a-CD34+CD31+) demonstrates that ETV2-OE enhances the formation of hemogenic endothelial cells. [Figure 3] Figure 1 shows that ETV2 overexpression (OE) enhances CD34+ cell formation during iPSC differentiation. Representative flow cytometry analysis and relative quantification of CD34+ cells demonstrates that ETV2-OE enhances CD34+ cell formation. [Figure 4A] Figure 1 shows that iPSC-derived HSCs derived by EHT of CD34+ cells (using Piezo1 activation in this example) undergo pre-T cell differentiation similar to bone marrow (BM)-HSCs. Figure 2 shows FACS plots of the differentiation efficiency of bone marrow (BM)-HSCs and iPSC-HSCs derived by EHT of CD34+ cells (using Piezo1 activation) into CD34+CD7+ pre-T cells. [Figure 4B] Figure 1 shows that iPSC-derived HSCs derived from EHT of CD34+ cells (in this example, using Piezo1 activation) undergo pre-T cell differentiation similar to bone marrow (BM)-HSCs. Quantification (%) of CD34+CD7+ cells derived from (1) BM-HSCs and (2) iPSC-HSCs (Piezo1 activation). Averages of three experiments are shown. [Figure 5A]Figure 1 shows that iPSC-derived HSCs generated during EHT of CD34+ cells (using Piezo1 activation in this example) can undergo T cell differentiation and be activated with CD3 / CD28 beads, similar to BM-HSCs. Figure 2 shows FACS plots of the activation efficiency (CD3+CD69+ expression) of T cells differentiated from BM-HSCs and iPSC-derived HSCs generated during EHT of CD34+ cells (using Piezo1 activation in this example). [Figure 5B] We show that iPSC-derived HSCs generated by EHT of CD34+ cells (in this example, using Piezo1 activation) can undergo T cell differentiation similar to BM-HSCs and can be activated with CD3 / CD28 beads. Quantification (%) of CD3+CD69+ cells derived from (1) BM-HSCs and (2) iPSC-HSCs (Piezo1 activation). Averages of three experiments are shown. [Figure 6] We demonstrate that iPSC-derived HSCs generated in EHT of CD34+ cells (in this example, using Piezo1 activation) can differentiate into functional T cells. IFNγ expression is a consequence of T cell activation after T cell receptor (TCR) stimulation via CD3 / CD28 beads. IFNγ expression in T cells differentiated from iPSC-derived HSCs (EHT of CD34+ cells generated upon Piezo1 activation) enhances their ability to further differentiate into functional T cells. Figure 6 shows the average of three experiments. [Figure 7A] Generation of three CD33-KO iPSC clones is shown. [Figure 7B] We show that CD33-KO does not affect the ability of cells to undergo endothelial-hematopoietic transition. [Figure 7C] Figure 1 shows that CD33-KO does not affect the ability of cells to generate self-renewing HSCs. [Figure 8] Figure 1 shows the generation of three CCR5 knockout (KO) iPSC clones. Figure 2 shows that CCR5-KO does not affect iPSC pluripotency. Figure 3 shows that CCR5-KO does not affect the ability of cells to undergo endothelial-hematopoietic transition. [Figure 9A]Figure 1 shows phenotypic analysis of triple knockout (HLA-edited) cells performed by FACS and immunofluorescence, demonstrating the overall expression of HLA class I molecules (HLA-A, HLA-B, and HLA-C) on the cell surface, with HLA-edited cells being positive for overall HLA class I expression to a similar extent as wild-type cells (gHSCs). [Figure 9B] Figure 1 shows phenotypic analysis of triple knockout (HLA-edited) cells performed by FACS and immunofluorescence. Cellular expression of HLA-A via immunofluorescence is shown, where HLA-A is not expressed in the HLA-edited clones. [Figure 10] We show that the HLA-edited clones retain their pluripotency (maintain tri-lineage differentiation) as illustrated by immunofluorescence, where ectodermal differentiation is indicated by NESTIN-488 and PAX6-594 staining, mesodermal differentiation is indicated by GATA-488 staining, and endodermal differentiation is indicated by CXCR4-488 and FOX2A-594 staining. [Figure 11] Immunocompatibility of HLA-edited HSCs is shown. HLA-edited HSCs and control HSCs (WT, B2M KO, and HLA class II null) were cocultured with peripheral blood mononuclear cells (PBMCs) bearing HLA-B and HLA-C matched but mismatched HLA-A. PBMC-mediated cytotoxicity was measured by Annexin V staining assay. [Figure 12] Figure 1 shows the in vivo engraftment potential of HLA-edited HSCs. Equal proportions of mCherry HLA-edited HSCs and wild-type HSCs (gHSCs) were mixed for competitive transplantation into mice, and bone marrow (BM) and peripheral blood samples were assessed by FACS to compare the relative amounts of each cell type present in the samples. [Figure 13A] Figure 1 shows that HLA-A deletion does not affect class I peptide presentation. Schematic diagram of immunopeptidome analysis. [Figure 13B] Figure 1 shows that HLA-A deletion does not affect class I peptide presentation. Figure 1 shows the results of immunopeptidome analysis, revealing little difference in the number of peptides and representative proteins presented by class I molecules in WT and HLA-edited cells. [Figure 14A] Figure 1 shows that deletion of HLA-DP and DQ does not affect class II peptide presentation. Figure 2 shows the immunopeptidome analysis scheme. [Figure 14B] Figure 1 shows that deletion of HLA-DP and DQ does not affect class II peptide presentation. Figure 2 shows that despite deletion of HLA-DP and DQ, cells retain the ability to present a wide range of peptides through HLA class II. [Figure 15] Schematic diagram of in vivo testing of antigen-mediated immune responses: delayed-type hypersensitivity assay (DTH), sensitization phase, and elimination phase, respectively. [Figure 16A] We demonstrate that HLA-edited HSCs reconstitute a functional immune system, as evidenced by DTH responses in immunodeficient mice. Figure 16 shows a delayed-type hypersensitivity assay performed on transplanted mice, an assay involving crosstalk between different types of immune cells. Mice were sensitized by subcutaneous injection of sheep red blood cells (antigen). A functional immune system results in swelling of the left paw, as measured with a microcaliper. As can be seen in Figure 16A, non-transplanted mice, being immunodeficient, did not exhibit swelling of the left paw. Conversely, mice transplanted with umbilical cord blood CD34+ cells exhibited tissue swelling, doubling the diameter of their left paw. [Figure 16B] 16A-B are graphical evaluations of the results shown in Figure 16A, showing that HLA-edited HSCs reconstitute a functional immune system as demonstrated by DTH responses in immunodeficient mice. [Figure 17] Figure 17 shows the differentiation potential of HSCs into T cell subtypes. After a 35-day differentiation period, pre-T cells were assessed by cell sorting for the presence of CD4+, CD8+, and AB+ T cell populations. Figure 17 compares the differentiation potential of bone marrow-derived CD34+ cells, embryoid body CD34+ cells, and HSCs prepared according to the present disclosure (e.g., using Piezo1 activation) ("gHSCs"). [Figure 18]

[0023] Figure 1 shows the degree of T cell-mediated cytotoxicity measured from co-culture of HSC-derived T cells with CD19+ lymphoma cells in the presence of an anti-CD3 / CD-19 bispecific antibody. T cells prepared from HSCs according to the present disclosure ("gHSCs") exhibit high levels of cytotoxicity against target cells. [Figure 19] These results demonstrate that HSC-derived T cells (pre-T cells) can be transduced with high efficiency. Pre-T cells were subjected to lentiviral (LV) transduction with an anti-CD19 chimeric antigen receptor (CAR) transgene (left), and the efficiency of LV transduction was measured by cell sorting based on anti-CD19 scFv staining (right). The results show that HSC-derived T cells achieved approximately 85% transduction efficiency. [Figure 20] We show that HSC-derived pre-T cells can be effectively matured into CD4+ / CD8+ T cells via CAR transduction. [Figure 21] Figure 1 shows the ability of HSC-derived T cells transduced with an anti-CD19 CAR (pre-CAR T cells) to function through receptor-mediated cytotoxicity. Luc NALM6 leukemia cells were co-cultured with pre-CAR T cells, and cell-mediated cytotoxicity was measured by luciferase assay. [Figure 22] 1 shows the ability of HSCs prepared according to the present disclosure (using Piezo1 activation) to evolve into pre-T cells, as measured by their CD34-CD7+ markers. [Figure 23A] 10 demonstrates increased expression of T cell-specific transcription factors and thymic engraftment molecules using pre-T cells derived from HSCs according to the present disclosure. TCF7 mRNA expression is shown. [Figure 23B] 10 demonstrates increased expression of T cell-specific transcription factors and thymic engraftment molecules using pre-T cells derived from HSCs according to the present disclosure. CCR7 mRNA expression is shown. [Figure 24A] 1 shows that HSC-derived pre-T cells engraft and differentiate in the thymus. Engraftment and analysis procedures are illustrated. [Figure 24B]Figure 1 shows that HSC-derived pre-T cells engraft and differentiate in the thymus. Figure 2 shows FACS analysis of the CD3 cell population of cells gated on the CD45+ cell population, demonstrating the superior engraftment and differentiation potential of HSC-derived pre-T cells in the thymus. [Figure 25] Figure 1 shows that HSC-derived T cells can be activated in vitro. The top panel shows FACS analysis of activated T cells from different sources, including from HSCs prepared according to the present disclosure. T cells of the present disclosure show comparable or superior activation, as measured by increased CD107 expression. The bottom panel shows Dynabeads activation, where activated T cells express inflammatory cytokines. HSC-derived T cells express higher levels of inflammatory cytokines, as exemplified by TNF-α and interferon gamma expression levels. [Figure 26] We show that CCR5 knockout HSCs are equally capable of differentiating into pre-T cells compared to their wild-type (gHSC) counterpart HSCs (in which CCR5 is retained). [Figure 27] We show that CCR5 knockout HSCs are equally capable of differentiating into double-positive (CD4+CD8+) T cells compared to their wild-type counterparts HSCs (in which CCR5 is retained). DETAILED DESCRIPTION OF THE INVENTION

[0022] The term "gHSC" is used herein to refer to the iPSC-derived hematopoietic stem cells of the present disclosure.

[0023] The terms "wild-type" (WT), "unedited," and "non-HLA edited" are used interchangeably herein to refer to non-gene-edited cells of the present disclosure.

[0024] EB34+ cells refer to embryonic body-derived CD34+ cells, which contain hemogenic endothelial cells.

[0025] The present disclosure in various aspects and embodiments relates to cell compositions (and methods for making or using the same) comprising hematopoietic stem cells (HSCs) that have one or more endogenous genes modified in their expression, thereby avoiding or reducing targeting of these cells by targeted therapies. In one aspect, the present disclosure provides such HSCs from gene-edited human induced pluripotent stem cells (iPSCs).

[0026] In one aspect, the present disclosure provides a method for preparing a population of HSCs and / or hematopoietic stem progenitor cells (HSPCs), which is useful for replenishing hematopoietic cells in a subject undergoing a therapy that targets one or more hematopoietic lineages for destruction. The method includes preparing a human iPSC population and modifying one or more endogenous genes within the iPSC population. Generally, the one or more endogenous genes include one or more cancer-associated antigens, and thus the present disclosure involves disrupting the expression of the cancer-associated antigen(s) in iPSC-derived HSCs. The method further includes differentiating the iPSC population into a CD34+ population (e.g., recovered from dissociated embryoid bodies) and inducing endothelial-to-hematopoietic transition (EHT) of the CD34+ population to prepare a population comprising HSCs and / or HSPCs with reduced expression of the cancer-associated antigen(s). EHT can be induced, for example, for at least two days and up to 12 days. When these HSCs and / or HSPCs (or cell populations derived therefrom) are administered in conjunction with cancer therapies that target cancer-associated antigens, targeting of the HSCs and / or HSPCs or their progeny can be reduced or avoided entirely.

[0027] One of the greatest complications in treating diseases such as cancer by targeting antigens associated with diseased cells is that most antigens are also present on normal cells. For example, when CD33 is targeted by T cell therapy (e.g., CAR-T cells) as a treatment for acute myeloid leukemia (AML), CD33 is also present on normal bone marrow cells, leading to their destruction. Therefore, administration of HSCs (or their progeny) described herein to reconstitute the hematopoietic lineage can circumvent this effect by reducing or eliminating CD33 expression. This same approach can be applied to various cancer-associated antigens that are useful for targeting using immunotherapy.

[0028] As used herein, a "cancer-associated antigen" or "tumor-associated antigen" is an antigen expressed in cancer or tumor cells. In some embodiments, the cancer-associated antigen is expressed on the surface of cancer or tumor cells. In some embodiments, the antigen is expressed at a higher level in cancer cells compared to non-malignant cells (i.e., normal cells). In other embodiments, the antigen is expressed in cell lineages, but at comparable levels in malignant and normal cells. Various cancer-associated antigens are described herein. In some embodiments, the cancer-associated antigen is expressed or overexpressed in hematological cancers. For example, the cancer-associated antigen may be expressed or overexpressed in one or more of cytogenetically normal acute myeloid leukemia (CN-AML), acute myeloid leukemia (AML), acute lymphocytic leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma. In embodiments, the cancer-associated antigen is expressed predominantly on immune cells (compared to other tissues). In some embodiments, the cancer-associated antigen is expressed predominantly on myeloid cells. That is, the cancer-associated antigen is expressed at a higher level on myeloid cells than on other cells and tissues. In some embodiments, the cancer-associated antigen is expressed predominantly on thymocytes. In some embodiments, the cancer-associated antigen is expressed predominantly in T cells or B cells. In some embodiments, the cancer-associated antigen is expressed in immune cells at all stages of cell differentiation. In some embodiments, the at least one endogenous gene (including the cancer-associated antigen) that is genetically modified is selected from CD33, CD19, CD7, CD123, and CD371.

[0029] In various embodiments, HSCs and / or HSPCs can have reduced expression of endogenous genes, or in some embodiments, expression of endogenous genes is eliminated. For example, according to embodiments, at least about 50%, or at least about 75%, or at least about 90%, or about 100% of HSCs (or their progeny) do not express the endogenous gene. Alternatively, at least about 50%, or at least about 75%, or at least about 90%, or about 100% of HSCs and / or HSPCs (or their progeny) express the endogenous gene at a reduced expression level. In some embodiments, all or part of the endogenous gene is deleted to eliminate its expression. Alternatively, cis- or trans-expression regulators can be genetically engineered to reduce, but not eliminate, expression. For example, expression can be reduced by at least 50% compared to HSCs (or their progeny) that do not include gene editing of cis- or trans-expression regulators. In some embodiments, the HSCs and / or HSPCs have a deletion or inactivation of only one copy of the endogenous gene (i.e., thereby eliminating expression of one copy of the endogenous gene), leading to reduced expression. Alternatively, the HSCs and / or HSPCs have a deletion or inactivation of both copies of the endogenous gene (thereby completely eliminating expression).

