Immunocompatible cells for allogeneic cell therapy to cover global, ethnic, or disease-specific populations

Abstract

In various aspects and embodiments, the present disclosure provides cell populations or cell "banks" (e.g., cell collections) thereof for providing immunocompatible allogeneic cell therapies covering global, ethnic, and disease-specific populations. In various aspects and embodiments, the cell banks and their progeny facilitate patient matching to prevent or reduce graft-versus-host disease (GVHD) or graft rejection while maintaining sufficient HLA class I and HLA class II function. The present disclosure further provides methods for generating cell banks by gene editing, and methods for cell therapy involving cells or tissues (including, but not limited to, hematopoietic stem cells, or "HSCs," their precursors, or progeny) derived from the cell banks.

Description

[Technical Field]

[0001] Priority This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 413,331, filed October 5, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted in XML format via EFS-Web and is incorporated herein by reference in its entirety. The XML copy, created on September 25, 2023, is named GRU-009 / 121145-5009_Sequence_Listing.xml and is 1,298,736 bytes in size. [Background technology]

[0003] Cell therapies based on allogeneic (derived from a healthy donor), autologous (derived from the patient), and / or induced pluripotent stem cells (iPSCs)-derived cells hold great promise for medical applications to regenerate cells and tissues. However, the use of allogeneic or autologous cells (generated from the intended recipient's cells) would be impractical in most cases. Meanwhile, transplantation of cells or tissues produced from allogeneic cells faces issues of immune rejection and / or graft-versus-host disease (GVHD), caused by, for example, significant HLA incompatibility. Similarly, autologous or allogeneic cell transplantation faces concerns regarding consistency, scalability, durability, and affordability. Furthermore, based on current HLA matching criteria, it is not feasible to prepare iPSC stocks representing sufficient HLA haplotypes to cover a significant portion of the population. There is a great need for cell banks that are HLA-modified to provide "off-the-shelf" cell therapies, offer ease of matching with a substantial portion of the population, and are consistent, scalable, and low-cost. In various aspects and embodiments, the present disclosure provides HLA-modified cells and collections thereof to meet these and other objectives. Summary of the Invention [Means for solving the problem]

[0004] In various aspects and embodiments, the present disclosure provides cell populations or cell "banks" (e.g., cell collections) thereof for providing immunocompatible allogeneic cell therapies covering global, ethnic, and disease-specific populations. In various aspects and embodiments, the cell banks and their progeny facilitate patient matching to prevent or reduce graft-versus-host disease (GVHD) or graft rejection while maintaining sufficient HLA class I and HLA class II function. The present disclosure further provides methods for generating cell banks by gene editing, and methods for cell therapy involving cells or tissues (including, but not limited to, hematopoietic stem cells, or "HSCs," their precursors, or progenies) derived from the cell banks.

[0005] In various aspects and embodiments, the present disclosure provides an HLA-A neg and (1) HLA-DPA1 neg and / or HLA-DPB1 neg , and / or (2) HLA-DQA1 neg and / or HLA-DQB1 neg In some embodiments, the cell population is homozygous for or comprises a single gene for HLA-C, HLA-DRB1, and optionally HLA-B. neg , HLA-DPB1 neg , and HLA-DQB1 neg and is homozygous for one or more (or all) of HLA-B, HLA-C, and HLA-DRB1. In various embodiments, the cell population has a haplotype described herein.

[0006] In various aspects and embodiments, the cell population is a human stem cell or human progenitor cell population. In some embodiments, the stem cells are pluripotent stem cells, which may be human induced pluripotent stem cells (hiPSCs). In various embodiments, the iPSCs are derived from peripheral blood CD34+ cells. In some embodiments, the stem cell population is a hematopoietic stem cell (HSC) population (e.g., differentiated from iPSCs), or a cell population derived therefrom. In some embodiments, the cell population includes cells of a hematopoietic cell lineage, such as a hematopoietic lineage selected from common lymphoid progenitor (CLP) cells, granulocyte-monocyte progenitor (GMP) cells, progenitor T cells, T lymphocytes, B lymphocytes, natural killer cells, neutrophils, monocytes, macrophages, erythrocytes, megakaryocytes, and platelets. In yet other embodiments, the cell population is a non-hematopoietic cell population, e.g., differentiated ex vivo from iPSCs. Exemplary cells include, but are not limited to, mesenchymal stem cells, neural stem cells, or epithelial stem cells. In some embodiments, the non-hematopoietic cells are selected from neurons, astrocytes, oligodendrocytes, cardiomyocytes, skeletal muscle cells, hepatocytes, pancreatic beta cells, and lung epithelial cells, or precursors thereof.

[0007] In another aspect, the present disclosure provides methods for cell therapy, comprising administering to a recipient in need thereof a cell population or tissue derived from a cell population disclosed herein. In various embodiments, the cell population is matched for retained classical HLA. For example, in embodiments in which the cell population retains HLA-B, HLA-C, and HLA-DRB1, the administered cell population or tissue is matched to the recipient at one or more (or all) of HLA-B, HLA-C, and HLA-DRB1.

[0008] In another aspect, the present invention provides methods for cell therapy (or use of cell compositions for cell therapy), comprising administering a cell population described herein or a pharmaceutically acceptable composition thereof to a human subject in need thereof. In various embodiments, the methods described herein are used to treat hematological (malignant and non-malignant), bone marrow, and immune disorders. In various embodiments, the human subject has a condition including one or more of lymphopenia, cancer, infectious disease (e.g., viral disease such as HPV or HIV), immunodeficiency, autoimmune disease, skeletal dysplasia, hemoglobinopathies, anemia, bone marrow failure syndromes, and genetic disorders (e.g., genetic disorders affecting the immune system).

[0009] In various embodiments, an HSC population is administered to the recipient, or in other embodiments, the cell population is a hematopoietic cell lineage differentiated (e.g., ex vivo) from the HSC population. In other embodiments, the cell population is a non-hematopoietic lineage differentiated from iPSCs described herein. In various embodiments, the subject has a condition selected from hematologic malignancies, aplastic anemia, hemoglobinopathies, inborn errors of metabolism, and severe immune deficiencies. Other conditions and disorders that may be treated are disclosed herein and include lymphopenia, cancer, immune deficiencies, autoimmune diseases, skeletal dysplasias, bone marrow failure syndromes, and genetic disorders affecting the immune system.

[0010] In some embodiments, the subject is a recipient of a tissue or organ transplant. In some embodiments, the subject is experiencing or at risk for GVHD. Transplantable organs include, for example, the heart, kidney, liver, lung, pancreas, intestine, and thymus, among others. Tissues for transplantation can include, for example, bone, tendon (both referred to as musculoskeletal grafts), bone marrow or HSCs, cornea, skin, heart valves, nerves, and / or veins.

[0011] In one aspect, the present disclosure provides a method for producing the cell population of the present disclosure, the method comprising providing an iPSC population and modifying the iPSC population to express HLA-A negand (1) HLA-DPA1 neg and / or HLA-DPB1 neg and / or (2) HLA-DQA1 neg and / or HLA-DQB1 neg and preparing an HLA-modified iPSC population comprising: HLA-B, HLA-C, and HLA-DRB1. In various embodiments, the iPSC population is homozygous for one or more (or all) of HLA-B, HLA-C, and HLA-DRB1, or contains a single gene for each. The method further comprises preparing pneumocystic bodies (EBs) from the iPSC population; dissociating the EBs and enriching for CD34+ cells to prepare a CD34+-enriched cell population; and inducing endothelial-hematopoietic transformation (EHT) of the CD34+-enriched cell population to prepare a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem progenitor cells (HSPCs). In some embodiments, the method may further comprise harvesting CD34+ cells from the population comprising HSCs and / or HSPCs to enrich for the population undergoing EHT. In some embodiments, the method further comprises differentiating the cell population undergoing EHT into a hematopoietic lineage.

[0012] In various embodiments, iPSCs are HLA-modified using CRISPR-Cas9, CRISPR-Cas12, STAR-CRISPR, CRISPR-CasX, CRISPR-associated transposases, zinc finger nucleases, RNA editors, isolated genomic domain platform editing, or combinations thereof. In various embodiments, iPSCs are HLA-modified by electroporation using CRISPR-Cas9 endonuclease and one or more guide RNAs (gRNAs) as ribonucleoproteins.

[0013] In various embodiments, CD34+ enrichment and endothelial-hematopoietic conversion are induced between days 7 and 15 of iPSC differentiation. In embodiments, induction of endothelial-hematopoietic conversion includes, but is not limited to, increasing dnmt3b expression or activity, such as by Piezol activation. Other methods for inducing EHT are described herein. In various embodiments, CD34+ enriched cells undergoing EHT are differentiated into one or more of common lymphoid progenitor (CLP) cells, granulocyte-monocyte progenitor (GMP) cells, precursor T cells, T lymphocytes, B lymphocytes, natural killer cells, neutrophils, monocytes, macrophages, erythrocytes, megakaryocytes, and platelets. In various embodiments, CD34+ enriched cells undergoing EHT are differentiated ex vivo into precursor T cells, T cells, or NK cells.

[0014] In various aspects, the present disclosure provides methods for producing an HLA-modified cell of the present disclosure, the method comprising contacting a cell with a Cas endonuclease and one or more guide RNAs (gRNAs) that target the Cas endonuclease to one or more HLA-specific or HLA allele-specific regions. In various embodiments, the Cas endonuclease comprises Cas9. In various embodiments, each gRNA is a single guide RNA (sgRNA). In various embodiments, the contacting comprises electroporating the Cas endonuclease and one or more gRNAs as a ribonucleoprotein. Exemplary gRNAs for targeting certain HLA haplotypes are described herein.

[0015] Other aspects and embodiments of the present disclosure will be apparent from the following detailed disclosure and examples. [Brief explanation of the drawings]

[0016] [Figure 1-1] Coverage of the most frequent haplotypes (based on HLA-C, HLA-B, and DRB1) in the United States is shown. Two haplotypes provide approximately 22% cumulative coverage, and approximately 50 haplotypes provide approximately 70% cumulative coverage of the US population. [Figure 1-2] Same as above. [Figure 2] 1 illustrates an HSC cell bank differentiated from iPSCs that have been gene-edited to knock out HLA genes. [Figure 3] Figure 1 shows that ETV2 overexpression (OE) does not affect pluripotency. Figure 2 shows a FACS plot showing the transduction efficiency of iPSCs with an adenoviral vector for overexpressing ETV2 and GFP sequences. ETV2 overexpression does not affect iPSC stemness, as shown by the expression of the TRA-1-60 stemness marker. [Figure 4] Figure 1 shows that ETV2 overexpression (OE) increases the yield of blood endothelial cells. Representative flow cytometry analysis of blood endothelial cells (denoted as CD235a-CD34+CD31+) and relative quantification demonstrate that ETV2-OE enhances the formation of blood endothelial cells. [Figure 5] 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 6A] Figure 1 shows that iPSC-derived HSCs induced by Piezo1 activation undergo pro-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 from Piezo1 activation into CD34+CD7+ pro-T cells. [Figure 6B] Figure 1 shows that iPSC-derived HSCs induced by Piezo1 activation undergo pro-T cell differentiation similar to bone marrow (BM)-HSCs. Quantification of CD34+CD7+ cells (%) derived from (1) BM-HSCs and (2) iPSC-HSCs (Piezo1-activated). Averages of three experiments are shown. [Figure 7A]We demonstrate that iPSC-derived HSCs generated by Piezo1 activation undergo T cell differentiation, and that such T cells can be activated with CD3 / CD28 beads in the same way as BM-HSC-derived T cells. Figure 1 shows FACS plots of the activation efficiency (CD3+CD69+ expression) of T cells differentiated from BM-HSCs and iPSC-derived HSCs generated by Piezo1 activation. [Figure 7B] We demonstrate that iPSC-derived HSCs generated by Piezo1 activation undergo T cell differentiation and that such T cells can be activated with CD3 / CD28 beads similar to T cells derived from BM-HSCs. Quantification of CD3+CD69+ cells (%) derived from (1) BM-HSCs and (2) iPSC-HSCs (Piezo1-activated). Averages of three experiments are shown. [Figure 8] We demonstrate that iPSC-derived HSCs can differentiate into functional T cells. IFNγ expression is a result of T cell activation after T cell receptor (TCR) stimulation via CD3 / CD28 beads. IFNγ expression in T cells differentiated from iPSC-derived HSCs is produced upon Piezo1 activation and enhances the HSC capacity to further differentiate into functional cells (e.g., cells). Averages of three experiments are shown. [Figure 9A] Generation of three CCR5 knockout (KO) iPSC clones is shown. [Figure 9B] Figure 1 shows that CCR5-KO does not affect iPSC pluripotency. [Figure 9C] Figure 1 shows that CCR5-KO does not affect the ability of cells to undergo endothelial-hematopoietic transition. [Figure 10A] Generation of three CD33-KO iPSC clones is shown. [Figure 10B] Figure 1 shows that CD33-KO does not affect the ability of cells to undergo endothelial-hematopoietic transition. [Figure 10C] Figure 1 shows that CD33-KO does not affect the ability of cells to generate self-renewing HSCs. [Figure 11A] Indicates the presence of the HLA gene located on the short arm of chromosome 6. [Figure 11B]Schematic representation of targeting of HLA-A, HLA-DQB1, and HLA-DPB1 genes using gRNAs. Exons are indicated by horizontal arrows, and vertical arrows indicate the location of gRNA targeting. Genomic coordinates are shown in parentheses. [Figure 12A] Phenotypic analysis of triple knockout (HLA-edited) cells performed by FACS and immunofluorescence shows 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. [Figure 12B] 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, which is not expressed in HLA-edited clones. [Figure 13] As illustrated by immunofluorescence, HLA-edited clones maintain their pluripotency (maintaining three germ layer differentiation), with ectodermal differentiation indicated by NESTIN-488 and PAX6-594 staining, mesodermal differentiation indicated by GATA-488 staining, and endodermal differentiation indicated by CXCR4-488 and FOX2A-594 staining. [Figure 14] Immunocompatibility of HLA-edited HSCs is shown. HLA-edited HSCs and control HSCs (WT, B2M KO, and HLA class II null) were cocultured with HLA-B and HLA-C-matched, but not HLA-A-matched, peripheral blood mononuclear cells (PBMCs). PBMC-CD8+ and NK cell-mediated cytotoxicity were measured by Annexin V staining assay. [Figure 15] We demonstrate the in vivo engraftment potential of HLA-edited HSCs. Equal proportions of mCherry HLA-edited and wild-type HSCs 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 16A]We show that WT and HLA-edited HSCs can differentiate into pro-T cells, as identified by the CD34-CD7+ and CD34+CD7+ marker combinations. [Figure 16B] The results shown in Figure 16A are presented graphically. [Figure 17] We show that WT and HLA-edited HSCs can differentiate into the NK cell lineage, as identified by the CD3-CD56+ marker. [Figure 18A] We show that WT and HLA-edited HSCs can differentiate into the monocyte / macrophage lineage, which also maintains the overall expression of both class I and class II molecules, as identified by the CD11b+CD14+ marker. [Figure 18B] Analysis of HLA-I and HLA-II in CD11b+CD14+ gated cells is shown. [Figure 19A] 1 shows that deletion of HLA-DQB1 and HLA-DPB1 does not affect the expression of other HLA class II molecules. 1 is a schematic showing differentiation of HLA-edited iPSCs into macrophages. [Figure 19B] 1 shows that deletion of HLA-DQB1 and HLA-DPB1 does not affect the expression of other HLA class II molecules. Immunofluorescence experiments confirming the specific deletion of DPB1 and DQB1 molecules. [Figure 19C] Figure 1 shows that deletion of HLA-DQB1 and HLA-DPB1 does not affect the expression of other HLA class II molecules, and that the same cells preserve class II DRB1 expression. [Figure 20A] Figure 1 shows that HLA-A deletion does not affect class I peptide presentation. Schematic representation of immunopeptidome analysis. [Figure 20B] We show that HLA-A deletion does not affect class I peptide presentation. We present the results of immunopeptidome analysis, which reveals little difference in the number of peptides and representative proteins presented by class I molecules in WT and HLA-edited cells. [Figure 21A]Figure 1 shows that deletion of HLA-DP and DQ does not affect class II peptide presentation. Immunopeptidome analysis scheme. [Figure 21B] 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 22] Schematic representation of in vivo testing of antigen-mediated immune responses: delayed-type hypersensitivity assay (DTH), sensitization phase, and elimination phase, respectively. [Figure 23A] This shows that HLA-edited HSCs reconstitute a functional immune system, as evidenced by DTH responses in immunodeficient mice. A delayed-type hypersensitivity assay was performed on the transplanted mice, which involves crosstalk between different types of immune cells. Specifically, the mice were sensitized by subcutaneous injection of sheep red blood cells (antigen). A functional immune system caused swelling of the left paw, which was measured with a microcaliper. As can be seen in Figure 23A, non-transplanted mice did not show swelling of the left paw due to their immunodeficiency. Conversely, mice transplanted with umbilical cord blood CD34+ cells showed tissue swelling, with the diameter of their left paw doubling. [Figure 23B] Figure 23A shows that HLA-edited HSCs reconstitute a functional immune system as demonstrated by DTH responses in immunodeficient mice. [Figure 24] 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. The T cells of the present disclosure demonstrate comparable or superior activation, as measured by increased CD107 expression. The bottom panel shows Dynabeads activation, and activated T cells express proinflammatory cytokines. HSC-derived T cells express higher levels of proinflammatory cytokines, as exemplified by TNF-α and interferon gamma expression levels. [Figure 25]We show that CCR5 knockout HSCs are equally capable of differentiating into pro-T cells compared to their wild-type (gHSC) counterpart HSCs (in which CCR5 is retained). [Figure 26] We show that CCR5 knockout HSCs are equally capable of differentiating into double-positive (CD4+CD8+) T cells compared to their wild-type (gHSC) counterpart HSCs (in which CCR5 is retained). [Figure 27] We demonstrate that HSCs generated according to the present disclosure (D8+7 iPSC-CD34+ cells, both with and without Yoda1 "Y") can be successfully differentiated into CD4+CD8+ ("double positive") T cells as well as TCRα / β T cells. The methods of the present disclosure substantially exceed T cell maturation from bone marrow CD34+ cells. [Figure 28] We show that HSCs generated according to the present disclosure (D8+7 iPSC-CD34+ cells, both with and without Y) successfully rearrange TCR and outperform bone marrow CD34+ cells. [Figure 29] Figure 29 shows the differentiation potential of HSCs into T cell subtypes. After a 35-day differentiation period, pro-T cells were assessed by cell sorting for the presence of CD4+, CD8+, and AB+ T cell populations. Figure 29 (right) 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). [Figure 30]

[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 demonstrate high levels of cytotoxicity against target cells. [Figure 31]These results demonstrate that HSC-derived T cells (pro T cells) can be transduced with high efficiency. Pro T cells were lentivirally transduced 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 32] We show that LV-transduced HSC-derived T cells (pro-T cells) can be effectively matured into CD4+ / CD8+ T cells via CAR transduction. [Figure 33] We demonstrate the ability of anti-CD19 CAR-transduced HSC-derived T cells (CAR pro-T cells) to function through receptor-mediated cytotoxicity. Luc+ NALM6 leukemia cells were co-cultured with CAR pro-T cells, and cell-mediated cytotoxicity was measured by luciferase assay. [Figure 34] Figure 1 shows the ability of HSCs to develop into pro-T cells as measured by their CD34-CD7+ markers. [Figure 35A] 10 demonstrates increased expression of T cell-specific transcription factors and thymic engraftment molecules using pro-T cells derived from HSCs according to the present disclosure. TCF7 mRNA expression is shown. [Figure 35B] 10 demonstrates increased expression of T cell-specific transcription factors and thymic engraftment molecules using pro-T cells derived from HSCs according to the present disclosure. CCR7 mRNA expression is shown. [Figure 36A] 1 shows that HSC-derived pro-T cells engraft and differentiate in the thymus. Engraftment and analysis procedures are shown. [Figure 36B] Figure 1 shows that HSC-derived pro-T cells engraft and differentiate in the thymus. FACS analysis of the CD3+ cell population of cells gated on the CD45+ cell population is shown, demonstrating the excellent engraftment and differentiation potential of HSC-derived pro-T cells in the thymus. DETAILED DESCRIPTION OF THE INVENTION

[0017] The term "gHSC" is used herein to refer to the iPSC-derived hematopoietic stem cells of the present disclosure.

[0018] 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.

[0019] EB34+ cells refer to embryonic body-derived CD34+ cells, which include blood endothelial cells.

[0020] In various aspects and embodiments, the present disclosure provides cell populations or cell "banks" and collections thereof for providing immunocompatible allogeneic cell therapies covering global, ethnic, and disease-specific populations. In various aspects and embodiments, the cell banks and their progeny facilitate patient matching to prevent or reduce graft-versus-host disease (GVHD) or graft rejection while maintaining sufficient HLA class I and HLA class II function. The present disclosure further provides methods for generating cell banks by gene editing, and methods for cell therapy involving cells or tissues (including, but not limited to, hematopoietic stem cells, or "HSCs," and their precursors and progeny) derived from the cell banks.