[0030] In embodiments, the one or more endogenous genes include CD33. In such embodiments, HSCs and / or HSPCs with reduced or eliminated expression of CD33 are suitable for myeloid differentiation. For example, the HSCs and / or HSPCs differentiate into myeloid and lymphoid progenitor cells and mature myeloid and lymphoid cells.

[0031] In embodiments, the one or more endogenous genes include CD19. In such embodiments, HSCs with reduced or eliminated expression of CD19 are suitable for B-lymphocyte differentiation. In embodiments, the HSCs and / or HSPCs differentiate into myeloid and lymphoid progenitor cells and mature myeloid and lymphoid cells.

[0032] In embodiments, the one or more endogenous genes include CD7. In such embodiments, HSCs and / or HSPCs with reduced or eliminated expression of CD7 are suitable for T cell progenitor differentiation. In embodiments, the HSCs and / or HSPCs differentiate into myeloid and lymphoid progenitor cells and mature myeloid and lymphoid cells.

[0033] In embodiments, the one or more endogenous genes include CD123. In such embodiments, HSCs with reduced or eliminated expression of CD123 are suitable for myeloid differentiation. In embodiments, the HSCs and / or HSPCs differentiate into myeloid and lymphoid progenitor cells and mature myeloid and lymphoid cells.

[0034] In embodiments, the one or more endogenous genes include CD371. In such embodiments, HSCs with reduced or eliminated expression of CD371 are suitable for myeloid differentiation. In embodiments, the HSCs and / or HSPCs differentiate into myeloid and lymphoid progenitor cells and mature myeloid and lymphoid cells therefrom.

[0035] In various embodiments, one, two, three, or four endogenous genes comprising tumor-associated antigens are genetically modified to reduce or eliminate expression, with one or two of such genes being selected from CD33, CD19, CD7, CD123, and CD371. Other exemplary tumor-associated antigens are CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDwl2, CD13, CD14, CD15, CD15u, CD15s, CD15su, CD16, CD16b, CD17, CD18, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD 29, CD30, CD31, CD32, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RA, CD45RB , CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58 , CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, C D69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85a, CD85d, CD85j, CD85k, CD86 , CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD104, CD105, CD 106, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a,CD121b、CD122、CD124、CD125、CD126、CD127、CD129、CD130、CD131、CD132、CD133、CD134、CD135、CD136、CD137、CD138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD144、CDw145、CD146、CD147、CD148、CDw149、CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CD156c、CD157、CD158e、CD158i、CD158k、CD159a、CD159c、CD160、CD161、CD162、CD163、CD164、CD165、CD166、CD167a、CD167b、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD181、CD182、CD183、CD184(CXCR4)、CD185、CD186、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CDw198、CD199、CD200、CD201、CD202b、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210、CDw210b、CD212、CD213a1、CD213a2、CD215、CD217a、CD218a、CD218b、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235b、CD236、CD236R、CD238、CD239、CD240CE、CD240DCE、CD240D、CD241、CD242、CD243、CD244、CD245、CD246、CD247、CD248、CD249、CD252、CD253、CD254、CD256、CD266、CD267、CD268、CD269、CD270、CD271、CD272、CD273、CD274、CD275、CD276、CD277、CD278、CD279、CD280、CD281、CD282、CD283、CD284, CD286, CD289, CD290, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300c, CD300e, CD301, CD302, CD303, CD304 , CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD308, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD3 22, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, and CD370.

[0036] In some embodiments, the at least one endogenous gene comprises a growth factor receptor such as one or more selected from ErbB1, ErbB2, ErbB3, or ErbB4, IGF1R, IGF2R, TβR I-II, VEGFR1, VEGFR2, VEGFR3, PDGFR(α / β), and FGFR1, 2, 3, or 4.

[0037] HSCs and / or HSPCs are derived from gene-edited iPSCs. In various embodiments, iPSCs are prepared by reprogramming somatic cells. The term "induced pluripotent stem cells" or "iPSCs" refers to cells derived from somatic cells, such as skin or blood cells, that have been reprogrammed back to an embryonic-like pluripotent state. In some embodiments, iPSCs are generated from somatic cells, such as (but not limited to) fibroblasts or PBMCs (or cells isolated therefrom). In some embodiments, iPSCs are derived from lymphocytes (e.g., T cells, B cells, NK cells, etc.), umbilical cord blood cells (including CD3+ or CD8+ cells derived from umbilical cord blood), PBMCs, CD34+ cells, or other primary human tissues. In some embodiments, iPSCs are derived from CD34+ cells isolated from peripheral blood. In various embodiments, iPSCs are autologous or allogeneic (e.g., HLA-matched at one or more loci) to the recipient (the subject in need of the treatment described herein). In various embodiments, iPSCs can be genetically edited to support HLA matching (such as deletion of one or more HLA class I and / or HLA class II alleles or their master regulators, including, but not limited to, beta-2-microglobulin (B2M), CIITA, etc.). For example, iPSCs can be genetically edited to delete one or more of HLA-A, HLA-B, and HLA-C, and one or more of HLA-DP, HLA-DQ, and HLA-DR. In certain embodiments, iPSCs retain expression of at least one HLA class I (e.g., HLA-A, HLA-B, HLA-C, and HLA-E) and at least one HLA class II complex. In certain embodiments, iPSCs are homozygous for at least one retained class I and class II locus.

[0038] In various embodiments, the HSC and / or HSPC population is derived from iPSCs that have been gene-edited to be one of: (i) HLA-A-B+C+DP-DR+DQ+, (ii) HLA-A-B+C+DP+DR+DQ-, (iii) HLA-A-B+C+DP-DR+DQ-, (iv) HLA-AB-C+DP-DR+DQ+, (v) HLA-AB-C+DP+DR+DQ-, or (vi) HLA-AB-C+DP-DR+DQ-. For retained HLA (e.g., HLA-B, HLA-C, and HLA-DR), the cells can be homozygous or can retain only a single copy of the gene. For example, the modified cells are identified as at least (a) HLA-C+ and HLA-DR+, and optionally as one or more of (b) HLA-B-, (c) HLA-DP-, and (d) HLA-DQ-. In an exemplary embodiment, the modified cells are HLA-B+, HLA-DP-, and HLA-DQ-.

[0039] In some embodiments, the HSC and / or HSPC population is HLA-A neg , homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg In some embodiments, the iPSCs are further homozygous for HLA-DRB1.

[0040] As used herein, the term "neg," (-), or "negative" with respect to a particular HLA class I or HLA class II molecule indicates that both copies of the gene have been disrupted in a cell line or population, and therefore the cell line or population does not exhibit significant functional expression of the gene. Such cells can be generated by complete or partial gene deletion, or alternatively, by other techniques such as siRNA. As used herein, the term "deletion" in the context of genetic modification of a targeted gene (i.e., gene editing) refers to the elimination of functional expression of the corresponding gene product (i.e., the corresponding polypeptide). Such gene editing includes complete or partial gene deletion or disruption, or deletion of critical cis-acting expression control sequences.

[0041] In certain embodiments, disruption of one or more endogenous genes (e.g., endogenous genes encoding cancer-associated antigens), and in some embodiments one or more HLA class I and / or HLA class II genes, or genes governing HLA expression or presentation capabilities, is generated by introducing a Cas9 endonuclease or a nucleic acid encoding the Cas9 endonuclease into iPSCs and a nucleic acid molecule encoding a guide RNA (gRNA) that directs the mutation or deletion of the nucleotide sequence of the endogenous gene by the Cas9 endonuclease.

[0042] In some embodiments, iPSCs are gene-edited using gRNAs that are 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more nucleotides in length. In some embodiments, the gRNAs comprise modifications at or near the 5' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5' end) and / or at or near the 3' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 3' end). In some embodiments, the modified gRNAs exhibit increased resistance to nucleases. In some embodiments, the gRNAs comprise two separate RNA molecules (i.e., "duplex gRNAs"). Duplex gRNAs comprise two separate RNA molecules, "crispr RNA" (or "crRNA") and "tracr RNA," and are well known to those of skill in the art.

[0043] Generally, various gene editing technologies are known that can be applied in accordance with various embodiments of the present disclosure, including, but not limited to, zinc finger (ZF), transcription activator-like effector (TALE), etc. Fusion proteins comprising one or more of these DNA binding domains and the cleavage domain of a Fokl endonuclease can be used to generate double-stranded breaks in desired regions of DNA within a cell (see, e.g., U.S. Patent Application Publication No. 2012 / 0064620, U.S. Patent Application Publication No. 2011 / 0239315, U.S. Patent No. 8,470,973, U.S. Patent Application Publication No. 2013 / 0217119, U.S. Patent No. 8,420,782, U.S. Patent Application Publication No. 2011 / 0301073, U.S. Patent Application Publication No. 2011 / 0145940, U.S. Patent No. 8,450,471, U.S. Patent No. 8,440,431, U.S. Patent No. 8,440,432, and U.S. Patent Application Publication No. 2013 / 0122581, the entire contents of which are incorporated herein by reference). In some embodiments, gene editing is performed using a CRISPR-associated Cas system (e.g., CRISPR-Cas9) known in the art. See, for example, US8,697,359, US8,906,616, and US8,999,641, each of which is incorporated herein by reference in its entirety. In various embodiments, gene editing employs a type II Cas endonuclease (such as Cas9) or a type V Cas endonuclease (such as Cas12a). Type II and type V Cas endonucleases are guide RNA dependent. Design of gRNAs to guide desired gene editing (while limiting or avoiding off-target editing) is known in the art. See, for example, Mohr SE, et al., CRISPR guide RNA design for research applications, FEBSJ. 2016 Sep; 283(17):3232-3238.In still other embodiments, non-canonical Type II or Type V Cas endonucleases with homology (albeit low primary sequence homology) to S. pyogenes Cas9 or Prevotella and Francisella 1 (Cpf1 or Cas12a) can be employed. Many such non-canonical Cas endonucleases are known in the art. Nidhi S, et al. Novel CRISPR-Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives, Int J Mol Sci. 2021 Apr;22(7):3327. In still other embodiments, gene editing employs base editing or prime editing to incorporate mutations without creating double-strand breaks. See, for example, Antoniou P, et al., Base and Prime Editing Technologies for Blood Disorders, Front. Genome Ed., 28 January 2021; Matsuokas IG, Prime Editing: Genome Editing for Rare Genetic Diseases Without Double-Strand Breaks or Donor DNA, Front. Genet., 9 June 2020. Various other gene editing processes are known, including the use of dead Cas (dCas) systems (e.g., Cas fusion proteins) to target DNA-modifying enzymes to desired targets using dCas as a guide RNA-dependent system. Brezgin S, Dead Cas Systems: Types, Principles, and Applications, Int J Mol Sci. 2019 Dec;20(23):6041.

[0044] Base editors that can introduce precise genome modifications without generating double-stranded DNA breaks can also be used for gene editing in cells (e.g., iPSCs) (e.g., designing gene therapy vectors). Base editors inherently cannot create DSBs and contain a catalytically inactive nuclease, such as Cas9 nickase (nCas9), fused to a nucleic acid base deaminase enzyme, possibly a DNA glycosylase inhibitor. Currently, there are two major categories of base editors: cytidine base editors (CBEs) and adenine base editors (ABEs), which catalyze C>T and A>G transversions. Base editors can be delivered, for example, via HDAd5 / 35++ vectors, to efficiently edit promoters and enhancers to activate or inactivate genes. Exemplary methods are described in U.S. Patent Nos. 9,840,699, 10,167,457, 10,113,163, 11,306,324, 11,268,082, 11,319,532, and 11,155,803. Also contemplated are prime editors comprising a reverse transcriptase conjugated to (e.g., fused with) a Cas endonuclease and a polynucleotide useful as a DNA synthesis template conjugated to (e.g., fused with) a guide RNA, as described in WO2020 / 191153.

[0045] Exemplary vectors that can be used for genome editing applications include plasmids, retroviral vectors, lentiviral vectors, adenoviral vectors (e.g., Ad5 / 35, Ad5, Ad26, Ad34, Ad35, Ad48), parvoviruses (e.g., adeno-associated virus (AAV) vectors), herpes simplex virus vectors, baculovirus vectors, negative-stranded RNA viruses such as coronaviruses, orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive-stranded RNA viruses such as picornaviruses and alphaviruses, and herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses. Examples of vectors that can be used include, but are not limited to, double-stranded DNA viruses, including canarypox, vaccinia, or modified vaccinia viruses. Vectors containing a nucleic acid molecule of interest can be delivered to cells (e.g., iPS cells, endothelial cells, hemogenic endothelial cells, HSCs (ST-HSCs or LT-HSCs)) via any method known in the art, including, but not limited to, transduction, transfection, infection, and electroporation. Any of these vectors may contain a transposable element (such as a piggyBac transposon or a Sleeping Beauty transposon). Transposons insert specific sequences of DNA into the vertebrate genome. A gene of interest can be integrated into the genome of a mammalian cell by transposase-catalyzed cleavage of a similar excision site present in the cell's nuclear genome.

[0046] For increased efficiency, in some embodiments, Cas and gRNA can be combined before delivery into cells. The Cas-gRNA complex is known as a ribonucleoprotein (RNP). Several methods have been developed for direct delivery of RNP into cells. For example, RNP can be delivered to cells in culture by lipofection or electroporation. Electroporation using a nucleofection protocol can be employed, which allows RNP to rapidly enter the cell's nucleus and immediately begin cleaving the genome. See, for example, Zhang S, Shen J, Li D, Cheng Y. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR / Cas9 genome editing. Theranostics. 2021 Jan 1;11(2):614-648, incorporated herein by reference in its entirety. In some embodiments, Cas9 and gRNA are electroporated into donor iPSCs and / or HSCs as RNP.

[0047] Generally, a protospacer adjacent motif (PAM) is required for Cas nuclease cleavage and is typically found 3-4 nucleotides downstream of the cleavage site. The PAM is a short DNA sequence (usually 2-6 base pairs in length) following the DNA region targeted for cleavage by a CRISPR system such as CRISPR-Cas9. In some embodiments, the PAM sequence, sgRNA, or base editing tool targeting a haplotype or polymorphism at an HLA locus does not contain four Gs, four Cs, a GC repeat, or a combination thereof.

[0048] In some embodiments, a CRISPR / Cas9 system specific for a unique HLA haplotype can be developed by using the gRNAs described herein to design a single gRNA that targets each of the donor-specific HLA-A, HLA-DPB1, and HLA-DQB1 genes (for example). To perform gene knockout, the gRNA targets the Cas9 protein to the appropriate site for editing. The Cas9 protein can then perform a double-strand break (DSB), which repairs the DNA through a non-homologous end joining (NHEJ) mechanism, generating an indel that results in a frameshift mutation and terminates the function of the resulting protein. However, off-target gene recombination can occur, altering the function of an otherwise intact gene. For example, the Cas9 endonuclease can generate DSBs at undesired off-target locations, even in the presence of some degree of mismatch. This off-target activity can result in genomic instability events such as point mutations and genomic structural perturbations. In various embodiments, an sgRNA targeting HLA-A can target a region of chromosome 6 defined as 29942532-29942626. In various embodiments, an sgRNA targeting HLA-DQB1 can target a region of chromosome 6 defined as 32665067-32664798. In various embodiments, an sgRNA targeting HLA-DPB1 can target a region of chromosome 6 defined as 33080672-33080935.

[0049] gRNAs can be used to develop clonal iPSCs. Such iPSC lines can be assessed for (i) on-target editing, (ii) off-target editing, and (iii) translocation editing, for example, using sequencing as described herein. Specifically, such assays can be performed by multiplex PCR using primers designed to target and enrich for the region of interest, followed by next-generation sequencing (e.g., Amplicon sequencing, AMP-seq). On-target and translocation panels can amplify the intended edited region and allow for the selection of iPSC clones with the expected edits that do not contain chromosomal translocations resulting from unintended DSB cleavage site fusions. Off-target panels can enrich for any potential off-target regions identified through sequencing, allowing for the selection of iPSC clones with negligible off-target mutations. Together, these assays enable the screening of iPSC clones to select clones with the desired edits while ruling out potential CRISPR / Cas9-related genome integrity issues.

[0050] In some embodiments, to further ensure the genomic stability and integrity of the reprogrammed and edited iPSCs, genetic and genomic assays can be performed to select clones that have not undergone translocation and mutation events and have not integrated the episomal vector. For example, whole genome sequencing (WGS) can be performed on CD34+ cells and iPSC clones after reprogramming, and the genomes can be compared for differences resulting from editing. These analyses provide an assessment of which iPSC clone genomes differ from the CD34+ starting material, allowing for the informed selection of iPSC clones that have not undergone mutations during reprogramming.

[0051] In some embodiments, karyotyping using a system such as the KARYOSTAT assay is used to select iPSC clones that did not develop indels or translocations during reprogramming, as described, for example, in Ramme AP, et al., "Supporting dataset of two integration-free induced pluripotent stem cell lines from related human donors," Data Brief. 2021 May 15;37:107140, incorporated herein by reference in its entirety. The KARYOSTAT assay allows visualization of chromosomal abnormalities with a resolution similar to G-banding karyotyping. The size of structural abnormalities that can be detected is >2 Mb for chromosome gains and >1 Mb for chromosome losses. The KARYOSTAT array is functionalized for balanced whole-genome coverage with low-resolution DNA copy number analysis, and the array covers all 36,000 RefSeq genes, including 14,000 OMIM targets. The assay allows for the detection of aneuploidy, submicroscopic abnormalities, and mosaic events.

[0052] In some embodiments, array comparative genomic hybridization (aCGH) analysis is used to select iPSC clones that have not developed copy number aberrations (CNAs) during reprogramming, as described, for example, in Wiesner et al. "Molecular Techniques," Editor(s): Klaus J. Busam, Pedram Gerami, Richard A. Scolyer, "Pathology of Melanocytic Tumors," Elsevier, 2019, pp. 364-373, ISBN 9780323374576, and Hussein SM, et al. "Copy number variation and selection during reprogramming to pluripotency," Nature. 2011 Mar 3;471(7336):58-62, which are incorporated herein by reference in their entireties. aCGH is a technique that analyzes the entire genome for CNAs by comparing sample DNA with reference DNA.

[0053] In some embodiments, a targeted hematologic malignancy NGS panel analysis is used to select iPSC clones that did not develop hematologic malignancy mutations during reprogramming. For example, a targeted hematologic malignancy NGS panel focuses on genes associated with myeloid leukemia, lymphoma, and / or other hematologic malignancies, generating smaller, more manageable datasets than more broad methods. A targeted hematologic malignancy NGS panel analysis involves the use of highly multiplexed PCR to amplify regions associated with hematologic malignancies, followed by next-generation sequencing.

[0054] In some embodiments, droplet digital PCR (ddPCR) is used to select iPSC clones that have not integrated the episomal vector and have been passaged sufficiently for episomal vector clearance. As discussed herein, iPSC reprogramming of CD34+ cells can be achieved by delivering episomal vectors encoding reprogramming factors. However, episomal vectors can randomly integrate into the cellular genome, although this is rare, which can disrupt developmental processes, homeostasis, and the like. Therefore, ddPCR can be used to detect residual episomal vectors in iPSC cultures, allowing for the selection of iPSC clones that have not integrated the episomal vector.

[0055] In some embodiments, after assessing that selected clones do not contain editing-related genomic abnormalities, the clones can be further tested for spontaneous mutations that may arise during expansion, such as mutations affecting hematologic malignancies, indels, translocations, and numerical abnormalities, as described for pre-edited reprogrammed clones. Analysis of spontaneous mutations can include whole genome sequencing (WGS), KARYOSTAT analysis, array comparative genomic hybridization (aCGH) analysis, targeted hematologic malignancies NGS panel AMP-Seq analysis, and / or droplet digital PCR (ddPCR).

[0056] Somatic cells can be reprogrammed by expression of reprogramming factors selected from Sox2, Oct3 / 4, c-Myc, Nanog, Lin28, and klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc, Nanog, Lin28, and klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc, and klf4. Methods for preparing iPSCs are described, for example, in U.S. Pat. Nos. 10,676,165, 9,580,689, and 9,376,664, which are incorporated by reference in their entireties. In various embodiments, reprogramming factors are expressed using well-known viral vector systems, such as lentivirus, Sendai, or measles virus systems. Alternatively, reprogramming factors can be expressed by introducing mRNA(s) encoding the reprogramming factors into somatic cells. Furthermore, iPSCs can be generated by introducing non-integrating episomal plasmids expressing reprogramming factors, i.e., for the generation of transgene-free and virus-free iPSCs. Known episomal plasmids that have limited replicative capacity and are therefore lost over several cell generations can be employed.

[0057] In some embodiments, the iPSCs are derived from T cells, e.g., with known or unknown TCR specificity. In some embodiments, the T cells have specificity for a cancer- or tumor-associated antigen, and in some embodiments, have a TCR that includes a cancer- or tumor-associated antigen whose expression is deleted or reduced in the iPSCs. For example, the iPSCs may be derived from CD3 +T-iPSCs can be prepared from cells, or in some embodiments, from T lymphocytes (e.g., CTLs). For example, T lymphocytes can be isolated with a desired antigen specificity (e.g., using cell sorting with HLA peptide ligands) and reprogrammed into T-iPSCs. These T-iPSCs are then redifferentiated into HSCs, or optionally into progenitor T cells or T cell lineages. When T-iPSCs are generated from antigen-specific T cells, they inherit rearranged T cell receptor (TCR) genes. In these embodiments, CTLs derived from HSCs exhibit the same antigen specificity as the original antigen-specific T cells.

[0058] In some embodiments, iPSCs can be further engineered by inserting at least one sequence encoding a transgene operably linked to an endogenous or exogenous promoter, where the transgene is inserted into a genomic safe harbor locus. A genomic safe harbor (GSH) locus refers to a locus that accommodates the insertion of exogenous DNA with either constitutive or conditional expression activity without significantly affecting the viability and ontogeny of somatic, progenitor, or germline cells. Well-known safe harbor loci include the AAVS1 adeno-associated virus insertion site on chromosome 19, the human homolog of the mouse Rosa26 locus, and the CCR5 chemokine receptor gene. Tools and techniques for inserting transgenes (i.e., exogenous DNA) into safe harbor loci are well known to those skilled in the art; see, for example, Papapetrou EP et al. Gene Insertion Into Genomic Safe Harbors for Human Gene Therapy. Mol Ther. (2016) 678-84.

[0059] Episomes are exogenous DNA that remain physically independent of a cell's endogenous chromosomes or chromosomal complement. Depending on their content and context, episomal DNA can replicate or synthesize messenger RNA (and indirectly proteins), thus conferring novel biological properties to the cell. Episomal concatemers derived from adeno-associated virus (AAV) vectors are considered non-replicating. Episomal iPSC reprogramming vectors or enhanced episomal vectors (EEVs) can be employed for non-integrating, non-viral gene expression. Because they replicate synchronously with the host cell, they are stably inherited and can be used for long-term expression of up to several months without modifying the host genome.

[0060] Alternatively, gene delivery systems may use non-viral delivery, using physical (carrier-free gene delivery) and chemical approaches (synthetic vector-based gene delivery). Such delivery systems are well known to those skilled in the art; see, for example, Zu, H., et al., Non-viral Vectors in Gene Therapy: Recent Development, Challenges, and Prospects. AAPS J 23, 78 (2021), which is incorporated herein by reference in its entirety.

[0061] In various embodiments, iPSCs are prepared and expanded using a culture system. The expanded iPSCs can be recovered from the culture for differentiation into embryoid bodies (EBs). EBs generated by differentiation of iPSCs are three-dimensional aggregates of iPSCs and contain three (or alternatively, two or one) embryonic germ cell layers depending on the differentiation method(s). EB preparation is described, for example, in US2019 / 0177695, which is incorporated herein by reference in its entirety. In some embodiments, EBs prepared by differentiation of iPSCs are expanded in a bioreactor, as described, for example, in Abecasis B. et al., "Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches." J. of Biotechnol. 246 (2017) 81-93. EBs can be used to generate any desired cell type. Other methods involving 3D suspension culture for the growth or differentiation of EBs are described in WO2020 / 086889, which is incorporated herein by reference in its entirety.

[0062] In some embodiments, the process for producing a population containing HSCs and / or HSPCs can include generating CD34+ enriched cells from pluripotent stem cells (e.g., EBs) and inducing endothelial and hematopoietic differentiation. HSCs containing a relatively high frequency of LT-HSCs can be generated from cell populations using a variety of stimuli or factors, including mechanical, biochemical, metabolic, and / or local stimuli, as well as factors such as extracellular matrix, niche factors, and cell-extrinsic factors, induction of cell-intrinsic properties, and including pharmacological and / or genetic means.

[0063] In some embodiments, the method includes preparing hematopoietic endothelial cells from iPSCs prior to induction of EHT. In some embodiments, combined overexpression of GATA2 / ETV2, GATA2 / TAL1, or ER71 / GATA2 / SCL can lead to the formation of hematopoietic endothelial cells from PSC sources. In some embodiments, the method includes overexpression of the E26 transformation-specific variant 2 (ETV2) transcription factor in iPSCs. After CD34+ enrichment, HSCs are then generated from the endothelial cells using mechanical, biochemical, pharmacological, and / or genetic stimulation or modification. ETV2 can be expressed by introduction of an encoding non-integrating episomal plasmid for constitutive or inducible expression of ETV2 and for the production of transgene-free hematopoietic ECs. In some embodiments, ETV2 is expressed from mRNA introduced into iPSCs. The mRNA can be introduced using any available method, including electroporation or lipofection. Differentiation of ETV2-expressing cells can include the addition of VEGF-A. See Wang K, et al., Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with mRNA. Sci. Adv. Vol. 6 (2020). Cells generated in this manner can be used to produce CD34+ cells and induce EHT according to embodiments of the present disclosure.

[0064] In embodiments, endothelial-to-hematopoietic transition (EHT) is induced between days 7 and 15 of iPSC differentiation. In various embodiments, the CD34+ population undergoing EHT can be harvested, i.e., separated from other cells. In some embodiments, endothelial-to-hematopoietic transition (EHT) generates a population of HSCs that includes one or more of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells, and hematopoietic stem and progenitor cells.

[0065] Traditionally, hematopoietic stem cells or hematopoietic lineages are prepared by differentiating iPSCs into embryoid bodies up to day 8 and harvesting CD34+ cells. CD34 is commonly used as a marker for hemogenic endothelial cells, hematopoietic stem cells, and hematopoietic progenitor cells. According to aspects and embodiments of the present disclosure, it has been discovered that inducing endothelial-to-hematopoietic transition (EHT) (e.g., over at least 2 days but not more than 12 days) of a CD34+ cell population that can be derived from iPSC embryoid bodies can be used for the ex vivo generation of superior HSCs and hematopoietic lineages.

[0066] In some embodiments, iPSC differentiation proceeds until the cells are at least about 10% CD34+, or at least about 20% CD34+, or at least about 25% CD34+, or at least about 30% CD34+. In some embodiments, CD34+ enrichment and EHT can be induced on days 8-14 of iPSC differentiation, such as on days 8, 9, 10, 11, 12, 13, or 14. Differentiation of iPSCs can be by known techniques. In some embodiments, iPSC differentiation involves factors such as, but not limited to, combinations of bFGF, Y27632, BMP4, VEGF, SCF, EPO, TPO, IL-6, IL-11, and / or IGF-1. In some embodiments, hPSCs are differentiated feeder-free, serum-free, and / or using GMP-compatible materials. In some embodiments, hPSCs are co-cultured with mouse bone marrow-derived feeder cells, such as the OP9 or MS5 cell line, in serum-containing medium. The culture can contain growth factors and cytokines to support differentiation into embryoid bodies or monolayers. The OP9 co-culture system can be used to generate multipotent HSPCs, which can optionally further differentiate into several hematopoietic lineages, including T lymphocytes, B lymphocytes, megakaryocytes, monocytes or macrophages, and erythrocytes. See Netsrithong R. et al., Multilineage differentiation potential of hematoendothelial progenitors derived from human induced pluripotent stem cells, Stem Cell Research & Therapy Vol. 11 Art. 481 (2020). Alternatively, a stepwise process using defined conditions in conjunction with specific signals can be used. For example, the expression of HOXA9, ERG, RORA, SOX4, and MYB in human PSCs favors direct differentiation into multipotent CD34+ / CD45+ progenitors. Furthermore, expression of factors such as HOXB4, CDX4, SCL / TAL1, or RUNX1a supports the hematopoietic program in human PSCs.See Doulatov S. et al., Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via re-specification of lineage-restricted precursors, Cell Stem Cell. 2013 Oct 3;13(4).