[0021] In one aspect, the present disclosure provides an HLA-A neg and (1) HLA-DPA1 neg and / or HLA-DPB1 neg , and / or (2) HLA-DQA1 neg and / or HLA-DQB1 neg Further, the cell population is homozygous for or contains a single gene for HLA-C, HLA-DRB1, and optionally HLA-B.

[0022] In some embodiments, the cell population is HLA-DPB1 neg and HLA-DQB1 negIn some embodiments, the cell population comprises deletions or inactivations of both DPB1 genes and both DQB1 genes.

[0023] In some embodiments, one or both DQB1 genes are deleted or retained. In some embodiments, one or both DQA1 genes are retained. In some embodiments, the cell population retains DQB1 unmodified. In other embodiments, the cell population contains both DQB1 genes, and optionally, both DQA1 genes deleted or inactivated. In these embodiments, the cell population is HLA-DQB1. neg is.

[0024] In some embodiments, one or both DPB1 genes are deleted or retained. In some embodiments, one or both DPA1 genes are retained. Thus, a cell population may lack both copies or retain HLA-DP, which may be single copy (one copy deleted or inactivated), or may retain HLA-DP as unmodified.

[0025] In some embodiments, the cell population comprises a deletion or inactivation of both DPB1 genes and both DQB1 genes. In these embodiments, DRB1 is retained and is either homozygous or single copy to support HLA-class II function.

[0026] In various embodiments, the cell population is homozygous or contains a single copy of HLA-B. Alternatively, the cell population may contain deletions or inactivations of both HLA-B genes. In these embodiments, class I antigen presentation may be primarily supported by HLA-C.

[0027] In various embodiments, the cell population retains one copy of HLA-DRB1, and the other copy of HLA-DRB1 is deleted or inactivated. In some embodiments, the cell population is homozygous or has only a single copy of one, two, three, or four of DRB2, DRB3, DRB4, and DRB5. Optionally, DRB2, DRB3, DRB4, and DRB5 are retained and unmodified.

[0028] In some embodiments, the cell population is homozygous for HLA-E, or one HLA-E gene is deleted or inactivated, hi some embodiments, the HLA-E is unmodified and can be homozygous or heterozygous.

[0029] In some embodiments, the cell population is homozygous for HLA-F, or one HLA-F gene is deleted or inactivated, hi some embodiments, HLA-F is unmodified and can be homozygous or heterozygous.

[0030] In some embodiments, the cell population is homozygous for HLA-G, or one HLA-G gene is deleted or inactivated, hi some embodiments, HLA-G is unmodified and can be homozygous or heterozygous.

[0031] In certain embodiments, the cell population is homozygous for both HLA-B and HLA-C. neg and HLA-DPB1 neg and HLA-DQB1 neg In some embodiments, the cell population is further homozygous for HLA-DRB1.

[0032] In various embodiments, the cell population is a stem cell population, such as pluripotent stem cells. In some embodiments, the cell population is human induced pluripotent stem cells (hiPSCs). As described in further detail herein, iPSCs can be derived from umbilical cord blood, bone marrow biopsies, mobilized peripheral blood-derived hCD34+ cells, human CD34+ cells, immune cells, immune progenitor cells, hematopoietic cells, non-hematopoietic cells (e.g., cells that can differentiate into cells such as fibroblasts, osteoblasts, chondrocytes, myocytes, endothelial cells, and neurons), and cells from banked organs. In various embodiments, as described further below, primary cells can be reprogrammed to generate human iPSC cell bank(s), which can be HLA-modified to generate off-the-shelf therapeutics containing immunocompatible allogeneic human cells.

[0033] In some embodiments, the stem cell population is a hematopoietic stem cell (HSC) population or a hematopoietic stem progenitor cell (HSPC) population, or a cell population derived therefrom. As described in more detail herein, the cell population may be of a hematopoietic cell lineage or may be used to derive a hematopoietic cell lineage. For example, the hematopoietic lineage may be selected from common lymphoid progenitor (CLP) cells, granulocyte-monocyte progenitor (GMP) cells, precursor T cells, T lymphocytes, B lymphocytes, natural killer cells, neutrophils, monocytes, macrophages, dendritic cells, erythrocytes, megakaryocytes, and platelets.

[0034] In some embodiments, the cell population may be human donor- or patient-derived cells, including, but not limited to, donor- or patient-derived HSCs, hematopoietic progenitors, and hematopoietic lineages (e.g., selected from CLP cells, GMP cells, progenitor T cells, T lymphocytes, B lymphocytes, natural killer cells, neutrophils, monocytes, macrophages, dendritic cells, erythrocytes, megakaryocytes, and platelets).

[0035] In yet other embodiments, the cell population is a non-hematopoietic stem cell population. The population may be derived from iPSCs or may be derived from a donor or patient. Exemplary non-hematopoietic stem cells include mesenchymal stem cells, neural stem cells, or epithelial stem cells. In still other embodiments, the cell population is, or is used to derive, a non-hematopoietic cell, such as a cell selected from fibroblasts, osteoclasts, chondrocytes, myocytes, cardiomyocytes, endothelial cells, neurons, astrocytes, oligodendrocytes, hepatocytes, pancreatic beta cells, and lung epithelial cells, or precursors thereof.

[0036] In some embodiments, the cell population(s) are stem or progenitor cells used to generate hCD34+ HSCs, hematopoietic precursors, immune cells, platelets, erythrocytes, other hematopoietic precursors, and lineages, as well as non-hematopoietic cells, including, but not limited to, neurons, astrocytes, oligodendrocytes, myocytes, cardiomyocytes, hepatocytes, pancreatic beta cells, lung epithelial cells, and the like.

[0037] According to aspects and embodiments of the present disclosure, the cell populations are hematopoietic progenitors derived from pluripotent stem cells (e.g., iPSCs), such as hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), or common lymphoid progenitors (CLPs), which can give rise to progenitors such as red blood cells / erythrocytes, platelets, mast cells, osteoclasts, granulocytes, monocytes-macrophages, dendritic cells, T cells / T lymphocytes, B cells / B lymphocytes, NK cells / natural killer cells, and dendritic cells.

[0038] In various aspects and embodiments of the present disclosure, cell populations and cell banks (whether allogeneic, autologous, or iPSC-derived cells) are developed according to HLA haplotype or polymorphism distributions based on region, ethnicity, and / or target disease. In embodiments, cell populations or cell lines are developed using suitable primary cell donors according to rank ordering of common or uncommon HLA haplotypes to cover the target population. For example, cumulative addition of cell lines to the bank can be based on HLA haplotypes and / or polymorphisms to cover a heterogeneous US population, a heterogeneous Caucasian population, a heterogeneous β-thalassemia population, or a heterogeneous global population (or other population). In various embodiments, cumulative addition of cell lines to the bank covers core HLA haplotypes and / or polymorphisms in a heterogeneous Asian population, a heterogeneous European population, a heterogeneous African population, a heterogeneous South American population, or a heterogeneous North American population.

[0039] In various embodiments, the cell population has a DRB1 haplotype selected from DRB1*03:01, DRB1*15:01, DRB1*01.02, DRB1*07.01, DRB1*04:01, DRB1*07:01, DRB1*01:01, DRB1*01:02, DRB1*04:04, DRB1*13:02, DRB1*13:01, DRB1*11:04, and / or DRB1*15:02, DRB1*03:02, DRB1*11:01, DRB1*15:03, DRB1*04:07, DRB1*08:04, DRB1*04:02, DRB1*14:01, and DRB1*10:01.

[0040] In some embodiments, the cell population has an HLA-C haplotype selected from C*07:01, C*04:01, C*07:02, C*06:02, C*03:04, C*05:01, C*02:02, C*12:03, C*03:03, and C*16:01.

[0041] In some embodiments, the cell population has an HLA-B haplotype selected from B*08:01 and B*07:02. For example, the cell population has an HLA-B haplotype selected from B*08:01, B*07:02, B*44:02, B*44:03, B*35:01, B*57:01, B*15:01, B*14:02, B*40:01, B*53:01, B*49:01, B*51:01, B*13:02, and B*38:01.

[0042] In some embodiments, the cell population comprises: C*07:01~B*08:01~DRB1*03:01、C*07:02~B*07:02~DRB1*15:01、C*05:01~B*44:02~DRB1*04:01、C*16:01~B*44:03~DRB1*07:01、C*04:01~B*35:01~DRB1*01:01、C*06:02~B*57:01~DRB1*07:01、C*06:02~B*13:02~DRB1*07:01、C*08:02~B*14:02~DRB1*01:02、C*3:04~B*40:01~DRB1*04:04、C*04:01~B*44:03~DRB1*07:01、C*03:04~B*40:01~DRB1*13:02、C*03:04~B*15:01~DRB1*04:01、C*05:01~B*18:01~DRB1*03:01、C*05:01~B*44:02~DRB1*13:01、C*07:02~B*07:02~DRB1*01:01、C*04:01~B*35:02~DRB1*11:04、C*12:02~B52:01~DRB1*15:02、C*03:03~B*15:01~DRB1*13:01、C*07:02~B*07:02~DRB1*07:01、C*05:01~B*44:02~DRB1*15:01、C*12:03~B*38:01~DRB1*13:01、C*17:01~B*42:01~DRB1*03:02、C*08:02~B*14:01~DRB1*07:01、C*01:02~B*27:05~DRB1*01:01、C*04:01~B*35:01~DRB1*11:01、C*06:02~B*50:01~DRB1*07:01、C*07:01~B*18:01~DRB1*11:04、C*04:01~B*53:01~DRB1~*13:02、C*12:03~B*18:01~DRB1*15:01、C*07:02~B*07:02~DRB1*11:01、C*05:01~B*44:02~DRB1*01:01、C*04:01~B*53:01~DRB1*15:03、C*07:02~B*07:02~DRB1*04:01、C*08:02~B*14:02~DRB1*13:02、C*04:01~B*35:01~DRB1*07:01、C*07:01~B*08:01~DRB1*15:01、C*07:02~B*39:05~DRB1*04:07、C*04:01~B*53:01~DRB1*08:04, C*07:02~B*07:02~DRB1*13:01, C*12:03~B*38:01~DRB1*04:02, C*03:03~ B*15:01~DRB1*04:01, C*04:01~B*35:01~DRB1*14:01, C*06:02~B*37:01~DRB1*10:01, C*07:01~B*49:01~D The HLA-C to HLA-B to DRB1 haplotypes are selected from among C*03:04 to B*40:01 to DRB1*04:01, C*15:02 to B*51:01 to DRB1*11:01, C*03:02 to B*58:01 to DRB1*03:01, C*03:03 to B*55:01 to DRB1*14:01, and C*04:01 to B*35:01 to DRB1*13:01.

[0043] In various embodiments, the cell lines are immunocompatible at 2, 4, 6, 8, 10, or 12 HLA loci by either matching at certain HLA haplotypes or mismatching at certain HLA haplotypes.

[0044] For example, a cell line may be immunocompatible in HLA-C by the cell line being homozygous for HLA-C (and HLA-C matched), or by having one copy of HLA-C matched and the other copy of HLA-C deleted or inactivated. A cell line may be immunocompatible in HLA-A by having both HLA-A genes deleted or inactivated (i.e., the cell line is HLA-A matched). neg The cell lines are also immunocompatible for HLA-DRB1 by being homozygous for HLA-DRB1 (and therefore HLA-DRB1 matched) or by having one copy of HLA-DRB1 matched and the other copy of HLA-DRB1 deleted or inactivated.

[0045] In various embodiments, the cell lines are immunocompatible for HLA-B, either by the cell line being homozygous for HLA-B or by having one copy of HLA-B matched and the other copy of HLA-B deleted or inactivated. In some embodiments, the cell line completely lacks HLA-B.

[0046] In some embodiments, the cell line is one in which both copies of DPB1 are deleted or inactivated (HLA-DPB1 neg Alternatively, the cell lines are homozygous for HLA-DPB1 (and DPB1 matched or mismatched), or one copy of HLA-DPB1 is matched and the other copy of HLA-DPB1 is deleted or inactivated.

[0047] In some embodiments, the cell line is one in which both copies of DQB1 are deleted or inactivated (HLA-QPB1 neg Alternatively, the cell lines are homozygous for HLA-DQB1 (and DQB1 matched or mismatched), or are matched for one copy of HLA-DQB1 and have the other copy of HLA-DQB1 deleted or inactivated.

[0048] Cell lines are immunocompatible at HLA-E either by the cell line being homozygous for HLA-E or by having one copy of HLA-E matched and the other copy of HLA-E deleted or inactivated, however, in some embodiments, HLA-E is kept unmodified and is either matched or mismatched.

[0049] In some embodiments, cell lines are developed by deleting or inactivating specific HLA haplotypes using gene editing techniques, including but not limited to CRISPR-Cas9, while preserving other HLA haplotypes. For example, cell lines can be derived from human primary cells from a donor that are homozygous (at one or more loci) and / or by deleting one copy of a mismatched haplotype. Non-limiting examples of sgRNAs for use with the CRISPR-Cas9 gene editing system are described herein. sgRNAs can be used alone or in combination to induce gene editing, such as a double-strand break in exon 1 and / or exon 2 of the target HLA, resulting in the inactivation, mutagenesis, or deletion of one or more bases (e.g., 5 or more, or 10 or more, or 50 or more, or 100 or more, or 500 or more bases) sufficient to functionally inactivate or eliminate the functional expression of the target gene. In some embodiments, the gRNA targeting domain is about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more double-stranded nucleotides in length. In some embodiments, the gRNA comprises a modification at or near the 5' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5' end) and / or a modification 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 gRNA exhibits increased resistance to nucleases. In some embodiments, the gRNA comprises two separate RNA molecules (i.e., a "dual gRNA"). Dual gRNAs comprise two separate RNA molecules, a "crispr RNA" (or "crRNA") and a "tracr RNA," and are well known to those of skill in the art.

[0050] The cell lines contain one or more HLA modifications (e.g., one or more HLA gene deletions) to facilitate HLA matching with the recipient and make the cell therapy available to diverse populations with a universal collection of HLA compatible cell line populations (i.e., compared to unmodified collections that are hindered by the vast diversity of HLA haplotypes in the population). In one aspect, the present disclosure provides a collection of cell lines (or "cell population") comprising at least two cell lines, wherein the cell lines in the collection represent at least two different HLA haplotypes. For example, each cell line contains one or more of (1) HLA-DQA1 and / or HLA-DQB1 and (2) HLA-DPA1 and / or HLA-DPB1, as well as a deletion or inactivation of an HLA-A gene. Other HLA modifications to class I and / or class II genes are made in accordance with the present disclosure to facilitate immunocompatibility matching with the recipient without compromising the safety or efficacy of the cell therapy.

[0051] The major histocompatibility complex (MHC) system, also referred to herein as human leukocyte antigen (HLA), consists of a polymorphic gene cluster located on the short arm of chromosome 6 (6p21.3). HLA includes regions designated as class I and class II. The primary function of HLA class I gene products is to present endogenous (i.e., intracellular) peptides to cognate CD8+ (cytotoxic) T cells. The primary function of HLA class II molecules is to present peptide antigens from exogenous proteins to CD4+ helper T cells. HLA class I gene products are important for detecting cells that develop harmful mutations and / or cancer, as well as for detecting and targeting cells that harbor intracellular pathogens, including viruses. HLA class II gene products are important for detecting the presence of pathogens in the tissue environment and orchestrating immune responses against pathogens. HLA class I gene products are expressed by most cells, while HLA class II genes are primarily expressed by professional antigen-presenting cells, such as dendritic cells, macrophages, and B cells. HLA class II molecules are also known to be expressed by, for example, some T cells and subsets of epithelial and endothelial cells (Kambayashi and Laufer, "Atypical MHC class II-expressing antigen-presenting cells: can anything replace a dendritic cell?" Nature Reviews Immunology, vol. 14: 719-730 (2014)).

[0052] HLA class I molecules include HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G, which vary substantially in their level of polymorphism. HLA class I molecules consist of a single polypeptide complexed with β2-microglobulin (B2M). In fact, knockout of B2M can eliminate functional expression of HLA class I gene products. There are approximately 7,453 identified HLA-A alleles, approximately 8,849 identified HLA-B alleles, approximately 7,393 identified HLA-C alleles, approximately 310 identified HLA-E alleles, approximately 50 identified HLA-F alleles, and approximately 102 identified HLA-G alleles. See hla.alleles.org. Natural killer (NK) cells recognize cells lacking HLA class I expression, a phenomenon frequently observed in a wide range of tumor types. Malmberg K., Immune selection during tumor checkpoint inhibition therapy paves the way for NK-cell “missing self” recognition, Immunogenetics vol. 69, pages 547-556 (2017). Generally, HLA-A and HLA-B show the highest expression among class I molecules.

[0053] HLA class II molecules contain two transmembrane polypeptide chains (α and β) that form an antigen-binding cavity. HLA molecules corresponding to class II include HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR, which have highly variable levels of polymorphism (see hla.alleles.org). HLA class II genes include the "classical" class II α and β chain genes of HLA-DP, -DQ, and -DR, as well as those with "non-classical" loci such as HLA-DM and -DO. DRB1 shows the highest diversity and is highly expressed among the class II genes. [Table 1-1] [Table 1-2] [Table 1-3]

[0054] For organ and tissue transplants (including hematopoietic stem cells) from allogeneic donors, the primary criterion for donor selection is HLA compatibility. Specifically, for HSC transplantation, a new lympho-hematopoietic system must be developed within the recipient to replace the recipient's diseased lympho-hematopoietic system. The immune response is essentially driven by T cells and includes the host-versus-graft (HVG) response, which refers to the patient's cellular reactivity against donor cells, and the graft-versus-host (GVH) response, which refers to the donor's lymphocyte reactivity against host tissues. The immunotherapeutic effect on neoplastic cells is often referred to as graft-versus-leukemia (GVL). GVH (or GVHD) can be associated with severe side effects in transplant recipients (e.g., HSCT) and is a major contributor to the morbidity and mortality associated with HSC transplantation.

[0055] Molecular HLA typing typically involves typing the α1 and α2 domains for class I and the α1 domain for class II. Donors are generally selected based on typing the HLA genes HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, and HLA-DPB1. For example, US standards traditionally contemplate matching eight loci (both alleles for HLA-A, HLA-B, HLA-C, and HLA-DRB1), while European standards involve matching 10 loci (both alleles for HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1).

[0056] In one aspect, the present disclosure provides a method for generating a ready-made cell population or "bank" of cells. The method includes (i) providing cells and / or pluripotent stem cells (e.g., iPSCs) from a donor or patient, and (ii) modifying in vitro one or more endogenous coding sequences in the genome of the cells (e.g., iPSCs), thereby knocking out one or more cells or mutating one or more cells to encode a non-functional protein in the cell population (e.g., iPSCs). In various embodiments, the modified cells are (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-A - B - C + DP - DR + DQ + , (v) HLA-A - B - C + DP + DR + DQ - , (vi) HLA-A - B - C + DP - DR + DQ -For retained HLA (e.g., HLA-B, HLA-C, and HLA-DR), the cells are homozygous or 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-.

[0057] As used herein, the term "neg" or (-) with respect to a particular HLA class I or HLA class II indicates that both copies of the gene are disrupted in a cell line or population, and therefore the cell line or population does not display 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.

[0058] According to aspects and embodiments of the present disclosure, cell populations or cell banks are provided to enable the generation (e.g., ex vivo expansion or differentiation) of hematopoietic stem cells (HSCs) and their precursors or progeny for harvesting from allogeneic donors or patients or for ready-to-use cell and tissue therapy. The cells are gene-edited to delete specific HLA genes (as described), thereby facilitating an immunocompatible match for the intended recipient. As used herein, the term "delete" in this context refers to genetic modification (i.e., gene editing) of a targeted gene that suppresses functional expression of the corresponding gene product (i.e., the corresponding polypeptide). Such gene editing includes complete or partial gene deletion or deletion of a critical cis-acting expression control sequence. According to embodiments of the present disclosure, expression of B2M is unchanged, nor is class II major histocompatibility complex transactivator (CIITA), as these modifications abolish HLA expression. B2M expression is important for functional expression of HLA class I, and CIITA is important for HLA class II expression. For example, alterations in B2M compromise natural killer (NK) cell responses, which may be detrimental to the proliferation of pathogen-infected or oncogenic cells. Alterations in CIITA compromise antigen-presenting capacity through class II HLA. For HSC transplantation (for example), functional class I and class II expression is required to reconstitute immune surveillance.

[0059] In various aspects and embodiments, the present disclosure provides HLA class I-modified cells and / or HLA class II-modified cells in which certain HLA gene(s) (as described) have been altered or deleted to make the cells suitable for cell-based therapy in subjects in need of such therapy without being hindered by concerns about the adverse effects of HLA mismatching or donor matching. Advantageously, these alterations in class I and class II molecules enhance the biocompatibility of these cells in diverse populations from Asia, Europe, Africa, South America, and North America, as they exhibit all of the characteristics of unmodified cells but advantageously eliminate or ameliorate the adverse or toxic effects of their HLA-unmodified counterpart cells in therapeutic applications.