[0067] When a cell population is described herein as having a particular phenotype, it is understood that the phenotype represents a substantial portion of the cell population, such as at least 25%, at least 40%, or at least about 50%, or at least about 60%, or at least about 75%, or at least about 80%, or at least about 90% of the cell population. Furthermore, in various steps, the cell population can be enriched for cells of the desired phenotype and / or depleted of cells of an undesired phenotype, such that the cell population contains at least about 75%, or at least about 80%, or at least about 90% of the desired phenotype. Such positive and negative selection methods are known in the art. For example, cells can be sorted based on cell surface antigens (including those described herein) using a fluorescence-activated cell sorter or magnetic beads that bind to certain cell surface antigens. Negative selection columns can be used to remove cells expressing undesirable cell surface markers. In some embodiments, cells are enriched for CD34+ cells (before and / or after undergoing EHT). In some embodiments, the cell population is cultured under conditions that promote the expansion of CD34+ cells, thereby producing an expanded population of stem cells.

[0068] In various embodiments, inducing endothelial-to-hematopoietic transition (EHT) comprises increasing the expression or activity of dnmt3b. For example, inducing endothelial-to-hematopoietic transition (EHT) can comprise applying cyclic stretch to iPSCs or cells derived from iPSCs, such as CD34+ cells, endothelial cells (ECs), and hemogenic endothelial cells (HECs). In embodiments, the cyclic stretch is 2D, 3D, or 4D cyclic stretch.

[0069] For example, a cell population can be introduced into a bioreactor that provides cyclic strain biomechanical stretching, as described in U.S. Patent No. 11,162,073, incorporated herein by reference in its entirety. Cyclic strain biomechanical stretching can increase the activity or expression of Dnmt3b and / or Gimap6. In these embodiments, the mechanical stretching means applies a stretching force to the cells or to a cell culture surface having cells (e.g., ECs or HECs) cultured thereon. For example, a computer-controlled vacuum pump system or other means for providing a stretching force (e.g., the FlexCell™ Tension System, CytoStretcher System) attached to a flexible, biocompatible and / or biomimetic surface can be used to apply cyclic 2D, 3D, or 4D stretching to cells ex vivo under defined and controlled cyclic strain conditions. For example, the applied cyclic stretch can be about 1% to about 20% cyclic strain (e.g., about 6% cyclic strain) for several hours or days (e.g., about 7 days). In various embodiments, the cyclic strain is applied for at least about 1 hour, at least about 2 hours, at least about 6 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 120 hours, at least about 144 hours, or at least about 168 hours.

[0070] Induction of EHT can be by any known process. In some embodiments, induction of EHT generates an HSC population including LT-HSCs. In some embodiments, EHT generates a cell population including HSPCs. In some embodiments, EHT generates HSCs and / or HSPCs through endothelial or hemogenic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological, and / or genetic means (e.g., via stimulation, inhibition, and / or genetic modification). In some embodiments, EHT generates a stem cell population including one or more of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs), and HSPCs. In various embodiments, EHT can be induced in culture for 2 to 12 days, such as about 4 to about 8 days (e.g., about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days). In some embodiments, EHT is induced in culture for about 5 to about 7 days.

[0071] In various embodiments, CD34+ cells (e.g., suspension and / or adherent cells) are harvested from cultures undergoing endothelial-hematopoietic transition between days 10 and 20 of iPSC differentiation, such as between days 10 and 17 or between days 12 and 15 of iPSC differentiation. In some embodiments, non-adherent cells are collected.

[0072] In some embodiments, HSC and / or HSPC populations or fractions thereof are differentiated into T cells or their progenitor or derivative cells, with or without the use of an agonist of a mechanosensitive receptor or mechanosensitive channel, such as Yoda1. In some embodiments, the use of an agonist of a mechanosensitive receptor or mechanosensitive channel (e.g., Yoda1) is optional. Thus, in some embodiments, CD34+ cells are enriched from a differentiated pluripotent stem cell population to prepare a CD34+ enriched population. Endothelial-hematopoietic transformation of the CD34+ enriched cell population is induced for at least 2 days but not more than 12 days, optionally with the use of an agonist of a mechanosensitive receptor or mechanosensitive channel, such as Yoda1, jedi1, jedi2, or ssRNA40. HSC and / or HSPC are differentiated (e.g., as described herein) into a progenitor T cell population or a T cell population. In some embodiments, endothelial-to-hematopoietic transformation of the CD34+ enriched cell population is induced for at least 2 days, optionally for an additional period of about 4 hours, or about 8 hours, or about 12 hours, or about 16 hours, or about 20 hours, or about 24 hours, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days. Full EHT differentiation proceeds over 12 days or less.

[0073] In embodiments, the induction of endothelial-hematopoietic transition (EHT) comprises Piezo1 activation. In embodiments, Piezo1 activation is by contacting iPSCs or cells derived from iPSCs with one or more Piezo1 agonists, optionally selected from Yodal, Jedil, Jedi2, or analogs or derivatives thereof. For example, the cells are contacted with an effective amount of an agonist of a mechanosensitive receptor or mechanosensitive channel (e.g., a Piezo1 agonist) that increases the activity or expression of Dnmt3b.

[0074] Yoda1 (2-[5-[[(2,6-dichlorophenyl)methyl]thio]-l,3,4-thiadiazol-2-yl]-pyrazine) is a small molecule agonist developed for the mechanosensitive ion channel Piezol. Syeda R, Chemical activation of the mechanotransduction channel Piezol. eLife (2015). Yoda1 has the following structure: [ka]

[0075] Derivatives of Yodal can be employed in various embodiments. For example, derivatives containing a 2,6-dichlorophenyl core are employed in some embodiments. Exemplary agonists are disclosed in Evans EL, et al., "Yoda1 analogue (Dooku1) which antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation," British Journal of Pharmacology 175(1744-1759):2018. Still other Piezo1 agonists include Jedi1, Jedi2, single-stranded (ss) RNA (e.g., ssRNA40), and derivatives and analogs thereof. See Wang Y., et al., "A lever-like transduction pathway for long-distance chemical- and mechano-gating of the mechanosensitive Piezo1 channel," Nature Communications (2018) 9:1300; and Sugisawa, et al., "RNA Sensing by Gut Piezo1 Is Essential for Systemic Serotonin Synthesis," Cell, Volume 182, Issue 3, 2020, Pages 609-624, which are incorporated herein by reference in their entireties. These Piezo1 agonists are commercially available. In various embodiments, the effective amount of Piezo1 agonist or derivative is in the range of about 1 μM to about 500 μM, or about 5 μM to about 200 μM, or about 5 μM to about 100 μM, or in some embodiments, about 25 μM to about 150 μM, or about 25 μM to about 100 μM, or about 25 μM to about 50 μM. Alternatively, single-stranded (ss) RNA (e.g., ssRNA) and derivatives and analogs thereof can be used for Piezo1 activation.

[0076] Alternatively, or in addition, EHT is stimulated by Trpv4 activation. Trpv4 activation can be by contacting cells (e.g., CD34-enriched cells, ECs, or HECs) with one or more Trpv4 agonists, optionally selected from GSK1016790A, 4α-PDD, or analogs and / or derivatives thereof. In embodiments, Trpv4 activation is by contacting iPSCs or cells derived from the iPSCs with one or more Trpv4 agonists, optionally selected from GSK1016790A, 4α-PDD, or analogs or derivatives thereof.

[0077] In various embodiments, pharmacological Piezo1 activation is applied to CD34+ cells (i.e., CD34+ enriched cells). In certain embodiments, pharmacological Piezo1 activation may be further applied to iPSCs, embryoid bodies (EBs), endothelial cells (ECs), hemogenic endothelial cells (HECs), HSCs, hematopoietic progenitor cells, and hematopoietic lineage(s). In certain embodiments, Piezo1 activation is applied to at least EBs generated from iPSCs, CD34+ cells isolated from EBs, and / or combinations thereof, which, according to various embodiments, allows for superior generation of HSCs compared to other methods for inducing EHT.

[0078] Alternatively, or in addition, Dnmt3b activity or expression can be increased directly in cells, for example, in CD34+ enriched cells. For example, Dnmt3b mRNA expression can be increased by, but not limited to, delivering a transcript encoding Dnmt3b to cells, or by introducing a transgene encoding Dnmt3b, or by transgene-free methods such as introducing a non-integrating episome into cells. In some embodiments, gene editing is employed to introduce genetic modifications into Dnmt3b-expressing elements in cells, such as, but not limited to, increasing promoter strength, ribosome binding, RNA stability, and / or affecting RNA splicing.

[0079] In some embodiments, the method includes increasing the activity or expression of Gimap6 in a cell, alone or in combination with Dnmt3b and / or other genes that are up- or down-regulated upon cyclic strain or Piezol activation. To increase the activity or expression of Gimap6, an mRNA transcript encoding Gimap6 can be introduced into the cell; a transgene-free approach can be employed, including, but not limited to, introducing an episome into the cell; or, alternatively, a transgene encoding Gimap6 can be introduced. In some embodiments, gene editing is employed to introduce genetic modifications (such as one or more modifications to increase promoter strength, ribosome binding, RNA stability, or to affect RNA splicing) into a Gimap6-expressing element in the cell.

[0080] In embodiments of the present disclosure employing mRNA delivery to cells, known chemical modifications can be used to circumvent the innate immune response in cells. For example, synthetic RNAs containing only canonical nucleotides can bind to pattern recognition receptors and induce a strong immune response in cells. This response can result in translation block, secretion of inflammatory cytokines, and cell death. RNAs containing certain non-canonical nucleotides can avoid detection by the innate immune system and can be translated into proteins with high efficiency. See US Pat. No. 9,181,319, incorporated herein by reference, particularly for nucleotide modifications to circumvent the innate immune response.

[0081] In some embodiments, expression of Dnmt3b and / or Gimap6 is increased by introducing a transgene into cells, which can direct the desired level of overexpression (with varying promoter strength or other selection of expression control elements). Various viral vectors or transfection reagents (including lipid nanoparticles) known in the art can be used to introduce the transgene. In some embodiments, expression of Dnmt3b and / or Gimap6 is increased by transgene-free methods (e.g., episomal delivery). In some embodiments, expression or activity of Dnmt3b and / or Gimap6, or other genes disclosed herein, is increased using gene editing techniques, for example, to introduce one or more modifications to increase promoter strength, ribosome binding, or RNA stability.

[0082] In yet another embodiment, CD34+ enriched cells are cultured with an inhibitor of histone methyltransferase EZH1 during EHT. Alternatively, EZH1 is partially or completely deleted or inactivated, or transiently silenced, in the stem cell population. See WO2018 / 048828, which is incorporated herein by reference in its entirety.

[0083] In various embodiments, CD34+ cells (e.g., suspension and / or adherent cells) are harvested from cultures undergoing endothelial-hematopoietic transition between days 8 and 15 of iPSC differentiation. In some embodiments, CD34+ suspension cells are harvested between days 8 and 15 of iPSC differentiation.

[0084] In various embodiments, the HSCs or CD34+ enriched cells are further expanded. For example, the HSCs or CD34+ enriched cells can be expanded according to the methods disclosed in US8,168,428, US9,028,811, US10,272,110, and US10,278,990, which are incorporated by reference in their entireties. In some embodiments, the ex vivo expansion of the HSCs or CD34+ enriched cells employs prostaglandin E2 (PGE2) or a PGE2 derivative. In some embodiments of the present disclosure, the HSCs comprise at least about 0.01% LT-HSCs, or at least about 0.05% LT-HSCs, or at least about 0.1% LT-HSCs, or at least about 0.5% LT-HSCs, or at least about 1% LT-HSCs.

[0085] Hematopoietic stem cells (HSCs), which give rise to erythroid, myeloid, and lymphoid lineages, can be identified based on the expression of CD34 and the absence of lineage-specific markers (referred to as Lin-). In some embodiments, a population of stem cells containing HSCs is enriched, for example, as described in US9,834,754, which is incorporated herein by reference in its entirety. For example, this process can include sorting a cell population based on the expression of one or more of CD34, CD90, CD38, and CD43. CD34 + , CD90 + , CD38 - , and CD43 - In some embodiments, a stem cell population for differentiation into hematopoietic lineages is selected for further differentiation from a fraction that is at least about 80% CD34 + , or at least about 90% CD34 + , or at least about 95% CD34 + is.

[0086] In some embodiments, stem cell populations, or CD34+ enriched cell or fraction thereof, or derivative populations are expanded as described in US 2020 / 0308540, which is incorporated herein by reference in its entirety. For example, the cells are expanded by exposing them to an aryl hydrocarbon receptor antagonist, including, for example, SR1 or an SR1 derivative. See also Wagner et al., Cell Stem Cell 2016;18(1):144-55, and Boitano A., et al., Aryl Hydrocarbon Receptor Antagonists Promote the Expansion of Human Hematopoietic Stem Cells. Science 2010 Sep 10;329(5997):1345-1348.

[0087] In some embodiments, CD34 + Compounds that promote cell proliferation include pyrimidoindole derivatives, including, for example, UM171 or UM729 (see US2020 / 0308540, incorporated herein by reference).

[0088] In some embodiments, the stem cell population or CD34+ enriched cells are further enriched for or modified to express periostin and / or platelet-derived growth factor receptor alpha (pdgfra), as described in WO2020 / 205969 (incorporated herein by reference in its entirety). Such expression can be by transgene-free methods, including but not limited to, by delivering an encoding transcript to the cells, by introducing an encoding transgene, or by introducing a non-integrating episome into the cells. In some embodiments, gene editing is employed to introduce genetic modifications into expression elements in the cells, such as to modify promoter activity or strength, ribosome binding, RNA stability, or affect RNA splicing.

[0089] In embodiments, HSCs are further differentiated into hematopoietic lineages for therapy. In embodiments, the hematopoietic lineages are selected from progenitor T cells, T lymphocytes, and natural killer cells. Other lineages that can be produced and used for therapy include common myeloid progenitors (CMPs) or common lymphoid progenitors (CLPs). CMPs give rise to progeny such as red blood cells / erythrocytes, platelets, mast cells, osteoclasts, granulocytes, monocytes-macrophages, and dendritic cells. CLPs give rise to progeny such as T cells / T lymphocytes, B cells / B lymphocytes, NK cells / natural killer cells, and dendritic cells.

[0090] For example, the HSC cell population or cells obtained therefrom may be characterized as CD7 + They can be cultured ex vivo with Notch ligand (partial or complete), SHH, extracellular matrix component(s), and / or combinations thereof to differentiate into precursor T cells and, optionally, into the T cell lineage or other lineages (e.g., NK cells).

[0091] As used herein, the term "Notch ligand" refers to a ligand capable of binding to a Notch receptor polypeptide present on the membrane of hematopoietic stem cells or progenitor T cells. Notch receptors include Notch-1, Notch-2, Notch-3, and Notch-4. Notch ligands typically have a DSL domain (D-Delta, S-Serrate, and L-Lag2) containing 20-22 amino acids at the amino terminus and 3-8 EGF repeats on the extracellular surface. In various embodiments, the Notch ligand comprises at least one of Delta-like 1 (DLL1), Delta-like 4 (DLL4), SFIP3, or a functional portion thereof. In vivo, a key signal delivered by thymic stromal cells to invading lymphoid progenitors is mediated by DL4, which is expressed by cortical thymic epithelial cells.

[0092] In some embodiments, the Notch ligand is DLL4 with one or more affinity-enhancing mutations, such as one or more (or all) of the following: for hDLL4, G28S, F107L, I143F, H194Y, L206P, N257P, T271L, F280Y, S301R, and Q305P. See Gonzalez-Perez, et al., Affinity-matured DLL4 ligands as broad-spectrum modulators of Notch signaling, Nature Chemical Biology (2022).

[0093] The earliest intrathymic progenitor cells express high levels of CD34 and CD7, do not express CD1a, and are triple negative (TN) for mature T cell markers: CD4, CD8, and CD3. Commitment to the T cell lineage is associated with expression of CD1a by CD7-expressing prothymocytes. Thus, immature stages of T cell development are typically characterized by CD34 + CD1a - (mostly immature) and CD34 + CD1a + Described as cells. CD34 by early thymocytes + CD7 + CD1a - from CD34 + CD7 + CD1a + Conversion to CD34 is associated with T cell involvement. + CD7 + CD1a + Following this stage, thymocytes progress to a CD4 immature mono-positive stage, at which point CD4 is expressed in the absence of CD8. A subset of cells then expresses CD4 + CD8 + Finally, following TCRα rearrangement, TCRαβ-expressing DP thymocytes undergo positive and negative selection and differentiate to the CD4 + CD8 - and CD4 - CD8 + Generate single positive (SP) T cells.

[0094] In some embodiments, progenitor T cells are isolated by enrichment for CD7 expression. In some embodiments, progenitor T cells are expanded as described in US2020 / 0308540, which is incorporated herein by reference in its entirety. For example, cells can be expanded by exposing them to an aryl hydrocarbon receptor antagonist, including, for example, SR1 or an SR1 derivative. See also Wagner et al., Cell Stem Cell 2016;18(1):144-55. In some embodiments, the proliferation-promoting compound comprises a pyrimidoindole derivative, including, for example, UM171 or UM729 (see US2020 / 0308540, which is incorporated herein by reference).

[0095] Differentiation into progenitor T cells can, in some embodiments, further include the presence of stem cell factor (SCF), Flt3L, and interleukin (IL)-7. In various embodiments, the generated CD7+ progenitor T cells express CD1a. The CD7+ progenitor T cells do not express CD34 or express reduced levels of CD34 compared to the HSC population. In some embodiments, the CD7+ progenitor T cells (or a portion thereof) further express CD5. Thus, the phenotype of the progenitor T cells is CD7. + CD1a + In some embodiments, the phenotype of the precursor T cells may be CD7 + CD5 + In some embodiments, the progenitor T cells are CD7 + CD1a + CD5 + , optionally CD34 + is.

[0096] In some embodiments, the Notch ligand is an anti-Notch (agonist) antibody that can bind to and participate in Notch signaling. In some embodiments, the antibody is a monoclonal antibody (including a human or humanized antibody), a single-chain antibody (scFv), a nanobody, or other antibody fragment or antigen-binding molecule that can activate the Notch signaling pathway.

[0097] In some embodiments, the Notch ligand is a Delta family Notch ligand. In some embodiments, the Delta family ligand is Delta-1 (Genbank Accession No. AF003522, Homo sapiens), Delta-like 1 (DLL1, Genbank Accession Nos. NM_005618 and NP_005609, Homo sapiens; Genbank Accession Nos. X80903, 148324, M. musculus), Delta-4 (Genbank Accession No. AF273454, BAB18580, Mus musculus; Genbank Accession Nos. AF279305, AAF81912, Homo sapiens), and / or Delta-like 4 (DLL4, Genbank Accession Nos. Q9NR61, AAF76427, AF253468, NM_019074, Homo sapiens; Genbank Accession No. NM 019454, Mus musculus). Notch ligands are commercially available or can be produced, for example, by recombinant DNA techniques.

[0098] In various embodiments, the Notch ligand is soluble and optionally immobilized on microparticles or nanoparticles that are optionally paramagnetic to enable magnetic enrichment or concentration processes. In yet other embodiments, the Notch ligand is immobilized on a 2D or 3D culture surface, optionally using other adhesion molecules such as VCAM-1. See US 2020 / 0399599, incorporated herein by reference in its entirety. In other embodiments, the beads or particles are constructed of biological materials, such as polymers (e.g., polystyrene or PLGA), gold, iron dextran, or particles formed from lipids and / or proteins. In various embodiments, the particles have a diameter or largest dimension of about 0.01 μm (10 nm) to about 500 μm (e.g., about 1 μm to about 7 μm). In still other embodiments, polymer scaffolds with conjugated ligands can be employed, as described in WO 2020 / 131582, incorporated herein by reference in its entirety. For example, the scaffold can be constructed from polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivatives, gelatin, collagen, agarose, hyaluronic acid, poly(lysine), polyhydroxybutyrate, poly-epsilon-caprolactone, polyphosphazine, poly(vinyl alcohol), poly(alkylene oxide), poly(ethylene oxide), poly(allylamine), poly(acrylate), poly(4-aminomethylstyrene), Pluronic® polyol, poloxamer, poly(uronic acid), poly(anhydride), poly(vinylpyrrolidone), and any combination thereof. In some embodiments, the scaffold comprises pores having diameters of about 1 pm to 100 pm.

[0099] In some embodiments, the C-terminus of the Notch ligand is conjugated to a support of choice. In some embodiments, this can involve adding a sequence to the C-terminus of the Notch ligand that can be enzymatically conjugated to the support, for example, via a biotin molecule. In another embodiment, the Notch ligand-Fc fusion is prepared such that the Fc segment can be immobilized by binding to Protein A or Protein G that has been conjugated to a support. Of course, any of the known protein conjugation methods can be employed.

[0100] Thus, in various embodiments, the Notch ligand can be immobilized, functionalized, and / or embedded in a 2D or 3D culture system. The Notch ligand can be incorporated with components of an extracellular matrix, such as one or more selected from fibronectin, retronectin, and laminin. In some embodiments, the Notch ligand and / or components of the extracellular matrix are embedded in an inert material that provides 3D culture conditions. Exemplary materials include, but are not limited to, cellulose, alginate, and combinations thereof. In some embodiments, the Notch ligand, components of the extracellular matrix, or combinations thereof, are in contact with culture conditions that provide cells with a topographical pattern and / or texture (e.g., roughness) that promotes differentiation and / or proliferation.

[0101] In some embodiments, populations comprising HSCs and / or HSPCs are differentiated into precursor T cells by culturing in medium containing TNF-α and / or an antagonist of the aryl hydrocarbon / dioxin receptor (SR1) and in the presence of a Notch ligand. See US2020 / 0390817, US2021 / 0169934, and US2021 / 0169935, which are incorporated by reference in their entireties. In some embodiments, HSCs are cultured in medium containing TNF-α, IL-7, thrombopoietin (TPO), Flt3L, and stem cell factor (SCF), and optionally SR1, in the presence of immobilized delta-like-4 ligand and fibronectin fragments. In some embodiments, cells are cultured with retronectin, a recombinant human fibronectin containing three functional domains: a human fibronectin cell-binding domain (C domain), a heparin-binding domain (H domain), and a CS-1 sequence domain. In some embodiments, cells are cultured in the presence of immobilized delta-like-4 ligand and retronectin. In some embodiments, cells are cultured in the presence of immobilized delta-like-4 ligand, TNF-α, and retronectin. In some embodiments, cells are cultured in the presence of immobilized delta-like-1 ligand and retronectin. In some embodiments, cells are cultured in the presence of SFIP3 and retronectin. In some embodiments, cells are cultured in the presence of immobilized delta-like-4 ligand and an SHH molecule and / or a functional derivative thereof. Exemplary fibronectin fragments include one or more of RGDS, CS-1, and heparin-binding motifs. The fibronectin fragments can be free in solution or immobilized on a culture surface or particles. In some embodiments, cells are cultured for 5 to 7 days to prepare CD7+ progenitor T cells.

[0102] In various embodiments, the method produces precursor T cells or T cell lineages by culturing a population of HSCs with a Notch ligand (including any of the embodiments described above), with or without a component(s) of the extracellular matrix, and optionally adding TNF-α to the culture at a particular stage of differentiation. Thus, in some embodiments, the cells produced are progenitor or precursor cells committed to the T cell lineage ("precursor T cells"). In some embodiments, the cells are CD7 + In some embodiments, the cells are CD25 + Immature T cells or cells that have undergone CD4 or CD8 lineage commitment. In some embodiments, the cells are CD4 + CD8 + Double positive (DP), CD4 - CD8 + , or CD4 + CD8 - In some embodiments, the cells are CD4 - CD8 + or CD4 + CD8 - and TCR hi In some embodiments, the cells are single positive (SP) cells that are TCRαβ + and / or TCRγΔ + In various embodiments, the cells are CD3 + is.

[0103] Adoptive transfer of precursor T cells is a strategy for enhancing T cell reconstitution. Precursor T cells are developmentally immature and undergo positive and negative selection in the host thymus. Thus, they become restricted to the recipient's major histocompatibility complex (MHC), giving rise to host-tolerant T cells that can circumvent the clinical challenges associated with graft-versus-host disease (GVHD). Importantly, engraftment with precursor T cells restores thymic architecture and improves subsequent thymic seeding by HSC-derived progenitor cells. In addition to their inherent regenerative medicine properties, precursor T cells can also be engineered (either via gene or mRNA delivery) with T cell receptors (TCRs) and chimeric antigen receptors (CARs) to confer specificity for tumor-associated antigens.

[0104] In various embodiments, the precursor T cells are further cultured under suitable conditions to generate cells of the desired T cell lineage containing one or more Notch ligands. For example, the cells can be cultured in the presence of one or more Notch ligands as described for a period of time sufficient to form cells of the T cell lineage. In some embodiments, stem cells or precursor T cells are cultured in suspension with soluble Notch ligand, or Notch ligand conjugated to particles or other supports, or Notch ligand-expressing cells. In some embodiments, precursor T cells or stem cells are cultured in suspension or with soluble or conjugated Notch ligand in suspension in an adherent form in a bioreactor, optionally a closed or automated closed bioreactor. One or more cytokines, extracellular matrix component(s), and thymic niche factor(s) that promote commitment and differentiation to the desired T cell lineage can also be added to the culture or reactor. Such cytokines or factors are known in the art. In various embodiments, the population comprising HSCs and / or HSPCs is cultured with a Notch ligand for about 4 to about 21 days, or about 6 to about 18 days, or about 7 to about 14 days to generate progenitor T cells. In some embodiments, the stem cell population or derivatives thereof is cultured for at least about 21 days, or at least about 28 days to generate mature T cell lineages or NK cells.

[0105] In various embodiments, the HSC population is cultured in artificial thymic organoids (ATOs). See Hagen, M. et al. (2019). ATOs involve culturing HSCs (or aggregates of HSCs) with Notch ligand-expressing stromal cell lines under serum-free conditions. Artificial thymic organoids are composed of naive CD3 + CD8 + and CD3 + CD4 + It is a 3D system that induces differentiation of hematopoietic progenitor cells into T cells.

[0106] In various embodiments, the methods include generating derivatives of progenitor T cells or generating T cell lineages from progenitor T cells. In certain embodiments, the progenitor T cells or derivatives of the T cell lineage express CD3 and a T cell receptor. In some embodiments, the T cell lineage expresses CD8 + and / or CD4 + For example, the T cell lineage is CD8 + CD4 - , CD8 - CD4 + , CD8 + CD4 + , and CD8 - CD4 - In some embodiments, iPSCs, CD34+ cells, or derivatives thereof are modified to express a chimeric antigen receptor (CAR) at the precursor T, T cell, and / or NK cell level.

[0107] In some embodiments, the derivative of the progenitor T cells is a natural killer (NK) cell. In some embodiments, the NK cell is generated from a progenitor T cell as described in US 10,266,805, which is incorporated herein by reference in its entirety. For example, the progenitor T cell can give rise to an NK cell when cultured with IL-15. In some embodiments, the NK cell expresses a CAR based on gene editing of iPSCs, embryonic bodies, hCD34+ cells, or NK cells, or via mRNA expression in the NK cell.

[0108] In some aspects and embodiments, the modified HSCs replace normal cells destroyed by antibodies, T cell therapy (e.g., CAR-T therapy), or NK cell therapy (e.g., CAR-NK). HSCs and their progenitor cells help the bone marrow recover and generate healthy cells, e.g., myeloid cells, in AML treatment. HSC rescue allows more targeted therapy to be given to patients so that more cancer cells are killed.

[0109] In various embodiments, the modified HSCs / HSPCs of the present disclosure may be used to treat or ameliorate a disease or disorder, such as treating a hematopoietic malignancy. Non-limiting examples of hematopoietic malignancies include cytogenetically normal acute myeloid leukemia (CN-AML), acute myeloid leukemia (AML), acute lymphocytic leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia or chronic lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, or refractory or relapsed forms thereof. In some embodiments, the subject has received, is receiving, or will receive a targeted therapy, e.g., a therapy that targets hematopoietic cells (or their lineages).

[0110] In embodiments, administration of HSCs and / or HSPCs is performed following a myeloablative, non-myeloablative, or immunotoxin-based (eg, anti-c-Kit, anti-CD45, etc.) conditioning regimen.

[0111] In various embodiments, the modified HSCs and / or HSPCs (or cell lineages derived therefrom) of the present disclosure are administered in conjunction with the therapeutic use of CAR-T therapy. Generally, the CAR-T cells target a cancer antigen that has reduced or eliminated expression in the modified HSCs. Exemplary CAR-T cells include CD33-specific CAR-T cells, CD7-specific CAR-T cells, CD8-specific CAR-T cells, CD19-specific CAR-T cells, CD20-specific CAR-T cells, CD22-specific CAR-T cells, CD123-specific CAR-T cells, CD125-specific CAR-T cells, CD133-specific CAR-T cells, and CD371-specific CAR-T cells. In some embodiments, the HSCs of the present disclosure are administered in conjunction with CAR-NK cell therapy.