[0060] In various aspects and embodiments, the present disclosure minimizes the HLA loci required for haplotype matching, including HSC transplantation. In various aspects and embodiments, the cells, or cells or tissues derived therefrom, exhibit functional class II antigen presentation (i.e., class II antigen presentation is not substantially impaired by lower class II expression and / or class II diversity compared to non-HLA-modified cells or tissues). In various aspects and embodiments, the cells, or cells or tissues derived therefrom, do not exhibit substantial susceptibility to carcinogenesis or viral infection (i.e., due to lack of class I expression or class I diversity). In various aspects and embodiments, the cells, or cells or tissues derived therefrom, are not substantially targeted by the recipient's innate immune system (e.g., NK cells) due to lack of HLA expression or diversity (i.e., compared to non-HLA-modified cells or tissues).

[0061] HLA haplotypes are designated according to convention herein. HLA alleles may be named by indicating the locus, antigen specificity, and molecular type allele group. The asterisk "*" symbol indicates that typing is performed by molecular methods, and the colon ":" is a field separator. For example, if A*03:01 is the allele of interest, the first field (A*03) refers to the group of alleles that code for the A3 antigen, and the second field (:01) refers to the specific allele that codes for the unique HLA protein A*03:01. Homozygous alleles may, in some embodiments, contain one or more polymorphisms in one or both copies.

[0062] The cell population or bank can be modified for one or more additional functionalities (described in more detail herein), including the deletion or insertion of additional genes. For example, cell lines can be modified to express or overexpress certain cytokines, suicide genes, T cell receptors, one or more chimeric antigen receptors (CARs), and / or combinations thereof. In some embodiments, the cell population or bank or their progeny is modified to delete, inactivate, or reduce the expression of certain endogenously expressed genes, such as, for example, genes encoding CCR5 or miR-155, or genes encoding cell surface markers, including, but not limited to, CD33, CLL, CD19, CD7, and / or CD38.

[0063] In various embodiments, the cells are introduced with nucleic acid encoding a CAR specific for a myeloma, leukemia, or lymphoma target, including but not limited to, CD19, CD33, and BCMA. In various embodiments, the cells are introduced with nucleic acid encoding a tandem CAR, including but not limited to, CD38 / IL3 and CD20 / CD19. In various embodiments, the cells are introduced with nucleic acid encoding a disease-specific dual CAR, quad CAR, or tandem CAR.

[0064] Thus, non-limiting examples include, but are not limited to: (i) cells deleted for CCR5 to generate a CCR5 deleted cell therapy for HIV-AIDS patients, (ii) cells deleted for CD33 to generate a CD33 deleted cell therapy to treat leukemia and / or lymphoma patients, and (iii) cells deleted for CD33 to generate a CD33 deleted cell therapy for use in conjunction with CAR-T, CAR-NK, CAR-T progenitor cells, or CAR-macrophage cells to treat leukemia and / or lymphoma patients.

[0065] The cell population or cells derived therefrom (e.g., progenitors) can be used in FDA-approved CAR-T therapies, such as tisagenlecleucel (also known as tisa-cel (Kymriah)), axicabtagene ciloreucel (also known as axi-cel (Yescarta)), brexcabtagene outrucel (also known as brexu-cel (Tecartas)), lisocabtagene maraleucel (also known as liso-cel (Breyanji)), idecabtagene bicrueucel (also known as ide-cel (Avecma)), ciltacabtagene outrucel (also known as cilta-cel (Carbicty)), or any other CAR-T therapy that damages normal cells during therapeutic use.

[0066] In one aspect, the disclosure provides a collection of cell populations (i.e., a cell bank comprising at least two populations of cells) of expanded primary cells, derivatives of iPSC cells, or stem cell lines, wherein the cell lines in the collection represent at least two different HLA-C haplotypes. Each cell population comprises a deletion or inactivation of both HLA-A genes. In various embodiments, the cell populations in the collection represent at least four, or at least six, or at least eight, or at least ten HLA-C haplotypes. In some embodiments, the cell populations in the collection represent at least 12 different HLA-C haplotypes, or at least 15 or at least 20 different HLA-C haplotypes. In various embodiments, the cell populations are either homozygous for the HLA-C gene or are edited to have only a single HLA-C gene (e.g., by deletion of one HLA-C gene).

[0067] In certain embodiments, the cell population or bank is represented by at least (i) HLA-C alleles and (ii) class II DRB alleles.

[0068] In some embodiments, the collection comprises a cell population or bank that comprises one or more of the HLA-C alleles selected from the following: C*01, C*02, C*03, C*04, C*05, C*06, C*07, C*08, C*12, C*14, C*15, C*16, C*17, C*18. In some embodiments, these HLA-C alleles match a DRB allele selected from DRB1*01, DRB1*03, DRB1*04, DRB1*07, DRB1*08, DRB1*09, DRB1*10, DRB1*11, DRB1*12, DRB1*13, DRB1*14, DRB1*15, DRB1*16, DRB2*01, DRB3*01, DRB3*02, DRB3*03, DRB4*01, DRB4*02, DRB4*03, DRB5*01, DRB5*02, DRB6*01, DRB6*02, DRB7*01, DRB8*01, and DRB9*01.

[0069] In some embodiments, these alleles match (or are HLA-B deleted) HLA-B alleles selected from B*07, B*08, B*13, B*14, B*15, B*18, B*27, B*35, B*37, B*38, B*39, B*40, B*41, B*42, B*44, B*45, B*46, B*47, B*48, B*49, B*50, B*51, B*52, B*53, B*54, B*55, B*56, B*57, B*58, B*59, B*67, B*73, B*78, B*81, B*82, and B*83.

[0070] In some embodiments, these matching Class II alleles are selected from DRA*01, DQA1*01, DQA1*02, DQA1*03, DQA1*04, DQA1*05, DQA1*06, DQB1*02, DQB1*03, DQB1*04, DQB1*05, DQB1*06, DPA1*01, DPA1*02, DPA1*03, DPA1*04, DPA2*01, and DPA2*02.

[0071] In some embodiments, these alleles are DPB1*01, DPB1*02, DPB1*03, DPB1*04, DPB1*05, DPB1*06, DPB1*08, DPB1*09, DPB1*10, DPB1*100, DPB1*101, DPB1*102, DPB1*103, DPB1*104, DPB1*105, DPB1*106, DPB1*107, DPB1*108, DPB1*109, DPB1*11, DPB1*110, DPB1*111, DPB1*112, DPB1*113, DPB1*114, DPB1*115, DPB1*116, DPB1*117, DPB1*118, DPB1*119, DPB1*120, DPB1*210, DPB1*211, DPB1*212, DPB1*213, DPB1*214, DPB1*215, DPB1*216, DPB1*217, DPB1*218, DPB1*219, DPB1*220, DPB1*221, DPB1*222, DPB1*223, DPB1*224, DPB1*225, DPB1*226, DPB1*227, DPB1*228, DPB1*229, DPB1*230, DPB1*231, DPB1*232, DPB1*233, DPB1*234, DPB1*235, DPB1*236, DPB1*237, DPB1*238, DPB1*239, DPB1*240, DPB1*241, DPB1*242, B1*116, DPB1*117, DPB1*118, DPB1*119, DPB1*120, DPB1*121, DPB1*122, DPB1*123, DPB1*124, DPB1*125, DPB1*126, DPB1*127, DPB1*128, DPB1*129, DPB1*13, DPB1*130, DPB1*131, DPB1*132, DPB1*133, DPB1*134, DPB1*135, DPB1*136, DPB1*137, DPB1*138, DPB1*139, DPB1*14, DPB1*140, DPB1*141, D PB1*142, DPB1*143, DPB1*144, DPB1*145, DPB1*146, DPB1*147, DPB1*148, DPB1*149, DPB1*15, DPB1*150, DPB1*151, DPB1*152, DPB1*153, DPB1*154, DPB1*155, DPB1*156, DPB1*157, DPB1*158, DPB1*159, DPB1*16, DPB1*160, DPB1*161, DPB1*162, DPB1*163, DPB1*164, DPB1*165, DPB1*166, DPB1*167, DPB1*168, DPB1*169, DPB1*17, DPB1*170, DPB1*171, DPB1*172, DPB1*173, DPB1*174, DPB1*175, DPB1*176, DPB1*177, DPB1*178, DPB1*179, DPB1*18, DPB1*180, DPB1*181, DPB1*182, DPB1*183, DPB1*184, DPB1*185, DPB1*186, DPB1*187, DPB1*188, DPB1*189, DPB1*19, DPB1*190, DPB1*191, DPB1*192,DPB1*193、DPB1*194、DPB1*195、DPB1*196、DPB1*197、DPB1*198、DPB1*199、DPB1*20、DPB1*200、DPB1*201、DPB1*202、DPB1*203、DPB1*204、DPB1*205、DPB1*206、DPB1*207、DPB1*208、DPB1*209、DPB1*21、DPB1*210、DPB1*211、DPB1*212、DPB1*213、DPB1*214、DPB1*215、DPB1*216、DPB1*217、DPB1*218、DPB1*219、DPB1*22、DPB1*220、DPB1*221、DPB1*222、DPB1*223、DPB1*224、DPB1*225、DPB1*226、DPB1*227、DPB1*228、DPB1*229、DPB1*23、DPB1*230、DPB1*231、DPB1*232、DPB1*233、DPB1*234、DPB1*235、DPB1*236、DPB1*237、DPB1*238、DPB1*239、DPB1*24、DPB1*240、DPB1*241、DPB1*242、DPB1*243、DPB1*244、DPB1*245、DPB1*246、DPB1*247、DPB1*248、DPB1*249、DPB1*25、DPB1*250、DPB1*251、DPB1*252、DPB1*253、DPB1*254、DPB1*255、DPB1*256、DPB1*257、DPB1*258、DPB1*259、DPB1*26、DPB1*260、DPB1*261、DPB1*262、DPB1*263、DPB1*264、DPB1*265、DPB1*266、DPB1*267、DPB1*268、DPB1*269、DPB1*27、DPB1*270、DPB1*271、DPB1*272、DPB1*273、DPB1*274、DPB1*275、DPB1*276、DPB1*277、DPB1*278、DPB1*279、DPB1*28、DPB1*280、DPB1*281、DPB1*282、DPB1*283、DPB1*284、DPB1*285、DPB1*286、DPB1*287、DPB1*288、DPB1*289、DPB1*29、DPB1*290、DPB1*291、DPB1*292、DPB1*293、DPB1*294、DPB1*295、DPB1*296、DPB1*297、DPB1*298、DPB1*299、DPB1*30、DPB1*300、DPB1*301、DPB1*302、DPB1*303、DPB1*304、DPB1*305、DPB1*306、DPB1*307、DPB1*308、DPB1*309、DPB1*31、DPB1*310、DPB1*311、DPB1*312、DPB1*313、DPB1*314、DPB1*315、DPB1*316、DPB1*317、DPB1*318、DPB1*319、DPB1*32、DPB1*320、DPB1*321、DPB1*322、DPB1*323、DPB1*324、DPB1*325、DPB1*326、DPB1*327、DPB1*328、DPB1*329、DPB1*33、DPB1*330、DPB1*331、DPB1*332、DPB1*333、DPB1*334、DPB1*335、DPB1*336、DPB1*337、DPB1*338、DPB1*339、DPB1*34、DPB1*340、DPB1*341、DPB1*342、DPB1*343、DPB1*344、DPB1*345、DPB1*346、DPB1*347、DPB1*348、DPB1*349、DPB1*35、DPB1*350、DPB1*351、DPB1*352、DPB1*353、DPB1*354、DPB1*355、DPB1*356、DPB1*357、DPB1*358、DPB1*359、DPB1*36、DPB1*360、DPB1*361、DPB1*362、DPB1*363、DPB1*364、DPB1*365、DPB1*366、DPB1*367、DPB1*368、DPB1*369、DPB1*37、DPB1*370、DPB1*371、DPB1*372、DPB1*373、DPB1*374、DPB1*375、DPB1*376、DPB1*377、DPB1*378、DPB1*379、DPB1*38、DPB1*380、DPB1*381、DPB1*382、DPB1*383、DPB1*384、DPB1*385、DPB1*386、DPB1*387、DPB1*388、DPB1*389、DPB1*39、DPB1*390、DPB1*391、DPB1*392、DPB1*393、DPB1*394、DPB1*395、DPB1*396、DPB1*397、DPB1*398、DPB1*399、DPB1*40、DPB1*400、DPB1*401、DPB1*402、DPB1*403、DPB1*404、DPB1*405、DPB1*406、DPB1*407、DPB1*408、DPB1*409、DPB1*41、DPB1*410、DPB1*411、DPB1*412、DPB1*413、DPB1*414、DPB1*415、DPB1*416、DPB1*417、DPB1*418、DPB1*419、DPB1*420、DPB1*421、DPB1*422、DPB1*423、DPB1*424、DPB1*425、DPB1*426、DPB1*427、DPB1*428、DPB1*429、DPB1*430、DPB1*431、DPB1*432、DPB1*433、DPB1*434、DPB1*435、DPB1*436、DPB1*437、DPB1*438、DPB1*439、DPB1*44、DPB1*440、DPB1*441、DPB1*442、DPB1*443、DPB1*444、DPB1*445、DPB1*446、DPB1*447、DPB1*448、DPB1*449、DPB1*45、DPB1*450、DPB1*451、DPB1*452、DPB1*453、DPB1*454、DPB1*455、DPB1*456、DPB1*457、DPB1*458、DPB1*459、DPB1*46、DPB1*460、DPB1*461、DPB1*462、DPB1*463、DPB1*464、DPB1*465、DPB1*466、DPB1*467、DPB1*468、DPB1*469、DPB1*47、DPB1*470、DPB1*471、DPB1*472、DPB1*473、DPB1*474、DPB1*475、DPB1*476、DPB1*477、DPB1*478、DPB1*479、DPB1*48、DPB1*480、DPB1*481、DPB1*482、DPB1*483、DPB1*484、DPB1*485、DPB1*486、DPB1*487、DPB1*488、DPB1*489、DPB1*49、DPB1*490、DPB1*491、DPB1*492、DPB1*493、DPB1*494、DPB1*495、DPB1*496、DPB1*497、DPB1*498、DPB1*499、DPB1*50、DPB1*500、DPB1*501、DPB1*502、DPB1*503、DPB1*504、DPB1*505、DPB1*506、DPB1*507、DPB1*508、DPB1*509、DPB1*51、DPB1*510、DPB1*511、DPB1*512、DPB1*513、DPB1*514、DPB1*515、DPB1*516、DPB1*517、DPB1*518、DPB1*519、DPB1*52、DPB1*520、DPB1*521、DPB1*522、DPB1*523、DPB1*524、DPB1*525、DPB1*526、DPB1*527、DPB1*528、DPB1*529、DPB1*53、DPB1*530、DPB1*531、DPB1*532、DPB1*533、DPB1*534、DPB1*535、DPB1*536、DPB1*537、DPB1*538、DPB1*539、DPB1*54、DPB1*540、DPB1*541、DPB1*542、DPB1*543、DPB1*544、DPB1*545、DPB1*546、DPB1*547、DPB1*548、DPB1*549、DPB1*55、DPB1*550、DPB1*551、DPB1*552、DPB1*553、DPB1*554、DPB1*555、DPB1*556、DPB1*557、DPB1*558、DPB1*559、DPB1*56、DPB1*560、DPB1*561、DPB1*562、DPB1*563、DPB1*564、DPB1*565、DPB1*566、DPB1*567、DPB1*568、DPB1*569、DPB1*57、DPB1*570、DPB1*571、DPB1*572、DPB1*573、DPB1*574、DPB1*575、DPB1*576、DPB1*577、DPB1*578m、DPB1*579、DPB1*58、DPB1*580、DPB1*581、DPB1*582、DPB1*583、DPB1*584、DPB1*585、DPB1*586、DPB1*587、DPB1*588、DPB1*589、DPB1*59、DPB1*590、DPB1*591、DPB1*592、DPB1*593、DPB1*594、DPB1*595、DPB1*596、DPB1*597、DPB1*598、DPB1*599、DPB1*60、DPB1*600、DPB1*601、DPB1*602、DPB1*603、DPB1*604、DPB1*605、DPB1*606、DPB1*607、DPB1*608、DPB1*609、DPB1*61、DPB1*610、DPB1*611、DPB1*612、DPB1*613、DPB1*614、DPB1*、 615、DPB1*616、DPB1*617、DPB1*618、DPB1*619、DPB1*62、DPB1*620、DPB1*621、DPB1*622、DPB1*623、DPB1*624、DPB1*625、DPB1*626、DPB1*627、DPB1*628、DPB1*629、DPB1*63、DPB1*630、DPB1*631、DPB1*632、DPB1*633、DPB1*634、DPB1*635、DPB1*636、DPB1*637、DPB1*638、DPB1*639、DPB1*64、DPB1*640、DPB1*641、DPB1*642、DPB1*643、DPB1*644、DPB1*645、DPB1*646、DPB1*647、DPB1*648、DPB1*649、DPB1*65、DPB1*650、DPB1*651、DPB1*652、DPB1*653、DPB1*654、DPB1*655、DPB1*656、DPB1*657、DPB1*658、DPB1*659、DPB1*66、DPB1*660、DPB1*661、DPB1*662、DPB1*663、DPB1*664、DPB1*665、DPB1*666、DPB1*667、DPB1*668、DPB1*669、DPB1*67、DPB1*670、DPB1*671、DPB1*672、DPB1*673、DPB1*674、DPB1*675、DPB1*676、DPB1*677、DPB1*678、DPB1*679、DPB1*68、DPB1*680、DPB1*681、DPB1*682、DPB1*683、DPB1*684、DPB1*685、DPB1*686、DPB1*687、DPB1*688、DPB1*689、DPB1*69、DPB1*690、DPB1*691、DPB1*692、DPB1*693、DPB1*694、DPB1*695、DPB1*696、DPB1*697、DPB1*698、DPB1*699、DPB1*70、DPB1*700、DPB1*701、DPB1*702、DPB1*703、DPB1*704、DPB1*705、DPB1*706、DPB1*707、DPB1*708、DPB1*709、DPB1*71、DPB1*710、DPB1*711、DPB1*712、DPB1*713、DPB1*714、DPB1*715、DPB1*716、DPB1*717, DPB1*718, DPB1*719, DPB1*72, DPB1*720, DPB1*721, DPB1*722, DPB1*723, DPB1*724, DPB1*7 25, DPB1*726, DPB1*727, DPB1*728, DPB1*729, DPB1*73, DPB1*730, DPB1*731, DPB1*732, DPB1*733, DPB1 *734, DPB1*735, DPB1*736, DPB1*737, DPB1*738, DPB1*739, DPB1*74, DPB1*740, DPB1*741, DPB1*742, DP B1*743, DPB1*744, DPB1*745, DPB1*746, DPB1*747, DPB1*748, DPB1*749, DPB1*75, DPB1*750, DPB1*751, DPB1*752, DPB1*753, DPB1*754, DPB1*755, DPB1*756, DPB1*757, DPB1*758, DPB1*759, DPB1*76, DPB1*7 60, DPB1*761, DPB1*762, DPB1*763, DPB1*77, DPB1*78, DPB1*79, DPB1*80, DPB1*81, DPB1*82, DPB1*83, D The DPB1 allele is selected from DPB1*84, DPB1*85, DPB1*86, DPB1*87, DPB1*88, DPB1*89, DPB1*90, DPB1*91, DPB1*92, DPB1*93, DPB1*94, DPB1*95, DPB1*96, DPB1*97, DPB1*98, DPB1*99, DPB2*01, DPB2*02, and DPB2*03. In some embodiments, DPB1 is deleted.

[0072] In some embodiments, the allele matches DMA*01, DMB*01, DOA*01, DOB*01, HFE*001, MICA*002, MICA*007, MICA*008, MICA*009, MICA*010, MICA*012, MICA*018, MICA*019, MICB*002, MICB*004, MICB*005, TAP1*01, TAP1*02, TAP1*03, TAP1*04, TAP1*05, TAP1*06, TAP2*01, or TAP2*02.

[0073] In some embodiments, the collection includes a population or bank with at least the following HLA-C haplotypes: C*07:01 and C*04:01. These HLA-C haplotypes are believed to be sufficient for approximately 46% of the US population.

[0074] In some embodiments, the collection includes cell populations or banked cell populations with one or more of the following HLA-C haplotypes: C*07:01, C*04:01, C*07:02, C*06:02, C*03:04, and C*05:01. These HLA-C haplotypes are believed to be sufficient for approximately 85% of the US population.

[0075] In some embodiments, the collection includes a cell population or bank with one or more of the following HLA-C haplotypes: C*07:01, C*04:01, C*07:02, C*06:02, C*03:04, C*05:01, C*02:02, C*12:03, C*03:03, or C*16:01. These HLA-C haplotypes are believed to be sufficient for approximately 95% of the US population.

[0076] In some embodiments, the collection comprises the following HLA-C alleles: C*01:02:01:01, C*01:02:01:02, C*01:02:01:03, C*01:02:01:04, C*01:02:01:05, C*01:02:01:06, C*01:02:01:07, C*01:02:01:08, C*01:02:01:09, C*01:02:01:10, C*01:02:01:11, C*01:02:01:12, C*01:02:01:13, C*01:02:01:14, C*01:02:01:15, C*01:02:01:16, C*01:02:01:17, C*01:02:01:18, C*01:02:01:19, C*01:02:01:20, C*01:02:01:21, C*01:02:01:22, C*01:02:01:23, C*01:02:01:24, C*01:02:01:25, C*01:02:01:26, C*01:02:01:27, C*01:02:01:28, C*01:02:01:29, C*01:02:01:30, C*01:02:01:31, C*01:02:01:32, C*01:02:01:33, C*01:02:01:34, C*01:02:01:35, C*01 1:02:01:10, C*01:02:01:11, C*01:02:01:12, C*01:02:01:13, C*01:02:01:14, C*01:02:01:15, C *01:02:01:16, C*01:02:01:17, C*01:02:01:18, C*01:02:01:19, C*01:02:01:20, C*01:02:01:21 C*01:02:01:22, C*01:02:01:23, C*01:02:01:24, C*01:02:01:25, C*01:02:01:26, C*01:02:01:27, C*01:02:01:28, C*0 1:02:01:29, C*01:02:01:30, C*01:02:01:31, C*01:02:01:32, C*01:02:01:33, C*01:02:01:34, C*01:02:01:35, C*01:02 :01:36, C*01:02:01:37, C*01:02:01:38, C*01:02:01:39, C*01:02:01:40, C*01:02:01:41, C*01:02:01:42, C*01:02:01:43, C*01:02:01:44, C*01:02:01:45, C*01:02:01:47, C*01:02:01:48, or C*01:02:01:50.

[0077] In some embodiments, the collection comprises the following HLA-DRB1 haplotype alleles: DRB1*01:01:01:01, DRB1*01:01:01:01, DRB1*01:01:01:02, DRB1*01:01:01:03, DRB1*01:01:01:04, DRB1*01:01:01:05, DRB1*01:01:02, DRB1*01:01:03, DRB1*01:01:04, DRB1*01:01:05, DRB1*01:01:06, DRB1*01:01:01:07, DRB1*01:01:08, DRB1*01:01:09, DRB1*01:01:10, DRB1*01:01:11, DRB1*01:01:12, DRB1*01:01:13, DRB1*01:01:14, DRB1*01:01:15, DRB1*01:01:16, DRB1*01:01:17, DRB1*01:01:18, DRB1*01:01:19 ... 1*01:01:07, DRB1*01:01:08, DRB1*01:01:09, DRB1*01:01:10, DRB1*01:01:10, DRB1*01:01:11, DRB1*01:01:12, DRB1*0 1:01:13, DRB1*01:01:14, DRB1*01:01:15, DRB1*01:01:16, DRB1*01:01:17, DRB1*01:01:18, DRB1*01:01:19, DRB1*01:0 1:20, DRB1*01:01:21, DRB1*01:01:22, DRB1*01:01:23, DRB1*01:01:24, DRB1*01:01:25, DRB1*01:01:26, DRB1*01:01:2 7, DRB1*01:01:28, DRB1*01:01:29, DRB1*01:01:30, DRB1*01:01:31, DRB1*01:01:32, DRB1*01:01:33, DRB1*01:01:34, D The present invention includes a cell population or bank having one or more of RB1*01:01:35, DRB1*01:01:36, DRB1*01:01:37, DRB1*01:01:38, DRB1*01:01:39, DRB1*01:01:40, DRB1*01:02:01:01, DRB1*01:02:01:02, DRB1*01:02:01:03, DRB1*01:02:02, DRB1*01:02:03, or DRB1*01:02:04.

[0078] In some embodiments, the cell populations in the collection retain at least one HLA-B gene and represent at least two different HLA-B haplotypes. For example, the cell populations in the collection may represent at least four, or at least six, or at least eight, or at least ten, or at least 12 different HLA-B haplotypes. In some embodiments, the cell lines in the collection represent at least 15 or at least 20 HLA-B haplotypes. In such embodiments, the cell lines are homozygous for the HLA-B gene or are edited to have only a single HLA-B gene.

[0079] In some embodiments, the collection comprises a cell population or bank having at least the following HLA-B haplotypes: B*08:01 and B*07:02.

[0080] In some embodiments, the collection comprises a cell population or bank having one or more of the following HLA-B haplotypes: B*08:01, B*07:02, B*44:02, B*35:01, B*14:02, or B*40:01.

[0081] In some embodiments, the collection comprises a cell population or bank having one or more of the following HLA-B haplotypes: B*08:01, B*07:02, B*44:02, B*44:03, B*35:01, B*57:01, B*15:01, B*14:02, B*40:01, or B*53:01.

[0082] In some embodiments, the collection comprises a cell population or bank having one or more of the following HLA-B haplotypes: B*08:01, B*07:02, B*44:02, B*44:03, B*35:01, B*57:01, B*15:01, B*14:02, B*40:01, B*53:01, B*49:01, B*51:01, B*13:02, or B*38:01.

[0083] In certain embodiments, the cell population or bank represents at least two, at least four, at least six, at least eight, or at least ten different HLA-C to HLA-B haplotypes. In some embodiments, the cell population in the collection represents at least 12 different HLA-C to HLA-B haplotypes. In some embodiments, the cell population in the collection represents at least 20 or at least 25 different HLA-C to HLA-B haplotypes.

[0084] In some embodiments, the collection includes a cell population or bank having the following haplotypes: C*07:01 to B*08:01 and C*07:02 to B*7:02. These haplotypes are believed to be sufficient to cover approximately 32% of the US population.

[0085] In some embodiments, the collection includes a cell population or bank having the following haplotypes: C*07:01 to B*08:01, C*07:02 to B*7:02, C*05:01 to B*44:02, C*16:01 to B*44:03, C*04:01 to B*35:01, C*03:04 to B*40:01, and C*08:02 to B*14:02. These haplotypes are believed to be sufficient to cover approximately 61% of the US population.

[0086] In some embodiments, the collection includes a cell population or bank having the following haplotypes: C*07:01 to B*08:01, C*07:02 to B*7:02, C*05:01 to B*44:02, C*16:01 to B*44:03, C*04:01 to B*35:01, C*03:04 to B*40:01, C*08:02 to B*14:02, C*06:02 to B*57:01, C*3:03 to B*15:01, and C*04:01 to B*53:01. These haplotypes are believed to be sufficient to cover approximately 69% of the US population.

[0087] In some embodiments, the collection comprises a cell population or bank having the following haplotypes: C*07:01 to B*08:01, C*07:02 to B*7:02, C*05:01 to B*44:02, C*16:01 to B*44:03, C*04:01 to B*35:01, C*03:04 to B*40:01, C*08:02 to B*14:02, C*06:02 to B*57:01, C*3:03 to B*15:01, C*04:01 to B*53:01, C*07:01 to B*49:01, C*15:02 to B*51:01, C*06:02 to B*13:02, and C*12:03 to B*38:01. These haplotypes are thought to be sufficient to cover approximately 76% of the US population.

[0088] In some embodiments, a cell population or bank represents at least two, or at least four, or at least six, or at least eight, or at least ten different DRB1 haplotypes. In some embodiments, the cell populations in a collection represent at least 12 or at least 15 different DRB1 haplotypes. The cell lines are either homozygous for the DRB1 gene or edited to have only a single DRB1 gene. In various embodiments, the cell populations are also homozygous for one or more isoforms of DR genes, such as the DRB2, DRB3, DRB4, and DRB5 genes, or edited to have only a single copy of one or more of the DRB2, DRB3, DRB4, and DRB5 genes. In yet other embodiments, DRB2, DRB3, DRB4, and DRB5 are retained and unmodified (and in some embodiments may be homozygous or heterozygous across cell lines).

[0089] In various embodiments, the collection comprises a cell population or bank having at least the following DRBI haplotypes: DRB1*03:01 and DRB1*15:01.

[0090] In some embodiments, the collection comprises a cell population or bank having at least the following DRBI haplotypes: DRB1*03:01, DRB1*15:01, DRB1*04:01, DRB1*07:01, and DRB1*01:01.

[0091] In some embodiments, the collection comprises a cell population or bank having the following DRBI haplotypes: DRB1*03:01, DRB1*15:01, DRB1*04:01, DRB1*07:01, DRB1*01:01, DRB1*01:02, DRB1*04:04, DRB1*13:02, DRB1*13:01, and DRB1*11:04.

[0092] In some embodiments, the collection comprises a cell population or bank having the following DRBI haplotypes: DRB1*03:01, DRB1*15:01, DRB1*04:01, DRB1*07:01, DRB1*01:01, DRB1*01:02, DRB1*04:04, DRB1*13:02, DRB1*13:01, DRB1*11:04, DRB1*15:02, DRB1*03:02, and DRB1*11:01.

[0093] In some embodiments, the collection comprises the following DRB1 haplotypes: DRB1*03:01, DRB1*15:01, DRB1*04:01, DRB1*07:01, DRB1*01:01, DRB1*01:02, DRB1*04:04, DRB1*13:02, DRB1*13:01, DRB1*11:04, DRB1*15:02, DRB1*03 :02, DRB1*11:01, DRB1*15:03, DRB1*04:07, DRB1*08:01, DRB1*08:03, DRB1*08:04, DRB1*08:06, DRB1*08:07, DRB1*08:11, DRB1*04:02, DRB1*14:01, DRB1*10:01.

[0094] In some embodiments, the collection includes a cell population or bank with the following DRBI haplotypes: DRB1*08:02:01 (Asia), DRB1*08:04:01 (Africa), DRB1*08:04:02 (North America), DRB1*08:04:04 (South America), and DRB1*16:01:01 (multi-ethnic).

[0095] In some embodiments, the cell populations or banks in the collection represent at least two, or at least four, or at least six, or at least eight, or at least 10 different HLA-C to DRB1 haplotypes. In some embodiments, the cell populations or banks in the collection represent at least 12 different HLA-C to DRB1 haplotypes. In various embodiments, the cell populations or banks in the collection represent at least 20 or at least 25 different HLA-C to DRB1 haplotypes.

[0096] In embodiments, the cell population or bank represents at least two, or at least four, or at least six, or at least eight, or at least ten different HLA-C to HLA-B to DRB1 haplotypes. In some embodiments, the cell population or bank in the collection represents at least 12 different HLA-C to HLA-B to DRB1 haplotypes. In various embodiments, the cell population in the collection represents at least 20 or at least 25 different HLA-C to HLA-B to DRB1 haplotypes.

[0097] In various embodiments, the collection includes a cell population or bank having at least the following DRBI haplotypes: C*07:01 to B*08:01 to DRB1*03:01 and C*07:02 to B*07:02 to DRB1*15:01. These haplotypes are believed to be sufficient to cover approximately 22% of the US population. See Figure 2.

[0098] In some embodiments, the collection comprises a cell population or bank having at least the following haplotypes: C*07:01 to B*08:01 to DRB1*03:01, C*07:02 to B*07:02 to DRB1*15:01, C*05:01 to B*44:02 to DRB1*04:01, C*16:01 to B*44:03 to DRB1*07:01, C*04:01 to B*35:01 to DRB1*01:01, C*06:02 to B*57:01 to DRB1*07:01, C*06:02 to B*13:02 to DRB1*07:01, and C*08:02 to B*14:02 to DRB1*01:02. These haplotypes are thought to be sufficient to cover approximately 40% of the US population.

[0099] In some embodiments, the collection comprises at least the following haplotypes: C*07:01 to B*08:01 to DRB1*03:01, C*07:02 to B*07:02 to DRB1*15:01, C*05:01 to B*44:02 to DRB1*04:01, C*16:01 to B*44:03 to DRB1*07:01, C*04:01 to B*35:01 to DRB1*01:01, C*06:02 to B*57:01 to DRB1*07:01, C*06:02 to B*13:02 to DRB1*07:01, C*08:02 to B*14:02 to DRB1*07:01, The present invention includes cell populations or banks having B1*01:02, C*3:04-B*40:01-DRB1*04:04, C*04:01-B*44:03-DRB1*07:01, C*03:04-B*40:01-DRB1*13:02, C*03:04-B*15:01-DRB1*04:01, C*05:01-B*18:01-DRB1*03:01, C*05:01-B*44:02-DRB1*13:01, C*07:02-B*07:02-DRB1*01:01, and C*04:01-B*35:02-DRB1*11:04. These haplotypes are believed to be sufficient to cover approximately 51% of the US population.

[0100] In some embodiments, the collection comprises at least the following haplotypes: C*07:01 to B*08:01 to DRB1*03:01, C*07:02 to B*07:02 to DRB1*15:01, C*05:01 to B*44:02 to DRB1*04:01, C*16:01 to B*44:03 to DRB1*07:01, C*04:01 to B*35:01 to DRB1*01:01, C*06:02 to B*57:01 to DRB1*07:01, C*06: 02~B*13:02~DRB1*07:01, C*08:02~B*14:02~DRB1*01:02, C*3:04~B*40:01~DRB1*04:04, C*04:01~B*44:03~DRB1*07:01 , C*03:04~B*40:01~DRB1*13:02, C*03:04~B*15:01~DRB1*04:01, C*05:01~B*18:01~DRB1*03:01, C*05:01~B*44:02~DRB 1*13:01, C*07:02~B*07:02~DRB1*01:01, C*04:01~B*35:02~DRB1*11:04, C*12:02~B52:01~DRB1*15:02, C*03:03~B*15: 01~DRB1*13:01, C*07:02~B*07:02~DRB1*07:01, C*05:01~B*44:02~DRB1*15:01, C*12:03~B*38:01~DRB1*13:01, C*17:0 The present invention includes cell populations or banks having haplotypes C*08:02~B*14:01~DRB1*07:01, C*01:02~B*27:05~DRB1*01:01, C*04:01~B*35:01~DRB1*11:01, C*06:02~B*50:01~DRB1*07:01, C*07:01~B*18:01~DRB1*11:04, and C*04:01~B*53:01~DRB1*13:02. These haplotypes are believed to be sufficient to cover approximately 60% of the US population.

[0101] In some embodiments, the collection comprises at least the following haplotypes: C*07:01 to B*08:01 to DRB1*03:01, C*07:02 to B*07:02 to DRB1*15:01, C*05:01 to B*44:02 to DRB1*04:01, C*16:01 to B*44:03 to DRB1*07:01, C*04:01 to B*35:01 to DRB1*01:01, C*06:02 to B*57:01 to DRB1*07:01, C*06:02 to B*13:02 to DRB1*07:01, C*08:02 to B*14:02 to DRB1*07:01, *01:02, C*3:04~B*40:01~DRB1*04:04, C*04:01~B*44:03~DRB1*07:01, C *03:04~B*40:01~DRB1*13:02, C*03:04~B*15:01~DRB1*04:01, C*05:01~ B*18:01~DRB1*03:01, C*05:01~B*44:02~DRB1*13:01, C*07:02~B*07:02 ~DRB1*01:01, C*04:01~B*35:02~DRB1*11:04, C*12:02~B52:01~DRB1*15: 02, C*03:03~B*15:01~DRB1*13:01, C*07:02~B*07:02~DRB1*07:01, C*05 :01~B*44:02~DRB1*15:01, C*12:03~B*38:01~DRB1*13:01, C*17:01~B*4 2:01~DRB1*03:02, C*08:02~B*14:01~DRB1*07:01, C*01:02~B*27:05~DR B1*01:01, C*04:01~B*35:01~DRB1*11:01, C*06:02~B*50:01~DRB1*07:01 , C*07:01~B*18:01~DRB1*11:04, C*04:01~B*53:01~DRB1~*13:02, C*12: 03~B*18:01~DRB1*15:01, C*07:02~B*07:02~DRB1*11:01, C*05:01~B*44 :02~DRB1*01:01, C*04:01~B*53:01~DRB1*15:03, C*07:02~B*07:02~DRB 1*04:01, C*08:02~B*14:02~DRB1*13:02, C*04:01~B*35:01~DRB1*07:01,C*07:01~B*08:01~DRB1*15:01, C*07:02~B*39:05~DRB1*04:07, C*04:01~B*53:01~DRB1*08:04, C*07:02~B*07:02~DR B1*13:01, C*12:03~B*38:01~DRB1*04:02, C*03:03~B*15:01~DRB1*04:01, C*04:01~B*35:01~DRB1*14:01, C*06:02~B The present invention includes cell populations or banks having haplotypes *37:01 to DRB1*10:01, C*07:01 to B*49:01 to DRB1*11:01, C*03:04 to B*40:01 to DRB1*04:01, C*15:02 to B*51:01 to DRB1*11:01, C*03:02 to B*58:01 to DRB1*03:01, C*03:03 to B*55:01 to DRB1*14:01, and C*04:01 to B*35:01 to DRB1*13:01. These haplotypes are considered sufficient to cover approximately 70% of the US population.

[0102] In various embodiments, the cell population or bank carries HLA-E, HLA-F, and HLA-G genes, which may be homozygous or heterozygous (e.g., unmodified) across the collection.

[0103] In some embodiments, the cell population or bank has deletion, inactivation, or mutagenesis of one or more HLA-DP genes, which may be selected from DPA1, DPA2, DPA3, DPB1, and DPB2. In some embodiments, the cell population or bank has deletion or inactivation of one or both HLA-DPA1 genes and / or one or both HLA-DPB1 genes. In some embodiments, the cell population or bank is homozygous for or retains at least one HLA-DPA1 gene and at least one HLA-DPB1 gene. In some embodiments, the cell line or bank retains DPA2, DPA3, and DPB2 (and these are unmodified and may be homozygous or heterozygous across the cell population or bank).

[0104] In some embodiments, the cell population or bank has deletion, inactivation, or mutagenesis of one or more HLA-DQ genes, which may be selected from DQA1, DQA2, DQB1, DQB2, and DQB3. In some embodiments, one or both HLA-DQA1 genes are deleted. In these or other embodiments, one or both HLA-DQB1 genes are deleted. In some embodiments, DQA2, DQB2, and DQB3 are retained.

[0105] In some embodiments, the cell population or bank has deletion or inactivation of both HLA-DQA1 and both HLA-DQB1 genes. Alternatively, the cell population or bank is homozygous for or carries at least one copy of the HLA-DQA1 and HLA-DQB1 genes. In some embodiments, the cell population or bank is unmodified (and may be homozygous and heterozygous across cell lines) at the HLA-DQA1 and HLA-DQB1 loci.

[0106] In exemplary embodiments, the cell population or bank has both HLA-A genes and both DPB1 genes and / or both DQB1 genes deleted. In such embodiments, the cell line is homozygous for, or retains only a single copy of, HLA-B, HLA-C, and HLA-DRB1. Both copies of all other HLA genes (not specifically annotated as pseudogenes) are retained, and these genes can be homozygous or heterozygous. In some embodiments, both copies of HLA-B are deleted.

[0107] In various embodiments, the cell population is an induced pluripotent stem cell (iPSC) line. In some embodiments, the cell population is a hematopoietic stem cell (HSC) line. For example, an HSC population may be prepared from iPSCs (having a desired gene deletion or inactivation) by the methods described herein. In various embodiments, the iPSC population is a human iPSC population derived from lymphocytes, umbilical cord blood cells, peripheral blood mononuclear cells, CD34+ cells, or human primary tissue, as described herein.

[0108] In some embodiments, cells are derived from HSCs for administration to a recipient, and the cells may be of any hematopoietic lineage. For example, the hematopoietic lineage may be selected from common lymphoid progenitor (CLP) cells, granulocyte-monocyte progenitor (GMP) cells, precursor T cells, T lymphocytes, B lymphocytes, natural killer cells, neutrophils, monocytes, macrophages, dendritic cells, erythrocytes, megakaryocytes, and platelets. The T cells may be CD4+ helper T cells, CD8+ cytotoxic T cells, or regulatory T cells (Tregs).

[0109] In some embodiments, cells including, but not limited to, HSCs, hematopoietic progenitors, and / or hematopoietic lineages (as described above) are derived from allogeneic donors, patients, or banked tissue or cells.

[0110] In some embodiments, the cells are non-hematopoietic stem or progenitor cells, or cells differentiated therefrom. Exemplary stem cells include mesenchymal stem cells, neural stem cells, and epithelial stem cells. In various embodiments, iPSC lines are used to generate a variety of non-hematopoietic cells and tissues, including those selected from neurons (including cortical, dopaminergic, and motor neurons), astrocytes, oligodendrocytes, cardiomyocytes, corneal, chondrocytes, skeletal muscle cells, hepatocytes, pancreatic beta cells, and lung epithelial cells. Protocols for deriving such cells and tissues are known in the art.

[0111] In various embodiments, each cell population within the bank is contained in a separate container suitable for maintaining the viability of the cell line or cell composition for expansion, differentiation, or administration. The cell compositions of the present disclosure may further comprise a pharmaceutically acceptable carrier or vehicle suitable for intravenous infusion or other administration routes, and the composition may include a suitable cryoprotectant. An exemplary, non-limiting carrier is DMSO (e.g., about 10% DMSO).

[0112] In another aspect, the present disclosure provides methods for cell therapy. The methods include administering a cell population derived from a cell population within a collection or bank of the present disclosure to a recipient in need thereof. In various embodiments, the administered cell population or tissue is matched to the recipient for HLA-C. In embodiments in which the administered cell population or tissue possesses at least one HLA-B gene, the cell population or tissue is further matched to the recipient for HLA-B. In some embodiments in which the administered cell population or tissue possesses at least one DRB1 gene, the cell population or tissue is further matched to the recipient for DRB1. In some embodiments in which the administered cell population or tissue possesses at least one HLA-C and DRB1 gene, the cell population or tissue is further matched to the recipient for HLA-C and DRB1. In some embodiments in which the administered cell population or tissue possesses at least one DQB1 gene, the cell population or tissue is further matched to the recipient for DQB1. In yet other embodiments, the cell population is not matched to the recipient for DQB1. In various embodiments, all other loci are mismatched.