[0112] In various embodiments, HSCs and / or HSPCs or cell lines derived therefrom (e.g., T cells or NK cells, including CAR-T cells or CAR-NK cells) are administered in connection with the treatment of a non-hematologic malignancy, and the target antigen is expressed (even at a low level) in normal hematopoietic cells or lineages. Such cancer-associated antigens include human epidermal growth factor receptor 2 (HER2) (e.g., for ovarian cancer, breast cancer, glioblastoma, colon cancer, osteosarcoma, and medulloblastoma), epidermal growth factor receptor (EGFR) (e.g., for non-small cell lung cancer, epithelial carcinoma, and glioma), mesothelin (e.g., for mesothelioma, ovarian cancer, and pancreatic adenocarcinoma), prostate-specific membrane antigen (PSMA) (e.g., for prostate cancer), carcinoembryonic antigen (CEA) (e.g., for pancreatic adenocarcinoma, breast cancer, and colon cancer), glypican-3 (e.g., for hepatocellular carcinoma), epidermal growth factor receptor variant III (EGFRvIII) (e.g., for glioblastoma), disialoganglioside 2 (GD2) (e.g., for neuroblastoma and melanoma), Carbonic anhydrase IX (CAIX) (e.g., for renal cell carcinoma), interleukin-13Ra2 (e.g., for glioma), fibroblast activation protein (FAP) (e.g., for malignant pleural mesothelioma), L1 cell adhesion molecule (L1-CAM) (e.g., for neuroblastoma, melanoma, and ovarian), cancer antigen 125 (CA125) (e.g., for epithelial ovarian cancer), cluster of differentiation 133 (CD133) (e.g., for glioblastoma and cholangiocarcinoma, adenocarcinoma), cancer / testis antigen 1B (CTAG1B) (e.g., for melanoma and ovarian cancer), mucin 1 (e.g., for seminal vesicle cancer), folate receptor-α (FR-α) (e.g., for ovarian cancer), and ErbB1, ErbB2, ErbB3, or ErbB4, IGF1R, IGF2R, TβR Growth factor receptors selected from one or more of I-II, VEGFR1, VEGFR2, VEGFR3, PDGFR(α / β), and FGFR1-4.

[0113] Modified HSCs according to the present disclosure can be used in connection with (e.g., in a method of treatment) an FDA-approved CAR-T therapy, such as tisagenlecleucel, also known as tisa-cel (KYMRIAH), axi-cel (YESCARTA), brexucabutagen siloreucel, also known as brexu-cel (TECARTUS), lisocabtagene malareucel, also known as liso-cel (BREYANZI), idecbutagen bicelueucel, also known as ide-cel (ABECMA), ciltacabtadine bicelueucel, also known as cilta-cel (CARVYKTI), or any other CAR-T therapy that damages normal cells (e.g., normal hematopoietic cells) during its therapeutic use.

[0114] In various embodiments, the modified HSCs of the present invention are administered to reduce normal cell killing or adverse effects caused by therapeutic applications of antibody therapy targeting cancer-associated antigens selected from one or more of growth factor receptors, ErbB1, ErbB2, ErbB3, or ErbB4, IGF1R, IGF2R, TβR I-II, VEGFR1, VEGFR2, VEGFR3, PDGFR(α / β), or FGFR1, FGFR2, FGFR3, or FGFR4, or antibodies targeting checkpoint proteins. Antibodies targeting such antigens include, but are not limited to, alefacept (AMEVIVE), alemtuzumab (CAMPATH), belimumab (BENLYSTA), cetuximab (ERBITUX), daclizumab (ZENAPAX, ZINBRYTA), denosumab (PROLIA, XGEVA), efalizumab (RAPTIVA), ipilimumab (YERVOY), natalizumab (TYSABRI), nivolumab (OPDIVO), olaratumab (LARTRUVO), panitumumab (VECTIBIX), pembrolizumab (KEYTRUDA), rituximab (RITUXAN), trastuzumab (HERCEPTIN), bevacizumab, or a combination thereof.Further, the antibodies include anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-p53 antibodies, anti-EGFR antibodies, anti-cathepsin D antibodies, anti-Bcl-2 antibodies, anti-E-cadherin antibodies, anti-CA125 antibodies, anti-CA15-3 antibodies, anti-CA19-9 antibodies, anti-c-erbB-2 antibodies, anti-P-glycoprotein antibodies, anti-CEA antibodies, anti-retinoblastoma protein antibodies, anti-ras oncoprotein antibodies, anti-Lewis antibodies, X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9 / p24 antibody, anti-CD1 antibody, anti-CD11c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody body, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD39 antibody, anti-CD41 antibody, anti-LCA / CD45 antibody, anti-CD The antibody may be selected from anti-CD45RO antibody, anti-CD45RA antibody, anti-CD71 antibody, anti-CD95 / Fas antibody, anti-CD99 antibody, anti-CD100 antibody, anti-S-100 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-lambda light chain antibody, anti-melanosome antibody, anti-prostate specific antigen antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratin antibody, and anti-Tn-antigen antibody, or any other antibody-based therapy that damages normal cells during their therapeutic use. In such embodiments, the targeted antigen has reduced expression or is deleted in HSCs or cell populations derived therefrom.

[0115] Compositions comprising modified HSCs and / or HSPCs of the present disclosure may further comprise a pharmaceutically acceptable excipient or carrier. Such excipient or carrier solutions may also contain buffers, diluents, and other suitable additives. A buffer refers to a solution or liquid whose chemical composition neutralizes acids or bases without significantly changing the pH. Examples of buffers contemplated by the present invention include, but are not limited to, physiological saline (0.9% NaCl), 5% dextrose in water (D5W), Dulbecco's phosphate-buffered saline (PBS), and Ringer's solution. The compositions may comprise a vehicle suitable for intravenous infusion or other administration routes and may include a suitable cryoprotectant. An exemplary carrier is DMSO (e.g., about 10% DMSO). The cell composition may be provided in an appropriate volume within an implantable device (e.g., a scaffold), or within a bag, vial, tube, or container, and may be stored frozen until use.

[0116] In other aspects, the present disclosure provides cell populations or pharmaceutically acceptable compositions thereof produced by the methods described herein. In various embodiments, the HSC and / or HSPC populations, as described herein, are derived from iPSCs, have genetically disrupted expression of one or more endogenous genes that are cancer-associated antigens, proliferate in vivo but do not exhibit uncontrolled proliferation or tumorigenesis in vivo, and differentiate in vivo to reconstitute hematopoietic lineages.

[0117] For example, in certain aspects, the present disclosure provides a population of HSCs having genetically disrupted expression of one or more endogenous genes that are tumor-associated antigens, and the iPSCs are expressed in a manner that inhibits expression of one or more endogenous genes that are tumor-associated antigens, such as HLA-A. neg , homozygous for both HLA-B and HLA-C, HLA-DPB1 neg , HLA-DQB1 neg, and homozygous for HLA-DRB1. As described elsewhere herein, the tumor-associated antigen is expressed or overexpressed in one or more of cytogenetically normal acute myeloid leukemia (CN-AML), acute myeloid leukemia (AML), acute lymphocytic leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma. In various embodiments, the endogenous gene is expressed on immune cells and may be expressed on myeloid cells, thymocytes (including T cells or B cells). In various embodiments, at least one of the endogenous genes is selected from CD33, CD19, CD7, CD123, and CD371.

[0118] In various embodiments, compositions for cell therapy (e.g., comprising the HSC populations described herein) comprise a desired cell population and a pharmaceutically acceptable vehicle. 2 cells, or at least about 10 3 or at least about 10 4 or at least about 10 5 or at least about 10 6 or at least about 10 7 or at least about 10 8 cells, or at least about 10 9 cells, or at least about 10 10 cells, or at least about 10 11 cells, or at least about 10 12 cells, or at least about 10 13 cells, or at least about 10 14 For example, in some embodiments, a pharmaceutical composition containing about 100,000 to about 400,000 T cell precursors per kilogram (e.g., about 200,000 cells / kg) is administered. In other embodiments, the cells are administered at a concentration of about 10 5 ~Approx. 5×10 5 cells (e.g., approximately 2.5 x 10 5 cells / kg), or approximately 10 per kilogram6 ~Approx. 5×10 6 cells (e.g., approximately 2.5 x 10 6 cells / kg), or approximately 5 x 10 cells per kilogram 6 ~about 10 7 cells (e.g., approximately 5 x 10 6 cells / kg), or approximately 10 per kilogram 7 ~about 10 8 cells (e.g., approximately 5 x 10 7 cells / kg), or approximately 10 per kilogram 8 ~about 10 9 cells (e.g., approximately 5 x 10 8 cells / kg), or approximately 10 per kilogram 9 ~about 10 10 cells, or approximately 10 per kilogram 10 ~about 10 11 cells, or approximately 10 per kilogram 11 ~about 10 12 cells, or approximately 10 per kilogram 12 ~about 10 13 cells, or approximately 10 per kilogram 13 ~about 10 14 It is administered in individual cells.

[0119] The route of administration of the cells (e.g., modified HSCs and / or HSPCs or their progeny) can be by any suitable means, including, but not limited to, parenteral routes. Parenteral infusions include intravenous and intra-arterial administration. In addition, the cells (e.g., modified HSCs and / or HSPCs or their progeny) can be suitably administered by pulse infusion, e.g., with decreasing doses of the cells (e.g., modified HSCs and / or HSPCs or their progeny). In some embodiments, the dosage is given by injection, e.g., via intravenous injection.

[0120] As used herein, the term "about" means ±10% of the associated numerical value.

[0121] Certain aspects and embodiments of the present disclosure are further illustrated with reference to the following examples. [Example]

[0122] Example 1 - ETV2 overexpression increases the yield of hemogenic endothelial cells and enhances CD34+ cell formation during iPSC differentiation, but does not affect pluripotency. method iPSCs were developed from hCD34+ cells by episomal reprogramming, as known in the art and essentially as described in Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells, Science 318, 1917-1920, (2007), and J. Yu, et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801, (2009). Embryoid bodies and hemogenic endothelial differentiation were performed essentially as described in R. Sugimura, et al., Hematopoietic stem and progenitor cells from human pluripotent stem cells. Nature 545, 432-438, (2017); C. M. Sturgeon, et al., Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat Biotechnol 32, 554-561, (2014); J. Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920, (2007); and J. Yu, et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801, (2009).

[0123] Briefly, hiPSCs were dissociated and resuspended in medium supplemented with L-glutamine, penicillin / streptomycin, ascorbic acid, human holo-transferrin, monothioglycerol, BMP4, and Y-27632. The cells were then seeded onto 10 cm dishes (EZSPHERE or low-attachment plates) for EB formation. On day 1, bFGF and BMP4 were added to the medium. On day 2, the medium was replaced with medium containing SB431542, CHIR99021, bFGF, and BMP4. On day 4, the cell medium was replaced with medium supplemented with VEGF and bFGF. On day 6, the cell medium was replaced with medium supplemented with bFGF, VEGF, interleukin (IL)-6, IGF-1, IL-11, SCF, and EPO. Cells were maintained in an incubator with 5% CO2, 5% O2, and 95% humidity. To harvest CD34+ cells, EBs were dissociated on day 8, cells were filtered through a 70 μm strainer, and CD34+ cells were isolated by CD34 magnetic bead staining.

[0124] result Induced pluripotent stem cells (iPSCs) were transduced using an adenoviral vector containing both ETV2 and GFP sequences under the control of the EF1A promoter. After transduction, approximately 45% of the iPSC cultures were observed to be GFP-positive, confirming ETV2 overexpression (ETV2-OE). It was further observed that ETV2-OE in iPSC cells retained the pluripotent properties of iPSCs, as indicated by the stemness marker expression TRA-1-60 (Figure 1). Figure 1 shows a FACS plot demonstrating the transduction efficiency of iPSCs using an adenoviral vector for overexpressing ETV2 and GFP sequences.

[0125] Next, ETV2-OE-iPSCs (along with control iPSCs transduced with a vector carrying the GFP sequence but without ETV2) were differentiated into embryoid bodies and then into hemogenic endothelial cells (Strugeon et al., 2014). The results showed that overexpression of ETV2 significantly increased the expression of CD235a. - CD34 in the population+ and CD31 + These results suggest that ETV2-OE enhances the formation of hemogenic endothelial cells, as evidenced by the expression of markers (Figure 2). Specifically, Figure 2 shows representative flow cytometry analysis and relative quantification of hemogenic endothelial cells (defined here as CD235a-CD34+CD31+), demonstrating that ETV2-OE enhances the formation of hemogenic endothelial cells compared to controls.

[0126] Furthermore, the results showed that ETV2-OE expresses CD34 + This suggests that ETV2-OE enhances CD34+ cell formation (Figure 3). Figure 3 shows representative flow cytometry analysis and relative quantification of CD34+ cells demonstrating that ETV2-OE enhances CD34+ cell formation.

[0127] Overall, these data indicate that ETV2 overexpression in iPSCs does not affect their pluripotent properties, but promotes their ability to undergo hemogenic endothelial and hematopoietic differentiation.

[0128] Example 2 - iPSC-derived HSCs generated by Piezo1 activation undergo T cell differentiation similar to bone marrow-derived HSCs. method To analyze EHT, EB-derived CD34+ cells were suspended in medium containing Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3. After the cells had adhered to the bottom of the well (by visual inspection) for approximately 4-18 hours, Yoda1 was added to the cultures for some experiments. After 4-7 days, cells were collected for analysis.

[0129] iPSCs were differentiated into embryoid bodies for 8 days. On day 8, CD34+ cells from the iPSC-derived embryoid bodies were harvested and cultured for an additional 5–7 days to induce endothelial-hematopoietic (EHT) transition. CD34+ cells were then harvested from the EHT cultures on days 5–7 for further hematopoietic lineage differentiation.

[0130] CD34+ cells harvested from EHT cultures on days 5–7 (or days 13–21 total of differentiation from iPSCs) were seeded onto 48-well plates pre-coated with rhDL4 and retronectin. T lineage differentiation was induced in medium containing aMEM, FBS, ITS-G, 2BME, ascorbic acid-2-phosphate, Glutamax, rhSCF, rhTPO, rhIL7, FLT3L, rhSDF-1α, and SB203580.

[0131] 80% of the medium was changed every other day from days 2 to 6. On day 7, cells were transferred to new coated plates and analyzed for the presence of pre-T cells (CD34+CD7+CD5+ / -).

[0132] 80% of the medium was changed every other day from days 8 to 13. On day 14, 100,000 cells / well were transferred to new coated plates, and cells were analyzed for the presence of pre-T cells (CD34-CD7+CD5+ / -).

[0133] 80% of the medium was changed every other day from days 15 to 20. Cells were harvested on day 21 and analyzed via FACS for CD3, CD8, CD5, CD7, TCRab expression on behalf of T cells, and / or activated using CD3 / CD28 beads to assess their functional properties.

[0134] After 21 days of differentiation, cells were harvested and approximately 80,000 cells were replated into new 96-well culture plates in RPMI 1640 (without L-glutamine, without phenol red) with FBS, L-glutamine, and IL-2, and then activated with 1:1 CD3 / CD28 beads. After 72 hours of activation with CD3 / CD28 beads, cells were analyzed for CD3, CD69, and CD25 expression by FACS and for IFN-γ expression using RT-qPCR. Supernatants were analyzed by ELISA.