[0113] In some embodiments, a method for treating a subject according to the present disclosure includes (a) expanding a population of pluripotent stem cells (e.g., iPSCs) according to the methods described herein, (b) preparing HSCs or their progenitors (as described herein), and (c) introducing a population of hematopoietic stem cells or their progenitors into a subject. Optionally, the hematopoietic stem cells can be differentiated into common megakaryocyte-erythroid progenitors, lymphoid progenitors, progenitor T and / or B cells, common myeloid progenitors, granulocytes, granulocyte-megakaryocyte progenitors, promyelocytes, basophils, eosinophils, neutrophils, erythrocytes, reticulocytes, platelets, megakaryoblasts, platelet-producing megakaryocytes, platelets, monocytes, macrophages, dendritic cells, microglia, osteoclasts, lymphocytes, NK cells, B cells, and / or T cells prior to administration.

[0114] In some embodiments, a cell composition comprises: (a) a medium optionally comprising cytokines and / or growth factors; and (b) one or more hematopoietic lineage cells or populations thereof derived from the cells of the present disclosure, wherein the hematopoietic lineage cells are (i) derivatives of a cell line(s) or bank of expanded primary cells (e.g., iPSCs) such as endothelial or hemogenic endothelial (HE), and the endothelial or HE cells are CD34+ and at least CD43− or CD93− or CXCR4− or CD73− or CXCR4−CD73− or CD34+CD45+;(ii) hematopoietic stem cells identified as CD34+CD31+, CD34+CD144+; (iii) T cell precursors derived from pluripotent stem cells, wherein the T cell precursor is CD34+CD45+CD7+, or CD34-CD45+CD7+, or CD34-CD7+CD5-CD1a-, and optionally comprises a thymic engraftment receptor such as CCR7; (iv) T cells derived from pluripotent stem cells, wherein the T cell is CD45+CD3+CD4+, or CD45+CD3+CD8+; (v) NK cell precursors derived from pluripotent stem cells, wherein the NK cell precursor is CD45+CD56+CD7+; (vi) NK cells derived from pluripotent stem cells, wherein the NK cell precursor is NK (vii) NK cells derived from pluripotent stem cells, wherein the cells are CD3-CD45+CD56+, and optionally further defined as NKp46+, CD57+, and CD16+; (vii) NKT cells derived from pluripotent stem cells, wherein the NKT cells are CD45+Valpha24Jα18+CD3+; (viii) B cells derived from pluripotent stem cells, wherein the B cells are CD45+CD19+; (ix) megakaryocytes derived from pluripotent stem cells, wherein the megakaryocytes are CD41 and / or CXCR4, and optionally they are CXCR4+ or glycoprotein V / CD42d+ or CXCR1 / IL-8RA+ or CXCR2 / IL-8RB+I or integrin alpha 2b / CD41+ or LIFRα or thrombopoietin R / Tpo (x) pluripotent stem cell-derived megakaryocytes that are CD41+ (GPIIb / IIIa), CD42a+ (GPIX), CD42b+ (GPIb), or CD61+; (xi) pluripotent stem cell-derived erythrocytes that are CD235a+; (xii) pluripotent stem cell-derived neutrophils that are CD15+, CD16+, and CD49d(-); and (xiii) pluripotent stem cell-derived granulocyte-monocyte precursors (GMPs);

[0115] In some embodiments, the cell populations are used to generate cell therapies for treating human diseases, including, but not limited to, hematological malignancies, aplastic anemia, hemoglobinopathies, inborn errors of metabolism, and severe immune deficiencies. For example, a subject may have acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, aplastic anemia, Krabbe disease, bone marrow failure syndromes, Hurler syndrome, leukodystrophy, myelodysplastic syndromes, POEMS syndrome, primary amyloidosis, myeloproliferative disorders, myelodysplastic syndromes, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, aplastic anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, thalassemia major, sickle cell disease, severe combined immunodeficiency (SCID), acquired immune deficiency syndrome (AIDS), or the like. (AIDS), Wiskott-Aldrich syndrome, hemophagocytic lymphohistiocytosis, congenital metabolic disorders, epidermolysis bullosa, severe congenital neutropenia, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, leukocyte adhesion deficiency, X-linked SCID, sickle cell disease, alpha thalassemia, beta thalassemia, delta thalassemia, hemoglobin E / thalassemia, hemoglobin S / thalassemia, hemoglobin C / thalassemia, hemoglobin D / thalassemia, chronic granulomatous disease, X-linked chronic granulomatous disease, autosomal recessive (AR) chronic granulomatous disease, chronic granulomatous disease AR I NCF1, chronic granulomatosis AR CYBA, chronic granulomatosis ARII NCF2, chronic granulomatosis AR III NCF4, X-linked severe combined immunodeficiency (SCID), IL7-RA SCID, CD3 SCID, Rag1 / Rag2 SCID, ADA SCID, Artemis SCID, CD45 SCID, Jak3 SCID, congenital agranulocytosis, congenital agranulocytosis-congenital neutropenia-SCN1, congenital agranulocytosis-congenital neutropenia-SCN2, familial hemophagocytic lymphohistiocytosis (FHL), familial hemophagocytic lymphohistiocytosis type 2 (FHL2, perforin mutation), agammaglobulinemia (X-linked agammaglobulinemia), Wiskott-Aldrich syndrome, Chediak-Higashi syndrome, hemolytic anemia due to pyruvate kinase deficiency, paroxysmal nocturnal hemoglobinuria, X-linked adrenoleukodystrophy (X-ALD), X-linked lymphoproliferative disorder, acquired sideroblastic anemia, systemic mastocytosis,von Willebrand's disease (VWD), congenital dyserythroid anemia type 2, cartilage hair hypoplasia, unicentric Castleman's disease, idiopathic multicentric Castleman's disease, congenital amegakaryocytic thrombocytopenia (CAMT) type 1, reticular dysplasia, hereditary spherocytosis, Blackfan-Diamond anemia, Shwachman-Diamond syndrome, mucopolysaccharidoses, Lesch-Nyhan syndrome, glycogen storage diseases, congenital mastocytosis, Omenn's syndrome, X-linked immune dysregulation, polyglandular autoimmune syndrome, thrombocytopenic radial deficiency syndrome, osteopetrosis, and childhood enteropathy (IPEX). is characterized by FOXP3, X-linked syndrome of polyendocrinopathy, immunodeficiency, and diarrhea (XPID), X-linked autoimmune-allergic dysregulation syndrome (XLAAD), IPEX-like syndrome, hyper IgM type 1, hyper IgM type 2, hyper IgM type 3, hyper IgM type 4, hyper IgM type 5, X-linked hyperimmunoglobulin M, bare lymphocyte syndrome type 1 or bare lymphocyte syndrome type 2, myasthenia gravis, rheumatoid arthritis, multiple sclerosis, type 1 diabetes, idiopathic inflammatory myopathy, systemic lupus erythematosus (SLE), myasthenia gravis, Graves' disease, skin Myositis, polymyositis, Crohn's disease, ulcerative colitis, gastritis, Hashimoto's thyroid inflammation, asthma, psoriasis, psoriatic arthritis, dermatitis, systemic sclerosis and sclerosis, inflammatory bowel disease (IBD), respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, atherosclerosis, leukocyte adhesion deficiency, Raynaud's syndrome, Sjogren's syndrome, reactive arthritis, Behcet's disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, immune-mediated thrombocytopenia (e.g., ITP), acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, hemolytic anemia, lupus nephritis, atopic dermatitis , pemphigus vulgaris, opsoclonus-myoclonus syndrome, pure red cell aplasia, mixed cryoglobulinemia, ankylosing spondylitis, hepatitis C cryoglobulinemic vasculitis, chronic regional encephalitis, bullous pemphigoid, hemophilia A, membranoproliferative glomerulonephritis, adult and juvenile dermatomyositis, adult polymyositis, chronic urticaria, primary biliary cholangitis, neuromyelitis optica, Graves' ophthalmopathy, bullous pemphigoid, membranoproliferative glomerulonephritis, Churg-Strauss syndrome, juvenile-onset diabetes, hemolytic anemia, atopic dermatitis, systemic sclerosis, Sjögren's syndrome and glomerulonephritis, dermatomyositis, ANCA,The patient may have a condition selected from aplastic anemia, autoimmune hemolytic anemia (AIHA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, Castleman's disease, Goodpasture's syndrome, solid organ transplant rejection, graft-versus-host disease (GVHD), autoimmune hepatitis, lymphocytic interstitial pneumonia, HIV, bronchiolitis obliterans (non-transplant), Guillain-Barré syndrome, large vasculitis, giant cell (Takayasu) arteries, medium-sized vasculitis, Kawasaki disease, polyarteritis nodosa, Wegener's granulomatosis, microscopic polyangiitis (MPA), Omenn's syndrome, Alzheimer's disease, chronic renal failure, acute infectious mononucleosis, or HIV- and herpes virus-related diseases.

[0116] In some embodiments, the cell populations are used to generate cell therapies to treat cancer, including but not limited to solid and liquid cancers, acquired diseases, congenital diseases, and non-hematopoietic diseases, including but not limited to diseases affecting neurons, astrocytes, oligodendrocytes, cardiomyocytes, skeletal muscle cells, hepatocytes, pancreatic beta cells, and lung epithelial cells.

[0117] In some embodiments, an immune cell lineage (derived from a cell population or bank described herein) is administered to a patient in need thereof. For example, the immune cell lineage may be a T cell, NK cell, B cell, or macrophage. In some embodiments, the T cell lineage is a T regulatory cell or a cytotoxic T cell. In some embodiments, the T cell expresses a heterologous TCR or a chimeric antigen receptor (CAR). In various embodiments, the recipient (patient) has one or more conditions selected from lymphopenia, cancer, immunodeficiency, autoimmune disease, skeletal dysplasia, bone marrow failure syndrome, genetic disorders affecting the immune system, heart failure, neurological disorders, immunodeficiency, blood disorders (e.g., thalassemia, anemia, sickle cell disease), heart disease, liver disease, multiple sclerosis, muscular dystrophy, skin and tissue regeneration, spinal cord degeneration, trauma, stroke, neurodegenerative diseases (e.g., Alzheimer's disease, dementia, Down's syndrome, or Parkinson's disease), metabolic disorders, hematopoietic stem cell transplantation (HPSCT), i.e., the administration of healthy hematopoietic stem cells to patients with dysfunctional or depleted bone marrow, thrombocytopenia, or cancer.

[0118] In various embodiments, the recipient undergoes lymphocyte-depleting, cytoreductive, or immunomodulatory therapy prior to administration of the cell therapy. In some embodiments, derivatives of the cell line(s) disclosed herein or expanded primary cell banks (e.g., HSCs and / or their derived progeny) are administered to reconstitute the recipient's hematopoietic system. Cell lines generated using the methods described herein are administered to the subject, for example, by intravenous infusion. In some embodiments, the methods can be performed following myeloablative, non-myeloablative, or immunotoxin-based (e.g., anti-cKit, anti-CD45, etc.) conditioning regimens.

[0119] In yet other embodiments, cells or tissues derived from the cell population are administered to a recipient in need thereof. Exemplary cells include mesenchymal stem cells, neural stem cells, corneal epithelium / endothelium and RPE, epithelial stem cells, neural cells (or their precursors) (including cortical, dopaminergic, and motor neurons, or their precursors), astrocytes (or their precursors), oligodendrocytes (or their precursors), cardiomyocytes (or their precursors), skeletal muscle cells (or their precursors), hepatocytes (or their precursors), pancreatic beta cells (or their precursors), and pulmonary epithelial cells (or their precursors). Such cells can be administered to treat or ameliorate any disease or condition (including genetic or acquired conditions) afflicting the relevant tissue or organ. Such tissues or organs include, but are not limited to, the central nervous system, skeletal muscle, heart, liver, pancreas, or lung.

[0120] In another aspect, the present disclosure provides methods for producing the cell populations of the present disclosure. The methods can include providing an HLA-modified iPSC population according to the present disclosure, enriching CD34+ cells from the differentiated iPSC population (e.g., embryoid bodies, or EBs) to prepare a CD34+-enriched population, and inducing endothelial-hematopoietic conversion (e.g., for at least 2 days but not more than 12 days) of the CD34+-enriched population to prepare a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem progenitor cells (HSPCs). In some embodiments, the methods further include harvesting the CD34+-enriched population undergoing endothelial-hematopoietic conversion. For example, this can include harvesting CD34+ suspension cells and / or adherent cells, but will generally include at least non-adherent cells.

[0121] Traditionally, hematopoietic lineages are prepared by differentiating iPSCs into embryoid bodies by day 8 and harvesting CD34+ cells. CD34 is commonly used as a marker for blood endothelial cells, hematopoietic stem cells, and hematopoietic progenitor cells. According to embodiments of the present disclosure, endothelial-hematopoietic transition (EHT) can be induced in the CD34+ cell population, which can be derived from iPSC-embryoid bodies and optionally used for ex vivo generation of hematopoietic lineages.

[0122] 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, bone marrow, cord blood, or peripheral blood cells that have been reprogrammed to an embryonic-like pluripotent state. In some embodiments, iPSCs are generated from somatic cells such as fibroblasts or PBMCs (or cells isolated therefrom). In some embodiments, iPSCs are derived from lymphocytes (e.g., T cells, B cells, NK cells, etc.), cord blood cells (including CD3+ or CD8+ cells from cord blood), PBMCs, CD34+ cells, or other primary human tissues. In some embodiments, iPSCs are derived from CD34+ cells isolated from peripheral blood.

[0123] 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-integrated episomal plasmids expressing reprogramming factors, i.e., for the generation of transgene-free and virus-free iPSCs. Conventional episomal plasmids have limited replicative capacity and are therefore lost over several cell generations. Alternative methods include minicircle vectors, PiggyBac transposons, and exosome integration.

[0124] In some embodiments, the iPSC population is genetically edited to delete or inactivate one or more HLA genes. The selection of HLA genes for deletion or inactivation is as previously described. Deletion or inactivation refers to genetic modification (i.e., gene editing) of a target gene to suppress functional expression of the corresponding gene product (i.e., the corresponding polypeptide). Such gene editing includes complete or partial gene deletions as well as deletion of critical cis-acting expression control sequences. For example, with respect to HLA class I genes, the deletion can include deletion of one or more extracellular domains, e.g., α1, α2, and α3. In some embodiments, the HLA class I deletion includes deletion of a transmembrane domain. With respect to HLA class II genes, the deletion can include deletion of one or more extracellular domains, e.g., α1 and / or α2, or β1 and / or β2. In some embodiments, the HLA class II deletion includes deletion of a transmembrane domain. In some embodiments, the HLA deletion includes deletion of the entire coding sequence or substantially the entire coding sequence. In some embodiments, the deletion is targeted to exon 1 and / or exon 2 of the HLA gene, and in various embodiments comprises a deletion of at least 50 base pairs, at least 100 base pairs, at least 250 base pairs, or at least 500 base pairs. In still other embodiments, the gene deletion or inactivation alters a critical expression control sequence, such as a promoter, a cis-acting sequence bound by a transcriptional activator, or a ribosomal binding sequence, thereby substantially reducing or eliminating expression.

[0125] 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. See Figure 11B.

[0126] The target HLA locus is deleted or inactivated using one or more genetic modification tools, such as CRISPR-Cas (e.g., CRISPR-Cas9, CRISPR-Cas12, STAR-CRISPR, CRISPR-CasX, CRISPR-associated transposase), RNA editors, isolated genomic domain platform editing, and combinations thereof. In some embodiments, the target HLA locus can also be deleted or inactivated using siRNAs, oligonucleotides, and / or zinc finger nucleases. In some embodiments, the HLA modification is performed by CRISPR-Cas9 and may use one or a combination of gRNAs (e.g., sgRNAs) containing spacer sequences listed in Tables 2A-17 for specific HLA genes and haplotypes.

[0127] Generally, various 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) and transcription activator-like effector (TALE) gene editing technologies. Fusion proteins comprising one or more of these DNA binding domains and the cleavage domain of a Fokl endonuclease can be used to create double-stranded breaks in desired regions of DNA within a cell (see, e.g., U.S. Patent Application No. 2012 / 0064620, U.S. Patent Application No. 2011 / 0239315, U.S. Patent No. 8,470,973, U.S. Patent Application No. 2013 / 0217119, U.S. Patent No. 8,420,782, U.S. Patent Application No. 2011 / 0301073, U.S. Patent Application 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 No. 2013 / 0122581, the contents of all 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 uses 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-directed. 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 with lower primary sequence homology) to S. pyogenes Cas9 or Prevotella and Francisella 1 (Cpf1 or Cas12a) can be used.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 yet other embodiments, gene editing uses base editing or prime editing to incorporate mutations without initiating double-strand breaks. See, e.g., 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., 09 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-directed system. Brezgin S, Dead Cas Systems: Types, Principles, and Applications, Int J Mol Sci. 2019 Dec;20(23):6041.

[0128] Base editors, which can introduce precise genomic changes without creating 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 catalytically damaging nucleases, such as Cas9 nickase (nCas9), fused to a nucleobase deaminase enzyme and, in some cases, 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 transitions. 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 (e.g., fused) to a Cas endonuclease and a polynucleotide useful as a DNA synthesis template conjugated (e.g., fused) to a guide RNA, as described in WO2020 / 191153A2.

[0129] 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 viruses), paramyxoviruses (e.g., measles and Sendai), positive-stranded RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses, including herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus). 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 can 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.

[0130] To increase efficiency, in some embodiments, Cas and gRNA are combined before delivery to cells. The Cas-gRNA complex is known as a ribonucleoprotein (RNP). Several methods have been developed for directly delivering RNPs to cells. For example, RNPs can be delivered to cells in culture by lipofection or electroporation. Electroporation using a nucleofection protocol is often preferred because it allows RNPs to rapidly enter the cell's nucleus, allowing genome cleavage to begin immediately. 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, which is incorporated herein by reference in its entirety. In some embodiments, Cas9 and gRNA are electroporated as RNPs into iPSCs and / or HSCs derived from donor PBMCs.

[0131] A protospacer adjacent motif (PAM) is required for Cas nuclease cleavage and is typically found 3–4 nucleotides downstream from the cleavage site. The PAM is a short DNA sequence (usually 2–6 base pairs in length) that follows the DNA region targeted for cleavage by a CRISPR system such as CRISPR-Cas9.

[0132] 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.

[0133] In some embodiments, a CRISPR / Cas9 system specific to a donor's unique HLA haplotype can be developed by using the gRNAs described herein to design a single gRNA that targets each of the donor's 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 the DNA repairs via a non-homologous end joining (NHEJ) mechanism, generating indels and resulting frameshift mutations, terminating the function of the resulting protein. However, off-target genetic modifications can occur, potentially altering the function of an otherwise intact gene. For example, the Cas9 endonuclease can create DSBs at undesired off-target locations, even with some degree of mismatching. This off-target activity can lead to genomic instability events such as point mutations and genomic structural polymorphisms.

[0134] Using gRNAs, clonal iPSCs can be developed from donor PBMCs. Such iPSC lines can be assessed for (i) on-target editing, (ii) off-target editing, and (iii) translocation editing, for example, using the sequencing assay described herein. Specifically, such assays can be performed by multiplex PCR using primers designed to target and enrich for the regions of interest, followed by next-generation sequencing (e.g., amplicon sequencing, AMP-seq). The on-target and translocation panels can amplify the intended edited region, allowing for the selection of iPSC clones with predicted edits that do not contain chromosomal translocations resulting from unintended DSB cleavage site fusions. The off-target panel 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 allow for the use of iPSC clone screens to select clones with the desired edits while ruling out potential CRISPR / Cas9-related genome integrity issues.

[0135] Although the differentiation potential of iPSCs has been demonstrated, some tissue-specific epigenetic memory from the starting material may hinder iPSC differentiation. Therefore, in some embodiments, CD34+ cells can be selected for iPSC reprogramming, as described, for example, in Tobin SC and Kim K, "Generating pluripotent stem cells: differential epigenetic changes during cellular reprogramming," FEBS Lett. 2012 Aug. 31;586(18):2874-81, which is incorporated herein by reference in its entirety. In some embodiments, to generate transgene-free and virus-free iPSCs, CD34+ cells are electroporated with an episomal vector to reprogram them into iPSCs. For example, using the oriP / EBNA1 vector, the episomal vector can contain five reprogramming factors (e.g., Oct4, Sox2, Lin28, Klf4, and L-Myc), which replicate extrachromosomally only once per cell cycle and are completely removed once iPSCs reach approximately 5-10 cell cycles. These embodiments do not include transient p53 suppression to maintain important safeguard checkpoints and reduce the risk of selecting clones with genomic instability. To further ensure the genomic stability and integrity of reprogrammed iPSCs, genetic and genomic assays can be performed, for example, to select clones that have not undergone translocation and mutation events and have not integrated the episomal vector.

[0136] In some embodiments, whole genome sequencing (WGS) is performed on CD34+ cells and iPSC clones after reprogramming, and the genomes are 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 did not acquire mutations during reprogramming.

[0137] In some embodiments, karyotyping analysis using a system such as the KARYOSTAT assay is used to select iPSC clones that did not generate 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. Detectable structural abnormalities are >2 Mb in size for chromosomal gains and >1 Mb for chromosomal losses. The KARYOSTAT array is functionalized for balanced whole-genome coverage with low-resolution DNA copy number analysis covering all 36,000 RefSeq genes, including 14,000 OMIM targets. The assay allows for the detection of aneuploidy, submicroscopic abnormalities, and mosaic events.

[0138] 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 (incorporated herein by reference in its entirety). aCGH is a technique that analyzes the entire genome for CNAs by comparing sample DNA with reference DNA.

[0139] In some embodiments, targeted hemolytic malignancy NGS panel analysis is used to select iPSC clones that did not develop hematologic malignancy mutations during reprogramming. For example, targeted hemolytic malignancy NGS panels focus on myeloid leukemia, lymphoma, and / or other hematologic malignancy-associated genes, generating smaller, more manageable datasets than broader methods. Targeted hemolytic malignancy NGS panel analysis involves using highly multiplexed PCR to amplify regions associated with hematologic malignancies, followed by next-generation sequencing.

[0140] 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, although rare, can randomly integrate into the cellular genome, potentially disrupting 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.

[0141] In some embodiments, iPSCs that have undergone one or more of these analyses and demonstrated successful reprogramming are used to construct a pre-edited iPSC seed bank.

[0142] In some embodiments, gene editing is performed on a pre-edited iPSC seed bank as described herein. Specifically, in embodiments, Cas9 and gRNA targeting each donor-specific HLA (e.g., HLA-A, HLA-DPB1, and HLA-DQB1) gene are electroporated into iPSCs, allowing a recovery period of approximately one day to one week. To ensure clonality (e.g., genetic homogeneity resulting from a cell population arising from a single modified cell), single cells can be seeded into individual wells, e.g., in a 384-well plate, using a single-cell printer. Such systems can be automated, require minimal user interface, and ensure clonality through imaging of each cell seeded in each well. After expanding the population from single cells to cell colonies, subcultures can be further expanded into culture trays with larger surface areas, e.g., 96-well plates, 12-well plates, etc., and a portion of the original clonal population of cells can be analyzed via on-target AMP-seq. Such analysis can guide the selection of clones with the desired edits. In some embodiments, a portion of cells with each expansion are used for off-target AMP-seq and / or translocation AMP-seq analysis to confirm genomic integrity throughout the operation.

[0143] In some embodiments, after assessing the absence of gRNA-associated genomic abnormalities in selected clones, the clones can be further tested for mutations that may arise during expansion. For example, mutations affecting hematologic malignancy genes, indels, translocations, and CNAs, as described for pre-edited reprogrammed clones. Mutation analysis can include whole genome sequencing (WGS), KARYOSTAT analysis, array comparative genomic hybridization (aCGH) analysis, targeted hematologic malignancy NGS panel AMP-Seq analysis, and / or droplet digital PCR (ddPCR).

[0144] In some embodiments, clones that are demonstrated to have preserved their genomic integrity are banked as a gene-edited iPSC seed bank.

[0145] In some embodiments, the cell line(s) are modified to express cytokines, suicide gene(s), T cell receptors, single, dual, quad, and / or tandem chimeric antigen receptors (CARs), and / or combinations thereof.

[0146] In some embodiments, cells of the present disclosure (e.g., iPSCs or HSCs, or their precursors or progenitors) can be modified such that certain endogenously expressed genes, such as genes encoding CCR5 or miR-155, or one or more genes encoding cell surface markers, including, but not limited to, CD33, CLL, CD19, CD7, and / or CD38, are deleted or mutated to abolish their expression, or to express non-functional proteins, or to underexpress them. Cell surface molecules that can be genetically modified in accordance with the present disclosure can be selected from any one of the cell surface molecules known to those of skill in the art, e.g., CD1 through CD371, provided that the genetic modification of the selected molecule provides an advantage in the therapeutic application of their wild-type counterparts (i.e., unmodified cells) by eliminating or ameliorating a deleterious or toxic function.

[0147] In various embodiments, the cell line(s) are transfected with a CAR specific for a myeloma, leukemia, or lymphoma target, including but not limited to, CD19, CD33, BCMA, etc.

[0148] In various embodiments, the cell line(s) are inserted with tandem CARs, including but not limited to CD38 / IL3, CD20 / CD19, etc.

[0149] In various embodiments, the cell line(s) are inserted with disease-specific dual CARs, quad CARs, tandem CARs, etc.

[0150] In various embodiments, the cell line(s) are engineered with a leucine-zipper system to incorporate multiple disease-modifying materials, including but not limited to dual CARs, quad CARs, tandem CARs, etc.

[0151] In various embodiments, the cell line(s) are engineered with a leucine-zipper system to incorporate multiple disease-modifying materials.

[0152] Non-limiting examples include, but are not limited to: (i) cell line(s) deleted for CCR5 to generate CCR5-deleted cell therapy for HIV-AIDS patients, (2) cell line(s) deleted for CD33 to generate CD33-deleted cell therapy to treat leukemia and / or lymphoma patients, (3) cell line(s) deleted for CD33 to generate CD33-deleted cell therapy together with CAR-T, CAR-NK, CAR-T progenitor cells, or CAR-macrophage cells to treat leukemia and / or lymphoma patients.

[0153] In various embodiments, the cell line(s) are administered to reduce normal cell killing or adverse effects caused by therapeutic applications of CAR-T therapy with 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, CD371-specific CAR-T cells, or any CAR-NK, CAR-T, or CAR-macrophage cells targeting the following tumor antigens: (i) Human epidermal growth factor receptor 2 (HER2) - ovarian cancer, breast cancer, glioblastoma, colon cancer, osteosarcoma, and medulloblastoma; (ii) epidermal growth factor receptor (EGFR)-positive malignancies, such as non-small cell lung cancer, epithelial carcinoma, cholangiocarcinoma, and glioma; (iii) mesothelin - mesothelioma, ovarian cancer, and pancreatic adenocarcinoma; (iv) prostate-specific membrane antigen (PSMA)-prostate cancer; (v) carcinoembryonic antigen (CEA)—pancreatic adenocarcinoma, breast cancer, and colorectal cancer; (vi) glypican-3-hepatocellular carcinoma; (vii) epidermal growth factor receptor variant III (EGFRvIII)-glioblastoma; (viii) didialoganglioside 2 (GD2) - neuroblastoma and melanoma; (ix) Carbonic anhydrase IX (CAIX) - Renal cell carcinoma; (x) Interleukin-13Ra2-glioma; (xi) fibroblast activation protein (FAP)-malignant pleural mesothelioma; (xii) L1 cell adhesion molecule (L1-CAM)—neuroblastoma, melanoma, and ovarian; (xiii) Cancer antigen 125 (CA125)-epithelial ovarian cancer; (xiv) Cluster of differentiation 133 (CD133) - glioblastoma and cholangiocarcinoma, adenocarcinoma; (xv) Cancer / Testis Antigen 1B (CTAG1B) - melanoma and ovarian cancer; (xvi) Mucin 1 - seminal vesicle cancer; and (xvii) Folate receptor-a (FR-a)—ovarian cancer. (xviii) EGFRvIII-glioblastoma. (xix) Claudin 18.2 - solid tumors, advanced gastric adenocarcinoma, pancreatic adenocarcinoma. (xx) Mesothelin - mesothelioma, metastatic pancreas, ovary, cervix, lung.

[0154] See, for example, Zhou Z et al., Chimeric antigen receptor T cells applied to solid tumors. Front Immunol. 2022 Oct 31; or Pooria et al, Novel antigens of CAR T cell therapy: New roads; old destination, Translational Oncology, Volume 14, Issue 7, 2021, each of which is incorporated herein by reference.

[0155] Alternatively, the cell population can be used to generate cell therapies (e.g., HSCs or immune lineages) for use in FDA-approved CAR-T therapies, such as tisagenlecleucel (also known as tisa-cel (Kymriah)), axicabtagene ciloreucel (also known as axi-cel (Yescarta)), brexcabtagene outrucel (also known as brexu-cel (Tecartas)), lisocabtagene maraleucel (also known as liso-cel (Breyangi)), idecabtagene bicrueucel (also known as ide-cel (Avecma)), ciltacabtagene outrucel (also known as cilta-cel (Carbicty)), or any other CAR-T therapy that damages normal cells during therapeutic use.

[0156] In some embodiments, pluripotent cells (e.g., iPSCs or HSCs, or their precursors or progenitors) of the present disclosure 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 within 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 germ 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.

[0157] According to aspects and embodiments of the present disclosure, methods are provided for generating HLA-modified cells of the present disclosure using CRISPR-Cas9 gene editing. Exemplary sgRNAs are disclosed herein that can be used alone, or in some embodiments, in combination, to generate multiple edits (e.g., double-strand breaks) in a target gene. Exemplary sgRNAs are disclosed herein for generating deletions in exon 1 and / or exon 2 of various HLA genes (including within the genomic coordinates shown in Figure 11B). In various embodiments, the method includes contacting a cell with Cas9 endonuclease (which can be delivered to the cell using any of the known processes) and one or more gRNAs (e.g., sgRNAs) that target the Cas9 endonuclease to an HLA-specific or HLA allele-specific region.

[0158] Tables 2A-17 list spacer sequences useful for targeting HLA alleles as indicated, which can be incorporated into gRNAs (e.g., sgRNAs) of CRISPR-Cas9 systems. In some embodiments, the sgRNA further comprises a scaffold sequence fused to the 3' end of the spacer sequence. In some embodiments, the gRNA further comprises a tracr-mate sequence fused to the 3' end of the spacer sequence.

[0159] In some embodiments, the cells are homozygous or heterozygous for HLA-A*01:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2A or Table 6.

[0160] In some embodiments, the cells are homozygous or heterozygous for HLA-A*02:05, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2B.

[0161] In some embodiments, the cells are homozygous or heterozygous for HLA-A*03:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2B or Table 6.

[0162] In some embodiments, the cells are homozygous or heterozygous for HLA-A*23:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2C.

[0163] In some embodiments, the cells are homozygous or heterozygous for HLA-A*29:02, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2E or Table 6.

[0164] In some embodiments, the cells are homozygous or heterozygous for HLA-A*25:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2D.

[0165] In some embodiments, the cells are homozygous or heterozygous for HLA-A*33:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2G or Table 6.

[0166] In some embodiments, the cells are homozygous or heterozygous for HLA-A*11:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2C or Table 7.

[0167] In some embodiments, the cells are homozygous or heterozygous for HLA-A*26:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2D or Table 7.

[0168] In some embodiments, the cells are homozygous or heterozygous for HLA-A*30:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2E.

[0169] In some embodiments, the cells are homozygous or heterozygous for HLA-A*30:02, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2F.

[0170] In some embodiments, the cells are homozygous or heterozygous for HLA-A*31:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2F.

[0171] In some embodiments, the cells are homozygous or heterozygous for HLA-A*24:02, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 10.

[0172] In some embodiments, the cells are homozygous or heterozygous for DQB1*02:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 4A and Table 8.

[0173] In some embodiments, the cells are homozygous or heterozygous for DQB1*06:02, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 8.

[0174] In some embodiments, the cells are homozygous or heterozygous for DQB1*03:03, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 4B or Table 8.

[0175] In some embodiments, the cells are homozygous or heterozygous for DQB1*05:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 4C or Table 8.

[0176] In some embodiments, the cells are homozygous or heterozygous for DQB1*06:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 4D or Table 9.

[0177] In some embodiments, the cells are homozygous or heterozygous for DQB1*06:03, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 9.

[0178] In some embodiments, the cells are homozygous or heterozygous for DQB1*02:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 4A.

[0179] In some embodiments, the cells are homozygous or heterozygous for DQB1*03:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 4A.

[0180] In some embodiments, the cells are homozygous or heterozygous for DQB1*03:02, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 4B.

[0181] In some embodiments, the cells are homozygous or heterozygous for DQB1*05:03, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 4C.

[0182] In some embodiments, the cells are homozygous or heterozygous for DQB1*06:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 4D.

[0183] In some embodiments, the cells are homozygous or heterozygous for DQB1*06:04, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 4E.

[0184] In some embodiments, the cells are homozygous or heterozygous for HLA-B*07:02, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 3A or Table 11.

[0185] In some embodiments, the cells are homozygous or heterozygous for HLA-B*13:02, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 3B.

[0186] In some embodiments, the cells are homozygous or heterozygous for HLA-B*44:03, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 3F or Table 11.

[0187] In some embodiments, the cells are homozygous or heterozygous for HLA-B*18:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 3C.

[0188] In some embodiments, the cells are homozygous or heterozygous for HLA-B*57:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 3G or Table 11.

[0189] In some embodiments, the cells are homozygous or heterozygous for HLA-B*08:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 3A or Table 11.

[0190] In some embodiments, the cells are homozygous or heterozygous for HLA-B*40:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 3E.

[0191] In some embodiments, the cells are homozygous or heterozygous for HLA-B*14:02, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 3B or Table 12.

[0192] In some embodiments, the cells are homozygous or heterozygous for HLA-B*50:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 3F or Table 12.

[0193] In some embodiments, the cells are homozygous or heterozygous for HLA-B*37:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 12.

[0194] In some embodiments, the cells are homozygous or heterozygous for HLA-B*52:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 3G or Table 12.

[0195] In some embodiments, the cells are homozygous or heterozygous for HLA-B*38:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 3D or Table 13.

[0196] In some embodiments, the cells are homozygous or heterozygous for HLA-B*35:01, and the cells are contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 3D or Table 13.

[0197] In some embodiments, the cells are homozygous or heterozygous for DRB1*03:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5A or Table 14.

[0198] In some embodiments, the cells are homozygous or heterozygous for DRB1*15:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5F or Table 14.

[0199] In some embodiments, the cells are homozygous or heterozygous for DRB1*07:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5C or Table 14.

[0200] In some embodiments, the cells are homozygous or heterozygous for DRB1*01:02, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5A or Table 14.

[0201] In some embodiments, the cells are homozygous or heterozygous for DRB1*10:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 15.

[0202] In some embodiments, the cells are homozygous or heterozygous for DRB1*15:02, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5F or Table 15.

[0203] In some embodiments, the cells are homozygous or heterozygous for DRB1*13:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5D or Table 15.

[0204] In some embodiments, the cells are homozygous or heterozygous for DRB1*01:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5A or Table 15.

[0205] In some embodiments, the cells are homozygous or heterozygous for DRB1*04:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5B.

[0206] In some embodiments, the cells are homozygous or heterozygous for DRB1*04:02, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5B.

[0207] In some embodiments, the cells are homozygous or heterozygous for DRB1*04:04, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5C.

[0208] In some embodiments, the cells are homozygous or heterozygous for DRB1*11:04, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5D.

[0209] In some embodiments, the cells are homozygous or heterozygous for DRB1*13:02, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5E.

[0210] In some embodiments, the cells are homozygous or heterozygous for DRB1*14:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5E.

[0211] In some embodiments, the cells are homozygous or heterozygous for HLA-DPB1*01:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 16.

[0212] In some embodiments, the cells are homozygous or heterozygous for HLA-DPB1*02:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 16.

[0213] In some embodiments, the cells are homozygous or heterozygous for HLA-DPB1*03:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 16.

[0214] In some embodiments, the cells are homozygous or heterozygous for HLA-DPB1*04:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 16.

[0215] In some embodiments, the cells are homozygous or heterozygous for HLA-DPB1*04:02, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 17.

[0216] In some embodiments, the cells are homozygous or heterozygous for HLA-DPB1*11:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 17.

[0217] In some embodiments, the cells are homozygous or heterozygous for HLA-DPB1*17:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 17.

[0218] In some embodiments, the cells are homozygous or heterozygous for HLA-DPB1*34:01, and the cells are contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 17.

[0219] In some embodiments, hiPSCs can be used to generate embryoid bodies (EBs), which can be used to generate (i.e., isolate or enrich for) CD34+ cells. For example, EBs can be dissociated and CD34+ hematopoietic progenitors isolated or enriched. In some embodiments, human iPSC aggregates are expanded in a bioreactor, e.g., as described 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.

[0220] Other bioreactors may include, but are not limited to, shear stress, mechanical strain, and pulsed electromagnetic field bioreactors, large-scale stirred tank bioreactors, automated bioreactors, rotating wall bioreactors (RWBs), and wave bioreactors, such as those found in organ-chip bioreactors. Other bioreactor configurations that allow for continuous perfusion operation, such as packed-bed bioreactors (PBBs), fluidized-bed bioreactors (FBBs), or PBBs and / or FBBs involving the use of microcarriers, CultiBag bioreactors, and membrane bioreactors such as hollow-fiber bioreactors (HFBs), may also be used. Such bioreactors are contemplated for producing the pluripotent cells of the present disclosure or precursors or progenitors derived therefrom. Bioreactor operation may require coupling to an internal or external cell retention device in a recirculation line by centrifugation, sedimentation, ultrasonic separation, or microfiltration by spin filters, alternating tangential flow (ATF) filtration, or tangential flow filtration (TFF).

[0221] In some embodiments, the process of generating a cell population comprising HSCs and / or HSPCs or their progeny can include generating CD34+ enriched cells from pluripotent stem cells (e.g., EBs) and inducing endothelial hematopoietic differentiation (e.g., for at least 2 days but not more than 12 days). In some embodiments, HSCs, including relatively high frequencies of LT-HSCs, can be generated from cell populations using a variety of stimuli or factors, including mechanical, biochemical, metabolic, and / or topographical stimuli, as well as factors such as extracellular matrix, niche factors, cell-extrinsic factors, induction of cell-intrinsic properties, and pharmacological and / or genetic means.

[0222] In some embodiments, the method involves preparing endothelial cells with hematopoietic potential from pluripotent stem cells prior to induction of EHT. In some embodiments, combined overexpression of GATA2 / ETV2, GATA2 / TAL1, or ER71 / GATA2 / SCL can result in the formation of endothelial cells with hematopoietic potential from a PSC source. In some embodiments, the method involves 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 the introduction of a non-integrated episomal plasmid encoding it 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. See PCT / US2021 / 062884, incorporated herein by reference in its entirety.

[0223] 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+, or at least 40% CD34+. In some embodiments, CD34+ enrichment and EHT can be induced between days 6 and 14 of iPSC differentiation, e.g., days 7, 8, 9, 10, 11, 12, 13, or 14. Differentiation of iPSCs can follow known techniques. In some embodiments, iPSC differentiation involves factors such as, but not limited to, a combination of bFGF, Y27632, BMP4, VEGF, SCF, EPO, TPO, IL-6, IL-11, and / or IGF-1. In some embodiments, hPSCs are differentiated using feeder-free, serum-free, and / or GMP-compatible materials. In some embodiments, hPSCs are co-cultured with mouse bone marrow-derived feeder cells, such as OP9 or MS5, or mouse embryonic fibroblast cell lines 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 that can be further differentiated 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 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).

[0224] Induction of EHT can be 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 via 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, e.g., 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.

[0225] In some embodiments, HSC and / or HSPC populations or fractions thereof are differentiated into T cells or precursors or derivatives thereof, with or without the use of agonists of mechanosensitive receptors or mechanosensitive channels, such as Yoda1. In some embodiments, the use of agonists of mechanosensitive receptors or mechanosensitive channels (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 conversion of the CD34+ enriched cell population is induced for at least 2 days but not more than 12 days, with the optional use of agonists of mechanosensitive receptors or mechanosensitive channels, such as Yoda1, jedi1, jedi2, or ssRNA40. HSC and / or HSPC are differentiated into progenitor T cell populations or T cell populations (e.g., as described herein). In some embodiments, endothelial-hematopoietic conversion of the CD34+ enriched cell population is induced for at least 2 days, or further for 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. Total EHT differentiation proceeds for 12 days or less.

[0226] In various embodiments, CD34+ cells (e.g., floating and / or adherent cells) are harvested from cultures undergoing endothelial-hematopoietic conversion between days 10 and 20 of iPSC differentiation, e.g., between days 10 and 17, or between days 12 and 15 of iPSC differentiation.

[0227] In some embodiments, the method comprises increasing the expression or activity of dnmt3b in PSCs, embryoid bodies, CD34-enriched cells, ECs, HECs, or HSCs, which may be by mechanical, genetic, biochemical, or pharmacological means. In some embodiments, the method comprises increasing the activity or expression of DNA (cytosine-5-)-methyltransferase 3 beta (Dnmt3b) and / or GTPase IMAP family member 6 (Gimap6) in the cells. See WO2019 / 236943 and WO2021 / 119061, which are incorporated by reference in their entireties. In some embodiments, the induction of EHT comprises increasing the expression or activity of dnmt3b.

[0228] In some embodiments, the cells are contacted with an effective amount of an agent, such as, but not limited to, an agonist of a mechanosensitive receptor or mechanosensitive channel, which increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezol. An exemplary Piezol agonist is Yodal.

[0229] In some embodiments, the mechanosensitive receptor is Trpv4. An exemplary Trpv4 agonist is GSK1016790A. 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).