[0135] result Figures 4A and 4B show that iPSC-derived HSCs derived from EHT of CD34+ cells (CD34+ cells harvested on day 8, EHT induced by Piezol activation) undergo pre-T cell differentiation, similar to bone marrow (BM)-HSCs. Furthermore, Figures 5A and 5B show that iPSC-derived HSCs generated from EHT of CD34+ cells (using Piezol activation) undergo T cell differentiation, similar to BM-HSCs, and can be activated with CD3 / CD28 beads. Figure 6 shows that iPSC-derived HSCs generated from EHT of CD34+ cells (using Piezol activation in this example) can differentiate into functional T cells, as demonstrated by INFγ expression upon stimulation with CD3 / CD28 beads. Together, these results demonstrate that EHT of CD34+ cells (optionally using Piezol activation) enhances the ability to further differentiate into hematopoietic lineages, such as progenitor and functional T cells, ex vivo.

[0136] iPSC-derived HSCs according to the present disclosure, as well as lineages derived therefrom, may be valuable therapies for reconstituting hematopoietic lineages, including cytotoxic T cells and natural killer cells, for neoplastic disease. When used in conjunction with therapies that target hematopoietic cells, such as for the treatment of hematopoietic malignancies, elimination or reduced expression of target cancer antigens in HSCs can provide a powerful therapeutic approach.

[0137] Example 3 - CD33 Deletion Figure 7A shows the generation of three CD33-KO iPSC clones. As shown in Figure 7B, CD33-KO does not affect the cells' ability to undergo endothelial-to-hematopoietic transition. Furthermore, CD33-KO does not affect the cells' ability to generate self-renewing HSCs (Figure 7C). When used in conjunction with therapies that target hematopoietic cells, such as for the treatment of hematopoietic malignancies, elimination or reduced expression of CD33 in HSCs (or cells derived therefrom) can provide a powerful therapeutic approach.

[0138] Example 4 - CCR5 deletion Figure 8A shows the generation of three CCR5 knockout (KO) iPSC clones. As shown in Figure 8B, CCR5-KO does not affect iPSC pluripotency. Furthermore, as shown in Figure 8C, CCR5-KO does not affect the ability of cells to undergo endothelial-to-hematopoietic transition.

[0139] Example 5 - Evaluation of off-target editing in HLA knockout HSCs HLA typing of triple knockout (HLA edited) HSC clones was performed to check for unwanted editing and ensure that major editing events, such as deletion(s), did not occur within other regions of chromosome 6. Sequencing methods and analyses were performed to assess the extent of gRNA off-target activity and select gRNAs that represent a low risk of affecting non-target HLA genes.

[0140] Sequencing was performed using in situ cleavage labeling in fixed and permeabilized cells by ligating full-length P5 sequencing adapters to end-prepared DSBs. Genomic DNA was extracted, fragmented, end-prepared, and ligated using chemically modified semi-functional P7 adapters. The resulting DNA library contained a mixture of functional DSB-labeled fragments (P5:P7) and non-functional genomic DNA fragments (P7:P7). Subsequent DNA sequencing of the DNA library enriched for the DNA-labeled fragments, eliminating all irrelevant non-functional DNA. Because the library preparation was PCR-free, each resulting sequencing read was equivalent to a single labeled DSB end from the cell. This generated a readout of DNA cleavage, enabling direct detection and quantification of genomic DSBs by sequencing without the need for error correction, and allowed for the unambiguous mapping of off-target mutations.

[0141] Table 1 below summarizes the results of the editing strategy in two representative HLA-edited clones relative to wild-type cells. [Table 1-1] [Table 1-2]

[0142] Table 2 provides non-limiting examples of gRNAs used in experiments that can be used to knock out expression of the indicated HLA genes. [Table 2]

[0143] The results show that the editing strategy was successful in selectively targeting the HLA-A, DPB1, and DQB1 genes without affecting other HLA genes or introducing major deletions elsewhere.

[0144] These results were confirmed by phenotypic analysis of the HLA-edited clones by FACS and immunofluorescence. As shown in Figures 9A and 9B, the HLA-edited cells tested positive for overall expression of HLA class I molecules, comparable to that of wild-type cells. Specific expression of HLA-A via immunofluorescence confirmed that HLA-A was not expressed in the HLA-edited cells, supporting the finding that the gene editing strategy successfully deleted only the HLA-A gene. Specifically, Figure 9A shows that all HLA-edited cells were positive for HLA-like class I to the same extent as wild-type cells (i.e., gHSCs). This result indicates that despite the deletion of HLA-A, other class I molecules, such as HLA-B and HLA-C, were expressed and were not affected by the gene editing strategy.

[0145] To confirm that the HLA-A gene had been deleted, the specific expression of HLA-A was analyzed by immunofluorescence. As can be seen in Figure 9B, HLA-A was not expressed in the HLA-edited clones, indicating that the gene editing strategy was effective in specifically deleting only the HLA-A gene. Such preservation of overall class I expression with deletion of HLA-A would facilitate patient matching while avoiding NK cell-mediated rejection.

[0146] Example 6 - Evaluation of pluripotency and immune compatibility of HLA-edited HSCs The ability of HLA-edited cells to preserve pluripotency was assessed. As shown in Figure 10, immunofluorescence evaluation of HLA-edited iPSC clones showed that they maintained tri-lineage differentiation, with ectoderm differentiation indicated by NESTIN-488 and PAX6-594 staining, mesoderm differentiation indicated by GATA-488 staining, and endoderm differentiation indicated by CXCR4-488 and FOX2A-594 staining.

[0147] HLA class I molecules are expressed on the surface of all nucleated cells, and if HLA class I molecules are mismatched between donor and recipient, the cells can be recognized and killed by CD8+ T cells. In addition, HLA mismatches can lead to cytokine release syndrome (CRS) and graft-versus-host disease (GVD). Conversely, complete deletion of HLA-I molecules via B2M KO would render the cells targets for NK cell-mediated cytotoxicity. Preservation of overall class I expression with deletion of HLA-A could facilitate patient matching while avoiding NK cell-mediated rejection. Therefore, the immune compatibility of HLA-edited HSCs was tested by coculture with peripheral blood mononuclear cells (PBMCs) to assess whether immune cells would reject grafts of HLA-edited and wild-type HSCs (gHSCs).

[0148] Wild-type (i.e., gHSCs) and HLA-edited HSCs were cocultured with PBMCs bearing HLA-B and HLA-C markers matched but mismatched HLA-A. B2M KO HSCs, which lack expression of HLA class I molecules, and CIITA KO HSCs, which lack expression of class II molecules, were used as controls to compare the degree of PBMC-mediated cytotoxicity for HLA-null and mismatched HLAs, respectively. Figure 11 shows the results of a PBMC-mediated cytotoxicity assay in the coculture, measured by Annexin V staining. The results show that deletion of HLA-A in HLA-edited HSCs protected the cells from PBMC-mediated cytotoxicity, while WT, B2M KO, and CIITA KO HSCs were susceptible to PBMC-mediated cytotoxicity. Coculture of HSCs with sorted CD8+ T cells from the same PBMC donor protected HLA-edited and B2M KO HSCs from CD8+ T cytotoxicity. Conversely, HSCs co-cultured with sorted NK cells protected only WT and HLA-edited cells from NK cell-mediated cytotoxicity.

[0149] In summary, the immunocompatibility results indicate that CD8+ T cells present in the PBMC samples were involved in killing cells bearing mismatched HLA molecules (WT) and CIITA KO, while NK cells present in the PBMCs were involved in killing HLA-null cells (B2M KO). However, HLA-edited HSCs were protected from CD8+ T cell-mediated cytotoxicity (because mismatched HLA-A had been knocked out) and from NK cell-mediated cytotoxicity (because HLA class I molecule expression was largely preserved).

[0150] Example 7 - Evaluation of in vivo engraftment potential of HLA-edited HSCs To evaluate the engraftment potential of HLA-edited HSCs, the ability of the cells to engraft in vivo was assessed by competitive transplantation against wild-type HSCs. Equal proportions of mCherry HLA-edited HSCs and wild-type HSCs (gHSCs) were mixed and transplanted into mice, from which bone marrow (BM) and peripheral blood samples were collected and evaluated by FACS to compare the relative amounts of each cell type present in the samples. As shown in Figure 12, both HLA-edited HSCs and wild-type HSCs contributed to approximately equal engraftment in the BM and peripheral blood samples. These results confirm that HLA-edited HSCs (prepared according to the present disclosure) are comparable to wild-type HSCs in their engraftment and reconstitution potential. Therefore, the properties of wild-type HSCs are expected to match those of the HLA-edited HSCs of the present disclosure for generating T cell lineages.

[0151] Example 8 - Differentiation of HLA-edited HSCs into CD4+ / CD8+ T cells Antigen-presenting cells (APCs) present antigens to helper CD4+ T cells through HLA-II molecules. Activation of helper CD4+ T cells promotes the generation of antigen-specific CD8+ T cells, which further evolve into antigen-specific CTLs. Similarly, HLA class I molecules are expressed on the surface of all nucleated cells and present peptide fragments of proteins to CD8+ CTLs from within the cell. CTLs induce cytotoxic killing of target (infected) cells upon recognition of HLA-I-peptide complexes expressed on the cell surface. Therefore, studies were performed to determine whether deletion of HLA-A affects class I peptide presentation of edited HSCs. As shown in Figures 13A and 13B, immunopeptidome analysis indicates that deletion of HLA-A does not affect overall class I peptide presentation. HLA-A-edited cells exhibited comparable peptide and protein presentation compared to wild-type (i.e., unedited) HSCs. Furthermore, as shown in Figure 14A and B, deletion of HLA-DQB1 and HLA-DPB1 does not affect overall class II peptide presentation by macrophages differentiated from HSCs. Together, these data suggest that despite the deletion of HLA-A, HLA-DQ, and HLA-DP molecules, the cells (and their derived lineages) retain the ability to present a wide range of class I and II peptides.

[0152] Example 7 - In vivo testing of antigen-mediated immune responses. Figure 15 is a schematic diagram of the delayed-type hypersensitivity reaction, showing the sensitization and elicitation stages of antigen presentation. Briefly, upon antigen injection, the antigen is processed by antigen-presenting cells (APCs) and presented by MHC class II molecules on the surface of the APCs. CD4+ T cells recognize peptide-MHC on the antigen-presenting cells (APCs). Upon antigen administration, CD4+ helper T cells are activated, and cytokines recruit macrophages and other immune cells that induce tissue swelling.

[0153] A delayed-type hypersensitivity assay was performed on transplanted mice. Specifically, mice were sensitized by subcutaneous injection of sheep red blood cells as an antigen. If the mouse has a functional immune system, APCs process the antigen and present the peptide antigen to CD4+ T cells. The mouse was then stimulated by subcutaneous injection of the same antigen in the left paw. At this point, T cells are activated and secrete cytokines that recruit macrophages and other immune cells to the antigen injection site, resulting in tissue swelling. In this assay, a functional immune system resulted in swelling of the left paw, as measured with a microcaliper.

[0154] As can be seen in Figures 16A and 16B, control (non-transplanted) mice, due to their immunodeficiency, did not show swelling of their left paws. Conversely, mice transplanted with cord blood CD34+ cells showed tissue swelling, doubling the diameter of their left paws. Similar immune system responses were found in both WT (non-edited HSCs) and HLA-edited HSCs (HLA-edited) transplanted mice.

[0155] Example 9 - Evaluation of differentiation and maturation of HSC-derived T cells (pre-T cells) Next, we tested the ability of HSC-derived T cells (pre-T cells) to differentiate into mature T cells. HSCs were generated via Peizo1 activation. After a 35-day differentiation period, pre-T cells were assessed by cell sorting for the presence of CD4+, CD8+, and AB+ T cell populations. As shown in Figure 17, pre-T cells differentiated into CD4+, CD8+, and αβ+ T cells more efficiently than bone marrow (BM)-derived CD34+ cells and embryonic body (EB)-derived CD34+ cells.

[0156] Next, to test their functional properties, each T cell population was co-cultured with a CD19+ lymphoma cell line and an anti-CD3 / CD19 bispecific antibody. In this experimental model, the bispecific antibody engaged both the CD3 receptor on T cells and the CD19 cell surface receptor on lymphoma cells, thus inducing T cell activation. The degree of activation was assessed by measuring subsequent T cell-mediated cytotoxicity compared to pan T cell controls. As shown in Figure 18, pre-T cells demonstrated statistically significant superiority in cytotoxicity compared to both BM CD34+ T cells and EB CD34+ T cells.

[0157] Because the overall differentiation process of pre-T cells is 35 days long, transduction experiments were performed to test whether the time required to differentiate HSCs could be shortened. Pre-T cells were cultured in activation medium (approximately 7 days) to increase the transduction efficiency of the cells. Next, the cells were transduced with lentiviral (LV) particles encoding an anti-CD19 CAR transgene. The cells were cultured for an additional 4-5 days (12 days total) to assess their maturation and killing capacity. As shown in Figure 19, HSC-derived pre-T cells could be transduced with high efficiency, with more than 80% of the cells expressing the anti-CD19 CAR, as evidenced by cell sorting.

[0158] Next, we evaluated the ability of pre-T cells to effectively mature into CD4+ / CD8+ T cells via CAR transduction. Pre-T cells were transduced with LVs using an anti-CD19 CAR, along with bone marrow (BM)-derived CD34+ cells and embryonic body (EB)-derived CD34+ cells (as well as Pan T cells as a positive control). T cell subsets were screened by cell sorting for the presence of CD4 or CD8 cell surface marker expression. As shown in Figure 20, the results indicated that CAR transduction promoted T cell maturation, and an increased degree of T cell maturation was observed in pre-T cells compared to bone marrow (BM)-derived CD34+ cells and embryonic body (EB)-derived CD34+ cells.

[0159] The ability of LV pre-transduced T cells to function via anti-CD19 receptor-mediated cytotoxicity was assessed. T cell subsets were cocultured with a CD19+ leukemia cell line (NALM6) expressing a luciferase reporter gene (Luc+) to measure the extent of T cell-mediated cytolysis, using untransduced cells and Pan T cells as negative and positive controls, respectively. As shown in Figure 21, the CAR pre-T cells effectively functioned via T cell-mediated lysis and exhibited a similar degree of cytotoxicity to CAR pre-T cells derived from BM CD34+ cells. Conversely, CAR pre-T cells derived from EB CD34+ cells did not exhibit the ability to kill target cells.

[0160] Example 10 - Evaluation of HSC characteristics evolving into pre-T cells. The ability of HSCs to evolve into pre-T cells was assessed by measuring the CD34-CD7+ marker on pre-T cells. As shown in Figure 22, FACS analysis demonstrated that HSCs produced according to the present disclosure successfully differentiated into CD34-CD7+ pre-T cells compared to bone marrow-derived CD34+ cells or EB-derived CD34+ cells.