[0230] Yoda1 has the following structure: [ka]

[0231] Derivatives of Yodali can be used in various embodiments. For example, derivatives containing a 2,6-dichlorophenyl core are used in some embodiments. Exemplary agonists are disclosed in Evans EL, et al., "Yodali analogue (Dooku1) which antagonizes Yodali-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; 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, an effective amount of a Piezo1 agonist or derivative is from about 1 μM to about 500 μM, or from about 5 μM to about 200 μM, or from about 5 μM to about 100 μM, or in some embodiments, from about 25 μM to about 150 μM, or from about 25 μM to about 100 μM, or from about 25 μM to about 50 μM. Alternatively, single-stranded (ss) RNA (e.g., ssRNA) and its derivatives and analogs can be used for Piezo1 activation.

[0232] In various embodiments, pharmacological Piezo1 activation is applied to CD34+ cells (i.e., CD34-enriched cells). In certain embodiments, pharmacological Piezo1 activation may further be applied to iPSCs, embryoid bodies, ECs, blood endothelial cells (HECs), HSCs, hematopoietic progenitors, 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. The use of Piezo1 activation for the generation of HSCs or their progeny is described in US2021 / 0222125 and US2022 / 00049221, which are incorporated herein by reference in their entireties.

[0233] 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 delivering a transcript encoding Dnmt3b into cells, or by introducing a transgene encoding Dnmt3b, or by a transgene-free method, including but not limited to introducing a non-integrating episome into cells. In some embodiments, gene editing is used to introduce genetic modifications into Dnmt3b-expressing elements in cells, including, but not limited to, to increase promoter strength, ribosome binding, RNA stability, and / or affect RNA splicing.

[0234] In some embodiments, the method involves increasing the activity or expression of Gimap6 in a cell, either alone or in combination with Dnmt3b and / or other genes that are up- or down-regulated upon repetitive strain or Piezol activation. To increase Gimap6 activity or expression, an mRNA transcript encoding Gimap6 can be introduced into the cell; transgene-free approaches can also be used, including, but not limited to, introducing an episome into the cell, or alternatively, introducing a transgene encoding Gimap6. In some embodiments, gene editing is used to introduce genetic modifications into Gimap6-expressing elements in the cell (such as one or more modifications to increase promoter strength, ribosome binding, RNA stability, or affect RNA splicing).

[0235] In embodiments of the present disclosure that use 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 trigger 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 9,181,319, which is incorporated herein by reference, particularly with respect to nucleotide modifications for circumventing the innate immune response.

[0236] 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 (by varying promoter strength or other selection of expression control elements). The transgene can be introduced using various viral vectors or transfection reagents (including lipid nanoparticles), as known in the art. In some embodiments, expression of Dnmt3b and / or Gimap6 is increased by transgene-free methods (e.g., episomal delivery or lipid nanoparticles carrying mRNA). 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.

[0237] In some embodiments, the method includes applying repeated 2D, 3D, or 4D stretch to cells. In various embodiments, the cells subjected to repeated 2D, 3D, or 4D stretch are selected from one or more of CD34-enriched cells, iPSCs, ECs, and HECs. For example, the cell population is introduced into a bioreactor that provides cyclic strain, biomechanical stretch, or cyclic strain biomechanical stretch. Cyclic strain biomechanical stretch is described in WO2017 / 096215, which is incorporated herein by reference in its entirety. Cyclic strain, biomechanical stretch, or cyclic strain biomechanical stretch can increase the activity or expression of Dnmt3b and / or Gimap6. In these embodiments, the mechanical 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, cyclic 2D, 3D, or 4D stretch can be applied to cells ex vivo under defined and controlled cyclic strain conditions using a computer-controlled vacuum pump system or other means for providing a stretching force attached to a flexible biocompatible and / or biomimetic surface (e.g., the FlexCell™ Tension System, Cytostretcher System). For example, the cyclic stretch applied can be about 1% to about 20% cyclic strain (e.g., about 6% cyclic strain) for several hours or several days (e.g., about 7 days). In various embodiments, the cyclic strain or mechanical stretch 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.

[0238] In various embodiments, the cyclic strain or mechanical stretch is applied for a period ranging from at least about 1 hour to about 2 hours, from at least about 6 hours to about 8 hours, from at least about 12 hours to about 24 hours, from at least about 48 hours to about 72 hours, from at least about 96 hours to about 120 hours, or from at least about 144 hours to about 168 hours.

[0239] Alternatively, or in addition, EHT is stimulated by Trpv4 activation, which can be achieved 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.

[0240] In some embodiments, the cells are contacted with an effective amount of an agent that (a) modulates histone acetylation, or (b) modulates histone methylation, or (c) modulates TGFbeta signaling, or (d) modulates the wnt and / or notch signaling pathways.

[0241] The modulator can be selected from inhibitors that modulate signal transduction via the TGF-beta pathway, the Wnt pathway, or the Notch pathway, or that modulate histone methylation and / or acetylation. Some proteins known to be acetylated include p53, HSP90, tubulin, NF-κB, HIF-1α, RUNX3, STAT-3, E2F1, Ku70, and c-MYC. Acetylation functions as a widespread post-translational modification that regulates protein function, including DNA binding, transcription factor activity, subcellular localization, and protein stability.

[0242] Non-limiting examples of modulators include, but are not limited to, histone methyltransferase EZH1, DNA methyltransferase inhibitors (DNMTi), histone deacetylase inhibitors (HDACi), suberoylanilide bishydroxamic acid (SBHA), tranylcypromine, LSD1 inhibitors such as IV RN-1, LSD1-C76, LSD1 inhibitor II S2101, LSD1 inhibitor III CBB1007, LSD1 inhibitor I, SNDX-275 (MS-275, entinostat), CI-994 (tacedinaline), MGCD-0103, valproic acid (VPA), sodium butyrate, phenyl butyrate (S-HDAC-42, AR-42), depsipeptide (romidepsin), apicidin, JNJ-26481585, suberoylanilide hydroxamic acid (SA HA, vorinostat), NVP-LAQ824 (dacinostat), CR-2408, RAS2410 (resminostat), trichostatin (TSA), LBH589 (panobinostat), ITF2357 (gibinostat), PXD101 (belinostat), ACY-1215 (rosilinostat), KD5170, and inhibitors of tubacin.

[0243] Some TGF-β R kinase inhibitors are designed to bind to the ATP-binding domain of TGF-β R kinase, inhibit ATP kinase activity, and block downstream signal transduction cascades. The TGF-β inhibitor can be selected from one or more of: galunisertib (LY21557299), ALK5 inhibitor II (E-616452), LY364947, A83-01, and DMH1, LY573636 (tasisuram), LY2109761, LY364937, Ki26894, LY580276, SB-431542, SB-505124, A83-01, SD-093 and SD-208, IN-1130, and bactosertib (TEW-7197). TGF-β inhibitors can also include antibodies such as, but not limited to, SRK181-mIgG1, fresolimumab, LY3022859, 264RAD, 1D11, 2G7, or pyrimidoindole derivatives, including, for example, UM171 or UM729.

[0244] In certain instances, the agent comprises a compound that inhibits a protein that propagates p38 signaling, such as SB203580. In additional embodiments, the one or more agents comprise a compound that inhibits a protein that promotes β-catenin degradation selected from one or more of lithium chloride, CHIR99021, ICG-001, XAV939, pyrvinium, BIO, C2 inhibitor, CRT-3, -5 and -14, stapled peptide StAx35R, and FGF2, or recombinant versions thereof.

[0245] In some embodiments, the methods use allosteric agonists, including but not limited to yoda1, jedi1, jedi2, docosahexaenoic acid or analogs thereof, or any agonist that modulates the activity of mechanosensitive Piezo channels.

[0246] In a non-limiting example, HSCs derived from iPSC cell line(s) are generated by CD34 enrichment from embryonic bodies and endothelial-hematopoietic conversion induced on days 8-15 of iPSC differentiation. CD34+ cells are harvested from cultures undergoing endothelial-hematopoietic conversion, including harvesting of CD34+ suspension and / or adherent cells. In some embodiments, induction of endothelial-hematopoietic conversion includes increasing the expression or activity of dnmt3b.

[0247] In various embodiments, inducing endothelial hematopoietic conversion comprises applying repeated stretch to the CD34-enriched population, where the repeated stretch is optionally 2D, 3D, or 4D repeated stretch. Alternatively, or in addition, inducing endothelial hematopoietic conversion comprises Piezo1 activation. Piezo1 activation can be achieved by contacting the CD34-enriched cells or a fraction thereof with one or more Piezo1 agonists, optionally selected from Yodal, Jedil, Jedi2, or analogs or derivatives thereof. In some embodiments, inducing endothelial hematopoietic conversion comprises Trpv4 activation, where Trpv4 activation is optionally by contacting the CD34-enriched cells with one or more Trpv4 agonists, optionally selected from one or more of GSK1016790A, 4α-PDD, or analogs or derivatives thereof. Optionally, any of the modulating agents provided in the present disclosure may be used in combination with any other agent(s) disclosed herein.

[0248] It is contemplated that one or more agents may be added to act simultaneously or to act on the same pathway or different pathways, for example, they may act simultaneously as inhibitors of TGF-β, or they may act independently to inhibit histone demethylase and TGF-β, respectively, which may be simultaneous or sequential.

[0249] When a cell population or bank is described herein as having a particular phenotype, it is understood that the phenotype represents a significant portion of the cell population, e.g., 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 a desired phenotype and / or depleted of cells of an undesired phenotype, resulting in a cell population containing 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 cells with 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.

[0250] In various embodiments, CD34+ cells (eg, suspension and / or adherent cells) are harvested from cultures undergoing endothelial-hematopoietic transition between days 8 and 15 of iPSC differentiation.

[0251] 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 US 8,168,428; US 9,028,811; US ​​10,272,110, and US 10,278,990. In some embodiments, the ex vivo expansion of the HSCs or CD34-enriched cells uses 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.

[0252] Hematopoietic stem cells (HSCs), which give rise to the erythroid, myeloid, and lymphoid lineages, can be identified based on the expression of CD34 and the absence of lineage-specific markers (called Lin-). In some embodiments, a population of stem cells containing HSCs is enriched, for example, as described in US9,834,754 (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, CD45, CD38, and CD43. The fraction can be enriched based on the expression of CD34+, CD90+, or CD45+. + and / or CD45 + , CD38 - , and CD43 - In some embodiments, stem cell populations for differentiation into hematopoietic lineages can be selected for further differentiation that are one or more of: + , or at least about 90% CD34 + , or at least about 95% CD34 + is.

[0253] In some embodiments, pluripotent hematopoietic stem cells (HSCs) self-renew and differentiate into two types of progenitor cells with specific lineage commitment. Similar to HSCs, human lineage-restricted progenitor cells express CD34 and Flt-3 / Flk-2. Myeloid progenitors (MPs) in humans and mice express IL-3Rα and give rise to cells of the myeloid lineage, including megakaryocytes, erythrocytes, granulocytes, and macrophages. Lymphoid progenitors (LPs) develop from HSCs and are responsible for generating B cells, T cells, and natural killer (NK) cells. Human bone marrow LPs are CD34+CD38+neprilysin+, while umbilical cord blood CLPs are CD34+CD38-CD7+.

[0254] In some embodiments, the cell population is differentiated into hematopoietic lineage cells for administration to a recipient. In various embodiments, the hematopoietic lineage cells are selected from common lymphoid progenitor (CLP) cells, granulocyte-monocyte progenitor (GMP) cells, progenitor T cells, T lymphocytes, B lymphocytes, natural killer cells, neutrophils, monocytes, macrophages, erythrocytes, megakaryocytes, and platelets. In some aspects and embodiments, the present disclosure provides methods for generating a CD7+ progenitor T cell population, or a derivative of this population. For example, the method includes generating a hematopoietic stem cell (HSC) population, including human long-term hematopoietic stem cells (LT-HSCs), from iPSCs (e.g., hiPSCs). The HSC population is derived by inducing endothelial-hematopoietic transformation of CD34+ cells (e.g., CD34+ cells derived from embryoid bodies). HSC populations (or cells isolated therefrom) are cultured with partial or complete Notch ligand, sonic hedgehog (SHH), retronectin (or other extracellular matrix component(s)), and / or combinations thereof to generate CD7+ progenitor T cell or derivative cell populations.

[0255] The Notch signaling pathway regulates the formation, differentiation, and function of precursor T cells, pre-T cells, and / or mature T lymphocytes. In vivo, T cell development progresses after lymphoid precursors differentiate from bone marrow hematopoietic stem cells and migrate to the thymus. Specialized thymic epithelial cells develop T cells along a regulated pathway. Notch signaling plays a key role in T lineage commitment in the thymus. As lymphoid precursors enter the thymus, they encounter high-density expression of Notch ligands on the thymic epithelium, which drives thymic lymphopoiesis. The present disclosure provides HSC populations generated ex vivo from iPSCs that respond to Notch ligands, SHH, and / or extracellular matrix components by robustly generating T precursor cells and T cell lineages ex vivo.

[0256] In some embodiments, the stem cell population, or CD34-enriched cell or fraction thereof, or derivative population, is expanded as described in US2020 / 0308540, the entire contents of which are incorporated herein by reference. 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.

[0257] In some embodiments, CD34 + Compounds that promote cell expansion include pyrimidoindole derivatives, including, for example, UM171 or UM729 (see US2020 / 0308540, incorporated herein by reference).

[0258] In some embodiments, the stem cell population or CD34-enriched cells are further enriched for cells that express periostin and / or platelet-derived growth factor receptor alpha (pdgfra) or are modified to express periostin and / or pdgfra, as described in WO 2020 / 205969 (incorporated herein by reference in its entirety). Such expression may be by delivering an encoding transgene to the cells, or by introducing an encoding transgene, or by transgene-free methods, including but not limited to introducing a non-integrated episome into the cells. In some embodiments, gene editing is used to introduce genetic modifications into expression elements in cells to modify promoter activity or strength, ribosome binding, RNA stability, or impact RNA splicing.

[0259] In yet another embodiment, the stem cell population or CD34-enriched cells are cultured with an inhibitor of histone methyltransferase EZH1. Alternatively, EZH1 is partially or completely deleted or inactivated, or transiently silenced, in the stem cell population. Inhibition of EZH1 can direct myeloid progenitor cells (e.g., CD34+CD45+) toward the lymphoid lineage. See WO2018 / 048828, the entire contents of which are incorporated herein by reference. In yet another embodiment, EZH1 is overexpressed in the stem cell population.

[0260] In various embodiments, the cell lines developed from primary cells or iPSC cell lines or cell lines derived from banks thereof are hematopoietic lineage cells selected from common lymphoid progenitor (CLP) cells, granulocyte-monocyte progenitor (GMP) cells, precursor T cells, T lymphocytes, B lymphocytes, natural killer cells, neutrophils, monocytes, macrophages, erythrocytes, megakaryocytes, and platelets.

[0261] In some embodiments, the HSC population or fractions thereof are differentiated ex vivo into progenitor T cells, T cells, NK cells, and / or fractions or analogs thereof.

[0262] In some embodiments, the HSC population or a fraction thereof is cultured with a partial or complete Notch ligand to produce a population comprising a CD7+ progenitor T cell or derivative cell population.

[0263] In some embodiments, the derivative cell population is a T cell, progenitor, subtype, and derivative, or NK cell population.

[0264] In some embodiments, the cell population is cultured with a partial or complete Notch ligand, SHH, extracellular matrix component(s), and / or combinations thereof to differentiate HSCs into CD7 + These cells are differentiated into precursor T cells and, optionally, into the T cell lineage or other lineages (e.g., NK cells). Furthermore, according to known processes, xenogeneic OP9-DL1 cells are often used for differentiation into T cells. The OP9-DL1 coculture system uses a bone marrow stromal cell line (OP9) transduced with the Notch ligand Delta-like-1 (DLL1) to support T cell development from a stem cell source. The OP9-DL1 system limits the potential of the cells for clinical applications. There is a need for a feeder-cell-free system capable of generating T lymphocytes from hiPSCs for clinical use, and in some embodiments, the present disclosure fulfills this objective.

[0265] 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 contain 20-22 amino acids at the amino terminus and have DSL domains (D-δ, S-Serrate, and L-Lag2) containing 3-8 EGF repeats on the extracellular surface. In various embodiments, Notch ligands include Delta-like-1 (DLL1), Delta-like-4 (DLL4), SFIP3, and Delta-like-5 (DLL5). Max(Disclosed in PCT / US2020 / 041765 and PCT / US2020 / 030977, which are incorporated herein by reference in their entireties), or a functional portion thereof. A key signal delivered by thymic stromal cells to incoming lymphocyte precursors in vivo is mediated by DL4, which is expressed by cortical thymic epithelial cells.

[0266] The earliest intrathymic precursors 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 typically involve the expression of CD34 + CD1a - (mostly immature) and CD34 + CD1a + Depicted as cells. CD34 by early thymocytes + CD7 + CD1a - from CD34 + CD7 + CD1a + Transition to CD34 is associated with T cell commitment. + CD7 + CD1a + The cells are likely T lineage restricted. Following this stage, thymocytes progress to a CD4 immature single-positive stage, at which point CD4 is expressed in the absence of CD8. A subset of cells then expresses CD4 + CD8 + Finally, after TCRα rearrangement, TCRαβ-expressing DP thymocytes undergo positive and negative selection and differentiate into CD4 + CD8 - and CD4 - CD8 + Obtain single positive (SP) T cells.

[0267] 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, compounds that promote expansion include pyrimidoindole derivatives, including, for example, UM171 or UM729 (see US2020 / 0308540, which is incorporated herein by reference).

[0268] 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 + and optionally CD34 + In some embodiments, the progenitor T cells are CD7 + CD1a - CD5, and optionally CD34 + is.

[0269] In some embodiments, the progenitor T cells exhibit reduced levels of CD34 expression, minimal CD34 expression (compared to the HSC population), or no CD34 expression, hi some embodiments, CD34 expression is reduced by at least about 50%, or at least about 75%, in the population relative to the HSC population.

[0270] In some embodiments, the Notch ligand is an anti-Notch (agonist) antibody capable of binding to and engaging 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 capable of activating the Notch signaling pathway.

[0271] In some embodiments, the Notch ligand is a Notch ligand of the Delta family, hi some embodiments, the Delta family ligand is Delta-1 (Genbank accession number AF003522, Homo sapiens), Delta-like 1 (DLL1, Genbank accession numbers NM_005618 and NP_005609, Homo sapiens; Genbank accession numbers X80903, 148324, Mus musculus), Delta-4 (Genbank accession number AF273454, BAB18580, Mus musculus; Genbank accession numbers AF279305, AAF81912, Homo sapiens), and / or Delta-like 4 (DLL4; Genbank accession numbers Q9NR61, AAF76427, AF253468, NM_019074, Homo sapiens; Genbank accession number NM019454, Mus musculus). Notch ligands are commercially available or can be produced, for example, by recombinant DNA techniques.

[0272] In some embodiments, the Notch ligand comprises an amino acid sequence that is at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 97% identical (e.g., about 100% identical) to a human DLL1 or DLL4 Notch ligand. Functional derivatives of Notch ligands (including fragments or portions thereof) are capable of binding to and activating Notch receptors. Binding to Notch receptors can be determined by a variety of methods known in the art, including in vitro binding assays and receptor activation / cell signaling assays.

[0273] In some embodiments, the Notch ligand is DLL4 with one or more affinity-enhancing mutations, for example, one or more (or all) of G28S, F107L, I143F, H194Y, L206P, N257P, T271L, F280Y, S301R, and Q305P relative to hDLL4. See Gonzalez-Perez, et al., Affinity-matured DLL4 ligands as broad-spectrum modulators of Notch signaling, Nature Chemical Biology (2022).

[0274] In various embodiments, the Notch ligand is immobilized on a soluble, optionally paramagnetic, microparticle or nanoparticle to enable magnetic enrichment or concentration processes. In yet other embodiments, the Notch ligand is immobilized on a 2D or 3D culture surface, optionally along with other adhesion molecules, such as VCAM-1. See US 2020 / 0399599, which is 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, polymeric scaffolds with conjugated ligands can be used, as described in WO 2020 / 131582, which is 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), polypropylene polyol, poloxamer, poly(uronic acid), poly(anhydride), poly(vinylpyrrolidone), and any combination thereof. In some embodiments, the scaffold comprises pores having diameters between about 1 pm and 100 pm.

[0275] In some embodiments, the C-terminus of the Notch ligand is conjugated to a support of choice. In some embodiments, this may involve adding a sequence to the C-terminus of the Notch ligand that can be enzymatically conjugated to the support via, for example, 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, which have been conjugated to a support. Of course, any of the known protein conjugation methods can be used.

[0276] Thus, in various embodiments, the Notch ligand is immobilized, functionalized, and / or embedded in a 2D or 3D culture system. The Notch ligand may be incorporated with an extracellular matrix component, such as one or more selected from fibronectin, retronectin, and laminin. In some embodiments, the Notch ligand and / or extracellular matrix component is 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, extracellular matrix component, or combinations thereof, contacts culture conditions that provide a topographical pattern and / or texture (e.g., roughness) for cells that contribute to differentiation and / or expansion.

[0277] In some embodiments, cell populations or banks are differentiated into progenitor 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 fragment may be free in solution or immobilized on a culture surface or particle. In some embodiments, cells are cultured for 5 to 7 days to prepare CD7+ progenitor T cells.

[0278] 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 extracellular matrix component(s), and optionally adding TNF-α to the culture at certain differentiation stages. Thus, in some embodiments, the cells produced are precursor or progenitor 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.