[0161] Next, we measured the expression of T cell-specific transcription factors and thymic engraftment molecules. Figure 23A shows increased TCF7 expression in pre-T cells derived from HSCs of the present disclosure, and Figure 23B shows increased CCR7 expression. Figure 24A shows a schematic diagram for determining whether HSC-derived pre-T cells engraft and differentiate in the thymus. Figure 24B shows FACS analysis of the CD3+ cell population of cells gated on the CD45+ cell population, demonstrating the superior engraftment and differentiation potential of HSC-derived pre-T cells in the thymus. Pre-T cells in this example were prepared from HSCs using Piezo1 activation, as previously described.

[0162] In vitro activation of HSC-derived T cells was also measured, as illustrated in Figure 25. The top panel of Figure 25 shows FACS analysis of activated T cells from different sources, including HSCs of the present disclosure (e.g., prepared using Piezol activation). T cells prepared from HSCs of the present disclosure showed comparable or superior activation, as measured by increased CD107 expression. The bottom panel shows Dynabeads activation, where activated T cells express inflammatory cytokines. T cells derived from HSCs according to the present disclosure (e.g., prepared using Piezol activation) expressed higher levels of inflammatory cytokines, as exemplified by TNF-α and interferon-gamma expression levels.

[0163] Example 10 - Evaluation of the properties of CCR5 knockout HSCs that evolve into pre-T cells. To determine whether CCR5 knockout (CCR5-KO) HSCs could differentiate into pre-T cells comparable to their wild-type counterparts from which they were derived (i.e., HSCs of the present disclosure prepared via Piezo1 activation), studies were performed in which CD34, CD7, and CD5 expression of HSCs and CCR5-KOs was measured. As can be seen in Figure 26, HSCs successfully differentiated into CD34+CD7+CD5+ pre-T cells comparable to bone marrow-derived CD34+ cells. Similarly, CCR5-KOs, like their wild-type counterparts, successfully differentiated into CD34+CD7+CD5+ pre-T cells.

[0164] Next, we evaluated the properties of CCR5 knockout HSCs that differentiated into double-positive (CD4+CD8+) T cells, which were comparable to those of the HSCs from which they were derived. As can be seen in Figure 27, CCR5 knockout HSCs differentiated into double-positive (CD4+CD8+) T cells comparable to their wild-type counterparts from which they were derived (i.e., the HSCs of the present disclosure prepared via Piezo1 activation).

Claims

1. 1. A method for preparing a population of hematopoietic stem cells (HSCs) and / or hematopoietic stem progenitor cells (HSPCs), comprising: Preparing a human induced pluripotent stem cell (iPSC) population; modifying one or more endogenous genes within said iPSC population that are tumor-associated antigens, thereby disrupting expression of said tumor-associated antigens; Inducing differentiation of the iPSC population into a CD34+ population; and inducing endothelial-hematopoietic transition (EHT) of the CD34+ population for at least 2 days and not more than 12 days to prepare a population comprising HSCs and / or HSPCs with reduced expression of the tumor-associated antigen.

2. 2. The method of claim 1, wherein the tumor-associated antigen is expressed or overexpressed in one or more of cytogenetically normal acute myeloid leukemia (CN-AML), acute myeloid leukemia (AML), acute lymphocytic leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma.

3. The method of claim 2 , wherein the endogenous gene is expressed on an immune cell.

4. The method of claim 2 , wherein the endogenous gene is expressed on bone marrow cells.

5. The method of claim 2 , wherein the endogenous gene is expressed on a thymocyte.

6. The method of claim 2 , wherein the endogenous gene is expressed in a T cell or a B cell.

7. The method of claim 2 , wherein the endogenous gene is expressed in immune cells at all stages of cell differentiation.

8. 8. The method of any one of claims 1 to 7, wherein at least 50% of the HSCs and / or HSPCs do not express the endogenous gene or express a reduced level of the endogenous gene.

9. The method of any one of claims 1 to 8, wherein the iPSCs have a deletion or inactivation of one or two copies of the endogenous gene.

10. The method of any one of claims 1 to 9, wherein at least one of the endogenous genes is selected from CD33, CD119, CD7, CD123, and CD371.

11. 11. The method of claim 10, wherein the one or more endogenous genes include CD33.

12. The method of claim 11 , wherein the HSCs are suitable for myeloid differentiation.

13. 13. The method of claim 12, wherein the HSCs and / or HSPCs differentiate into myeloid and lymphoid progenitor cells and mature myeloid and lymphoid cells therefrom.

14. 11. The method of claim 10, wherein the one or more endogenous genes include CD19.

15. 15. The method of claim 14, wherein the HSCs and / or HSPCs are suitable for B lymphocyte differentiation.

16. 16. The method of claim 15, wherein the HSCs and / or HSPCs differentiate into myeloid and lymphoid progenitor cells and mature myeloid and lymphoid cells therefrom.

17. 11. The method of claim 10, wherein the one or more endogenous genes include CD7.

18. 18. The method of claim 17, wherein the HSCs and / or HSPCs are suitable for T cell progenitor differentiation.

19. 19. The method of claim 18, wherein the HSCs and / or HSPCs differentiate into myeloid and lymphoid progenitor cells and mature myeloid and lymphoid cells therefrom.

20. 11. The method of claim 10, wherein the one or more endogenous genes include CD123.

21. 21. The method of claim 20, wherein the HSCs and / or HSPCs are suitable for myeloid differentiation.

22. 22. The method of claim 21, wherein the HSCs and / or HSPCs differentiate into myeloid and lymphoid progenitor cells and mature myeloid and lymphoid cells therefrom.

23. 11. The method of claim 10, wherein the one or more endogenous genes include CD371.

24. 24. The method of claim 23, wherein the HSCs and / or HSPCs are suitable for myeloid differentiation.

25. 25. The method of claim 24, wherein the HSCs and / or HSPCs differentiate into myeloid and lymphoid progenitor cells and mature myeloid and lymphoid cells therefrom.

26. 26. The method of any one of claims 1 to 25, wherein the iPSC population is derived from lymphocytes, umbilical cord blood cells, peripheral blood mononuclear cells, CD34+ cells, or primary human tissue.

27. 27. The method of claim 26, wherein the iPSC population is derived from CD34+ enriched cells isolated from peripheral blood.

28. 28. The method of any one of claims 1 to 27, wherein the iPSCs are homozygous for one or more HLA class I and / or HLA class II genes.

29. 29. The method of claim 28, wherein the iPSCs are homozygous for HLA-DRB1.

30. 29. The cell composition of claim 28, wherein the iPSCs are homozygous for one or both of HLA-B and HLA-C.

31. 29. The method of Claim 28, wherein the iPSCs have been gene-edited to delete one or more HLA class I genes, delete one or more class II genes, and / or delete one or more genes that govern HLA or MHC expression or presentation capacity.

32. 32. The method of claim 31, wherein the iPSCs comprise a deletion of HLA-A.

33. 33. The method of claim 31 or 32, wherein the iPSCs comprise a deletion of HLA-DPB1 and / or HLA-DQB1.

34. The method according to claim 31, wherein the one or more genes controlling the ability to express or present HLA or MHC are β2-microglobulin and / or CIITA.

35. The iPSCs are HLA-A ｎｅｇ , homozygous for both HLA-B and HLA-C, HLA-DPB1 ｎｅｇ , HLA-DQB1 ｎｅｇ and homozygous for HLA-DRB1.

36. 36. The method of any one of claims 1 to 35, wherein the disruption of the one or more endogenous genes is produced by introducing (a) a Cas9 endonuclease or a nucleic acid encoding a Cas9 endonuclease, and (b) a nucleic acid molecule encoding a guide RNA (gRNA) that directs the mutation or deletion of a nucleotide sequence of the endogenous gene by the Cas9 endonuclease.

37. 37. The method of any one of claims 1 to 36, further comprising harvesting cells from the CD34+ population undergoing EHT.

38. 38. The method of any one of claims 1 to 37, wherein the endothelial-hematopoietic transition (EHT) is induced between days 7 and 15 of iPSC differentiation.

39. 39. The method of claim 38, wherein EHT is induced for 5 to 7 days.

40. 40. The method of claim 38 or 39, wherein the endothelial-to-hematopoietic transition (EHT) generates a HSC population comprising one or more of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells, and hematopoietic stem progenitor cells.

41. 41. The method of claim 40, wherein the HSC population comprises long-term hematopoietic stem cells (LT-HSCs).

42. The method of any one of claims 1 to 41, wherein the induction of endothelial-hematopoietic transition (EHT) comprises increasing the expression or activity of dnmt3b.

43. 43. The method of any one of claims 1 to 42, wherein the induction of endothelial-hematopoietic transition (EHT) comprises applying cyclic stretch to the iPSCs or cells derived from the iPSCs, such as CD34+ cells, endothelial cells (ECs), and hemogenic endothelial cells (HECs).

44. 44. The method of claim 43, wherein the cyclic stretching is 2D, 3D, or 4D cyclic stretching.

45. The method of any one of claims 1 to 44, wherein the induction of endothelial-hematopoietic transition (EHT) comprises Piezo1 activation.

46. 46. ​​The method of claim 45, wherein the Piezo1 activation is by contacting the iPSCs or cells derived from the iPSCs with one or more Piezo1 agonists optionally selected from Yoda1, Jedi1, Jedi2, ssRNA, or analogs or derivatives thereof.

47. 47. The method of any one of claims 1 to 46, wherein the induction of endothelial-hematopoietic transition (EHT) comprises Trpv4 activation.

48. 48. The method of claim 47, wherein said Trpv4 activation is by contacting said iPSCs or cells derived from said iPSCs with one or more Trpv4 agonists optionally selected from GSK1016790A, 4α-PDD, or analogs or derivatives thereof.

49. 49. The method of any one of claims 1 to 48, wherein the HSCs and / or HSPCs are differentiated into hematopoietic lineages.

50. 50. The method of claim 49, wherein the hematopoietic lineage is selected from progenitor T cells, T lymphocytes, and natural killer cells.

51. A cell population prepared according to the method of any one of claims 1 to 50.

52. A population of HSCs, (i) derived from iPSCs by EHT of CD34+ cells dissociated from embryoid bodies; (ii) have genetically disrupted expression of one or more endogenous genes that are tumor-associated antigens; (iii) proliferate in vivo but do not exhibit uncontrolled growth or tumor formation in vivo; (iv) HSC populations that differentiate in vivo to reconstitute hematopoietic lineages.

53. 53. The HSC population of claim 52, wherein the iPSCs are homozygous for one or more HLA class I and / or HLA class II genes.

54. 54. The HSC population of claim 53, wherein the T cell population is homozygous for both HLA-B and HLA-C.

55. 55. The HSC population of claim 53 or 54, wherein the T cell population is homozygous for HLA-DRB1.

56. 56. The HSC population of any one of claims 52-55, wherein the iPSCs have been gene-edited to delete one or more HLA class I genes, one or more class II genes, and / or one or more genes that govern HLA or MHC expression or presentation capacity.

57. 57. The HSC population of claim 56, wherein the iPSCs comprise a deletion of HLA-A.

58. 58. The HSC population of claim 56 or 57, wherein the iPSCs comprise a deletion of HLA-DPB1 and / or HLA-DQB1.

59. 57. The HSC population of claim 56, wherein the one or more genes controlling the ability to express or present HLA or MHC are β2-microglobulin and / or CIITA.

60. The iPSCs are HLA-A ｎｅｇ , homozygous for both HLA-B and HLA-C, HLA-DPB1 ｎｅｇ , HLA-DQB1 ｎｅｇ 60. The HSC population of any one of claims 52 to 59, wherein the HSC population is homozygous for HLA-DRB1 and HLA-DRB2.

61. A population of HSCs having genetically disrupted expression of one or more endogenous genes that are tumor-associated antigens, wherein the iPSCs are HLA-A ｎｅｇ , homozygous for both HLA-B and HLA-C, HLA-DPB1 ｎｅｇ , HLA-DQB1 ｎｅｇ , as well as an HSC population that is homozygous for HLA-DRB1.

62. 62. The HSC population of claim 61, wherein the tumor-associated antigen is expressed or overexpressed in one or more of cytogenetically normal acute myeloid leukemia (CN-AML), acute myeloid leukemia (AML), acute lymphocytic leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma.

63. 63. The HSC population of claim 61 or 62, wherein the endogenous gene is expressed on an immune cell.

64. 64. The HSC population of claim 63, wherein the endogenous gene is expressed on bone marrow cells.

65. 64. The HSC population of claim 63, wherein the endogenous gene is expressed on thymocytes.

66. 64. The HSC population of claim 63, wherein the endogenous gene is expressed in T cells or B cells.

67. 64. The HSC population of claim 63, wherein the endogenous gene is expressed in immune cells at all stages of cell differentiation.

68. 64. The HSC population of claim 63, wherein at least one of the endogenous genes is selected from CD33, CD119, CD7, CD123, and CD371.

69. 64. The HSC population of claim 63, wherein the one or more endogenous genes include CD33.

70. 70. The HSC population of claim 68 or 69, wherein the HSCs are suitable for myeloid differentiation.

71. 70. The HSC population of claim 68 or 69, wherein the HSCs differentiate into myeloid and lymphoid progenitor cells and mature myeloid and lymphoid cells therefrom.

72. 69. The HSC population of claim 68, wherein the one or more endogenous genes include CD19.

73. 73. A method for treating a subject having cancer and undergoing therapy targeting cancer cells that express a tumor-associated antigen, the method comprising administering to the subject a therapeutically effective dose of a cell population described in claim 51 or an HSC population described in any one of claims 52 to 72, wherein the HSCs have reduced expression of the tumor-associated antigen.

74. 74. The method of claim 73, wherein the therapy targeting cells expressing the tumor-associated antigen is T cell therapy, CAR-T therapy, CAR-NK cell therapy, antibody-based therapy, or an antibody drug conjugate.

75. 75. The method of claim 73 or 74, wherein the tumor-associated antigen is CD33, CD19, CD7, CD123, or CD371.

76. 76. The method of any one of claims 73 to 75, wherein the disease or disorder is a hematopoietic malignancy.

77. 77. The method of claim 76, wherein the hematopoietic malignancy is selected from cytogenetically normal acute myeloid leukemia (CN-AML), acute myeloid leukemia (AML), acute lymphocytic leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia or chronic lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, or refractory or relapsed forms thereof.

78. 78. The method of any one of claims 73 to 77, wherein the cell population is an HSC population.

79. 79. The method of any one of claims 73 to 78, wherein said administering is performed according to a myeloablative, non-myeloablative, or immunotoxin-based (e.g., anti-c-Kit, anti-CD45, etc.) conditioning regimen.

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