[0279] 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 are restricted to the recipient's major histocompatibility complex (MHC), resulting in host-tolerant T cells that can bypass 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 precursors. 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.

[0280] 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 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 with soluble or conjugated Notch ligand in a bioreactor, optionally a closed or closed automated 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 may also be added to the culture or reactor. Such cytokines or factors are known in the art. In various embodiments, the HSC population 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 precursor T cells. In some embodiments, the stem cell population or derivatives thereof are cultured for at least about 21 days, or at least about 28 days to generate mature T cell lineage or NK cells.

[0281] In various embodiments, a cell population, such as a population of HSCs, is cultured in artificial thymic organoids (ATOs). See Hagen, M. et al. (2019). ATOs involve culturing HSCs (or aggregates of HSCs) with a Notch ligand-expressing stromal cell line under serum-free conditions. Artificial thymic organoids are a 3D system that contains naive CD3 + CD8 + and CD3 + CD4 + Induces differentiation of hematopoietic precursors into T cells.

[0282] In various embodiments, the methods include generating derivatives of precursor T cells or generating T cell lineages from precursor T cells. In certain embodiments, the precursor T cells or derivatives of the T cell lineage express CD3 and T cell receptors. 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, the 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.

[0283] In some embodiments, the T cell lineage is a regulatory T cell. T regulatory cells (or T regs) are CD4 + CD25 + Tregs are defined as Tregs that regulate immune responses to self and foreign antigens and help prevent autoimmune disease. In some embodiments, differentiation of precursor T cells into Tregs involves ectopic expression of FOXP3 and culturing the precursor T cells or Treg precursors with one or more growth factors, such as, but not limited to, IL-2.

[0284] In some embodiments, expanded primary cell populations or banks, or derivatives of iPSCs (e.g., HSC populations), are differentiated into B lymphocytes ("B cells"). For example, culturing CD34+ or CD34+CD43+ cells with MS5 stromal cells or S17 stromal cells (e.g., for 15-25 days, or about 21 days) can generate B lymphocyte identity with expression of CD19, CD45, and CD10. See Carpenter L. et al., "Human induced pluripotent stem cells are capable of B-cell lymphopoiesis," Blood 117(15):4008-4011. Dubois F. et al., "Toward a better definition of hematopoietic progenitors suitable for B cell differentiation," Plos One December 15, 2020. In various embodiments, B cells produced according to the present disclosure express surface IgM (sIgM) and undergo VDJ rearrangement. In various embodiments, B cells produced in accordance with the present disclosure engraft in the subject's spleen and secondary lymphoid tissues for maturation.

[0285] In some embodiments, the cell population is differentiated into monocytes, macrophages, or neutrophils. For example, erythroid myeloid progenitors (EMPs) (CD43+CD45+) can be generated by culture with IL-6, IL-3, thyroid peroxidase (TPO), SCF, FGF2, and VEGF, followed by differentiation into monocytes. Monocyte differentiation utilizes culture with M-CSF, IL-3, and IL-6. See Cao X et al., "Differentiation and Functional Comparison of Monocytes and Macrophages from hiPSCs with Peripheral Blood Derivatives," Stem Cell Reports. 2019 Jun 11;12(6):1282-1297. Monocyte and macrophage lineages prepared according to the present disclosure are CD14+ and exhibit endocytosis and phagocytosis. In some embodiments, macrophages are polarized ex vivo toward an M1 (pro-inflammatory) or M2 (immunosuppressive) phenotype. In some embodiments, differentiation of iPSC-derived hCD34+ cells generates CD45+ hematopoietic cells with phagocyte markers such as CD33 and CD11b, and optionally subsequently generates cells with neutrophil-specific markers such as CD66b, CD16b, and GPI-80. These processes can use differentiation media containing a mixture of cytokines and growth factors, including, but not limited to, SCF, IL3, FLT3, IL6, GM-CSF, G-CSF, EPO, TPO, and / or combinations thereof.In some embodiments, neutrophils and their precursors are derived from the neutrophils described in Saeki L., et al., A Feeder-Free and Efficient Production of Functional Neutrophils from Human Embryonic Stem Cells, Stem Cells Vol. 27, Issue 1, 2009, Pages 59-67; Morishima T. et al., Neutrophil differentiation from human-induced pluripotent stem cells. J. Cell. Physiol. 226:1283-1291, 2011; Yokoyama Y. et al., Derivation of functional mature neutrophils from human embryonic stem cells. Blood 2009 Jun 25;113(26):6584-92; and Sweeney CL et al., Generation of functionally mature neutrophils from induced pluripotent stem cells. Methods Mol Biol 2014;1124:189-206.

[0286] In some embodiments, HSC populations or fractions thereof are differentiated into megakaryocytes or platelets. For example, megakaryocytes (as a renewable source of platelets) can be prepared from HSCs or fractions thereof by culturing them with SCF, IL-11, and TPO for several days (e.g., about 5 days). Alternatively, other cytokines and growth factors, such as IL-3, IL-6, SDF-1, and FGF-4, can be used. Megakaryocytes will be CD42b+CD61+. See Liu L., Efficient Generation of Megakaryocytes From Human Induced Pluripotent Stem Cells Using Food and Drug Administration-Approved Pharmacological Reagents, Stem Cells Transl Med. 2015 Apr;4(4):309-319. Platelets can be further generated from megakaryocytes by culturing them with IL-11 in serum-free medium. CD41+CD42a+ platelet-like particles are collected from the culture medium.

[0287] 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, the entire contents of which are incorporated herein by reference. 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.

[0288] In some embodiments, the HSC population or fraction thereof is differentiated into red blood cells or derivatives thereof. Red blood cells produced in accordance with the present disclosure can be administered or used in therapy for the treatment of, for example, inherited or acquired red blood cell disorders, bone marrow failure disorders, high altitude-related physiological and pathological conditions, conditions associated with chemical or radiation exposure, and / or subjects undergoing HSC transplantation. In further embodiments, red blood cells prepared in accordance with the present disclosure are provided as pharmaceutically acceptable compositions that deliver or encapsulate drugs (including, but not limited to, enzymes), oxygen carriers, or other suitable materials for treating human diseases or physiological or pathological conditions.

[0289] In another aspect, the present disclosure provides a cell population according to the present disclosure, or a pharmaceutically acceptable composition thereof. In some embodiments, the cell population is a lymphocyte population capable of engrafting in the thymus, spleen, or secondary lymphoid organs upon administration to a subject in need thereof. In various embodiments, a composition for cell therapy is prepared comprising the desired cell population and a pharmaceutically acceptable vehicle.

[0290] In some embodiments, the cell composition comprises HLA-A neg , HLA-DPB1 neg , and HLA-DQB1 neg The present invention also includes an iPSC or HSC cell population (or a population differentiated therefrom) that is a hematopoietic lineage comprising: a) a hematopoietic stem cell lineage (e.g., a hematopoietic stem cell lineage) derived from a hematopoietic stem cell ...

[0291] In various embodiments, the cells of the HSC composition are at least about 50% CD34+, or at least about 60% CD34+, or at least about 75% CD34+, or at least about 80% CD34+, or at least about 85% CD34+, or at least about 90% CD34+, or at least about 95% CD34+. Additionally, in embodiments, at least about 50%, or at least about 60%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% of the cells in the composition are one or more of CD90+ and CD45+.

[0292] The pharmaceutical composition comprises at least about 10 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, pharmaceutical compositions containing about 100,000 to about 400,000 HSCs per kilogram of recipient body weight (e.g., about 200,000 cells / kg) are administered. In other embodiments, the cells are administered in a concentration of about 10 5 ~Approx. 5×10 5 cells / kg (e.g., approximately 2.5 x 10 5 cells / kg), or approximately 10 6 ~Approx. 5×10 6 cells / kg (e.g., approximately 2.5 x 10 6 cells / kg), or approximately 5 × 10 6 ~about 10 7 cells / kg (e.g., approximately 5 x 10 6 cells / kg), or approximately 107 ~about 10 8 cells / kg (e.g., approximately 5 x 10 7 cells / kg), or approximately 10 8 ~about 10 9 cells / kg (e.g., approximately 5 x 10 8 cells / kg), or approximately 10 9 ~about 10 10 cells / kg, or approximately 10 10 ~about 10 11 cells / kg, or approximately 10 11 ~about 10 12 cells / kg, or approximately 10 12 ~about 10 13 cells / kg, or approximately 10 13 ~about 10 14 Dose is given in cells / kg.

[0293] The cell compositions of the present disclosure may further comprise a pharmaceutically acceptable carrier or vehicle suitable for intravenous infusion or other routes of administration, and the compositions may include a suitable cryoprotectant. An exemplary carrier is DMSO (e.g., about 10% DMSO). The cell compositions are provided in unit vials or bags and can be stored frozen until use. In certain embodiments, the volume of the composition is about 1 fluid ounce to 1 pint.

[0294] In some embodiments, the present disclosure provides CD7+ progenitor T cells, or pharmaceutically acceptable compositions thereof, produced by the methods disclosed herein. In various embodiments, the progenitor T cells are capable of engrafting in the thymus or spleen of the recipient. The progenitor T cells have the potential to reduce the risk of recurrence of leukemia or other types of cancer in bone marrow transplant patients and to reduce the number of post-transplant infections that cause significant morbidity and mortality in patients. In another aspect, the present disclosure provides derivatives of progenitor T cells or T cell lineages produced by the methods disclosed herein, or pharmaceutically acceptable compositions thereof.

[0295] In some embodiments, the cell population is a T cell population (or a precursor T cell population) or an NK cell population and is useful for adoptive cell therapy, e.g., in a human subject having a condition selected from lymphopenia, cancer, immunodeficiency, viral infection, autoimmune disease (particularly when the T cell population includes Tregs), skeletal dysplasia, bone marrow failure syndrome, or a genetic disorder that impairs T cell development or function. Exemplary genetic disorders may affect the immune system manifesting as an immunodeficiency state, or an autoimmune or pro-inflammatory state. In some embodiments, the subject has cancer, which is optionally a hematological malignancy or solid tumor. In some embodiments, the T cells are CAR-T cells.

[0296] In some embodiments, the cell population is a B lymphocyte population and is capable of engrafting in the subject's spleen or secondary lymphoid tissues. B cell populations according to the present disclosure have the potential to partially reconstitute humoral immunity in immunocompromised patients, providing protection from or treatment for infectious diseases, including, for example, viral, bacterial, fungal, or parasitic infections. In various embodiments, B cells according to the present disclosure can be differentiated into plasma cells to produce antigen-specific antibodies in vivo. In other embodiments, B cells produced according to the present disclosure can be used for cancer immunotherapy. In some embodiments, chimeric antigen B cells (CAR B cells) are prepared by genetic modification at the iPSC, embryoid body, hCD34+ cells, hematopoietic progenitor cells, or B cell level. CAR B cells express a surface BCR and / or secrete recombinant monoclonal antibodies that recognize a target antigen, such as a cancer antigen or an infectious disease antigen. In yet other embodiments, B cells produced according to the present disclosure are used for ex vivo production of antibodies (e.g., vaccine antibodies to provide protection from infectious agents).

[0297] In some embodiments, the cell population is a monocyte or macrophage cell population, and the cell population is capable of engrafting and maturing in various tissues of the subject, including tumors. In various embodiments, the monocyte or macrophage cell population is capable of forming tissue-resident macrophages in the subject. In various embodiments, the macrophages are predominantly of the M1 (pro-inflammatory) or M2 (immunosuppressive) phenotype. In various embodiments, the subject in need of treatment has cancer, either of various tissues or organs, inflammatory disease of the liver or kidney, or bacterial infection (e.g., sepsis or infection, or colonization of an indwelling medical device).

[0298] In some embodiments, the cell population is a megakaryocyte population or platelets developed therefrom, and these cells or platelets are useful for treating inherited platelet disorders, for example, affecting the coagulation pathway.

[0299] In some embodiments, the cell population is red blood cells.

[0300] In yet other embodiments, iPSCs are differentiated into non-hematopoietic stem or progenitor cells, or cells or tissues differentiated therefrom, including mesenchymal stem cells, neural stem cells, epithelial stem cells, neural cells (or their precursors) (including cortical, dopaminergic, and motor neurons, or their precursors), astrocytes (or their precursors), oligodendrocytes (or their precursors), cardiomyocytes (or their precursors), skeletal muscle cells (or their precursors), hepatocytes (or their precursors), pancreatic beta cells (or their precursors), and lung epithelial cells (or their precursors).

[0301] The cell compositions 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 disclosure include, but are not limited to, normal / physiological saline (0.9% NaCl), 5% dextrose in water (D5W), Dulbecco's phosphate buffered saline (PBS), and Ringer's solution. Pharmaceutically acceptable carriers may be selected from, but are not limited to, fillers (e.g., polyacrylates, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, lactose and other sugars, calcium hydrogen phosphate, etc.), binders (e.g., polyvinylpyrrolidone, pregelatinized corn starch, or hydroxypropyl methylcellulose, etc.), disintegrants (e.g., sodium starch glycolate, starch, etc.), or wetting agents (e.g., sodium lauryl sulfate (SDS)), lubricants (e.g., talc, silica, magnesium stearate, stearic acid, stearic acid colloidal silicon dioxide, metal stearates, corn starch, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, etc. Other suitable pharmaceutically acceptable carriers for the compositions of the present disclosure include, but are not limited to, Examples of suitable carriers include, but are not limited to, alcohol, water, silicic acid, salt solutions, gelatin, polyethylene glycol, amylose, magnesium stearate, talc, hydroxymethylcellulose, viscous paraffin, polyvinylpyrrolidone, etc. Additionally, a vehicle suitable for intravenous infusion or other administration routes (intra-arterial or intravenous injection, subcutaneous injection, collagen sponge seeding with fibrillar adhesive seeding, nanoparticles) may be included, and the composition may contain a suitable cryoprotectant. An exemplary carrier is DMSO (e.g., about 10% DMSO). Other carriers may include dimethoxyethane (DME), N,N-dimethylformamide (DMF), or dimethylacetamide, including mixtures or combinations thereof. The cell composition may be provided in an appropriate volume in an implantable device (e.g., a scaffold) or bag, vial, tube, or container and stored frozen until use.

[0302] Pharmaceutical compositions for use in the disclosed methods may also contain additional therapeutic agents for the treatment of a particular targeted disorder. For example, the pharmaceutical compositions may also contain cytokines and growth factors (e.g., interleukins, interferons, FGF, VEGF, PDGF, PIGF, STAT, etc.). Such additional factors and / or agents may be included in the pharmaceutical compositions to produce the benefits of the therapeutic approaches disclosed herein, i.e., to reduce systemic toxicity and provide improved therapeutic efficacy.

[0303] The route of administration of the cell line(s) or bank derivatives of primary cells or iPSC cells or bank derivatives thereof (HSCs or their progenitors) can be by any suitable means, including, but not limited to, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, and, if desired for local immunosuppressive treatment, intralesional administration. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, cells (e.g., HLA-modified iPSCs or their precursors or progenitors) can be suitably administered by pulse infusion, e.g., with decreasing doses of cells (e.g., HLA-modified iPSCs or HSCs and their precursors or progenitors). In some embodiments, administration is given by injection, e.g., via intravenous or subcutaneous injection, depending in part on whether administration is short-term or chronic.

[0304] Derivatives of cell line(s) or banks of expanded primary cells (e.g., HSCs and their progeny) can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disease or disorder being treated, the particular mammal (e.g., human) being treated, the clinical condition of the individual patient, the cause of the disease or disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to physicians. The therapeutically effective amount of cells (precursors or progeny derived from HSCs or cell line(s) or banks of expanded primary cells, or iPSC cells or banks thereof) administered will be governed by such considerations.

[0305] Administration may be a single dose, or multiple doses delivered at intervals of 3-4 hours, 1-4 times daily, 1-4 times weekly, 1-4 times monthly, 1-7 times monthly, or administration may occur once every 3 or 4 weeks over several months.

[0306] One may administer other compounds, such as cytotoxic agents, immunosuppressants, and / or cytokines or growth factors (e.g., stem cell factor, thrombopoietin, transforming growth factor (TGF)-α or β, fibroblast growth factor (FGF), the angiopoietin (Ang) family of growth factors, insulin-like growth factor, granulocyte-macrophage colony-stimulating factor, TNF-α or β, VEGF, interleukins (e.g., IL-2, 6, 7, 8, 10, 12, 15, etc.), and interferons (e.g., INF-α or γ) together with the cells herein (e.g., HLA-modified iPSC-derived HSCs or their precursors or progenitors). Concomitant administration includes simultaneous administration using separate formulations or a single pharmaceutical formulation, and sequential administration in any order, preferably with a period during which both (and all) active agents simultaneously exert their biological activities.

[0307] Therapeutic formulations of cells (e.g., derivatives of cell line(s) or banks of expanded primary cells, such as HSCs and their progeny) used in accordance with the present disclosure are prepared for storage by mixing cells (e.g., iPSC-derived HSCs or their precursors or progeny) having a desired purity with any pharmaceutically acceptable carrier, excipient, or stabilizer, such acceptable carriers, excipients, or stabilizers disclosed in Remington: The Science and Practice of Pharmacy, Twenty Third Edition: Elsevier (2020), which is incorporated herein by reference in its entirety, in the form of a lyophilized formulation or aqueous solution. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride); hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pengol; and m-cresol; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; polyvinyl alcohol. amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes); and / or TWEEN®, PLURONICS®, or polyethylene glycol (PEG), e.g., PEG chains having a molecular weight of 1,000 to 15,000 daltons, or 2,000 to 10,000 daltons, or 2,000 to 5,000 daltons.Other hydrophilic polymers that may be suitable include polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0308] Lyophilized formulations adapted for subcutaneous administration are also contemplated by the present disclosure. Such lyophilized formulations may be reconstituted with a suitable diluent to an optimal concentration, and the reconstituted formulation may be administered subcutaneously to a mammal treated herein.

[0309] The formulations herein may also contain two or more active agents as needed for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to additionally provide a cytotoxic agent, cytokine, or immunosuppressant. The effective amount of such other agents will depend on the amount of acceptable carriers, excipients, or stabilizers present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These will generally be used in the same dosages and by the same route of administration as used herein, or at about 1-99% of the dosages previously used.

[0310] Agents such as hormones, growth factors, and cytokine antibodies that may be co-administered with derivatives of the cell line(s) or expanded primary cell banks of the present disclosure (e.g., iPSC-derived HSCs or their precursors or progenitors) include molecules such as renin; growth hormones, including human growth hormone and bovine growth hormone; growth hormone-releasing factor; parathyroid hormone; thyroid-stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle-stimulating hormone; calcitonin; luteinizing hormone; glucagon; vmc factor, factor I; Coagulation factors such as factor X, tissue factor (TF), and von Willebrand factor; anticoagulants such as protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activators such as urokinase or human urinary or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factors-α and -β; enkephalinase; RANTES (normally expressed and secreted by T cells upon activation); human macrophage inflammatory protein (MIP-1-α); and human serum albumin. serum albumin; Muellerian inhibitory substance; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide; microbial proteins such as β-lactamase; DNase; IgE; cytotoxic T lymphocyte-associated antigen (CTLA) such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); hormone or growth factor receptor; protein A or D; rheumatoid factor; bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4, NT-5, or NT-6) neurotrophic factors such as nerve growth factors such as NGF-β or platelet-derived growth factor (PDGF); fibroblast growth factors such as aFGF and bFGF; fibroblast growth factor receptor 2 (FGFR2), epidermal growth factor (EGF); transforming growth factors (TGFs) such as TGF-α and TGF-β (including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5); bone morphogenetic proteins (BMPs) and BMP receptor 2, including BMP1, BMP6, and BMP7; insulin-like growth factors-I and -II (IGF-I and IGF-II);des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding protein, hepatocyte growth factor (HGF), EpCAM, GD3, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, CEA, TENB2, EphA receptors, EphB receptors, folate receptors, FOLR1, mesothelin, cripto, alphavbeta6, integrins, VEGF, VEGFR, EGFR, tarnsferin (tar nsferrin) receptors, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5; CD proteins, e.g., CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80. antibodies that bind to one or more tumor-associated antigens or cell surface receptors; erythropoietin; bone morphogenetic factors; immunotoxins; bone morphogenetic proteins (BMPs); interferons, e.g., interferon-α, -β, and -γ; colony-stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-2, IL-6, IL-12, IL-23, IL-12 / 23p40, IL-17, IL-15, IL-21, IL-1a, IL-1b, IL-18 , IL-8, IL-4, IL-3, and IL-5; superoxide dismutase; T cell receptors; surface membrane proteins; decay-accelerating factors; viral antigens such as portions of the HIV envelope; transport proteins; homing receptors; addressins; regulatory proteins; integrins, e.g., CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, and VCAM; tumor-associated antigens such as HER2, HER3, or HER4; endoglin, c-Met, c-kit, 1GF1R, PSGR, NGEP, PSMA, PSCA, LGR5, B7H4, TAG72 (tumor-associated glycoprotein 72), and fragments of any of the above.

[0311] Examples of antibodies or fragments thereof that can be administered include anti-PD-L1 antibodies, abciximab (Reopro), adalimumab (Humira, Amjevita), alefacept (Amevive), alemtuzumab (Campath), basiliximab (Simulect), belimumab (Benlysta), bezlotoximab (Zinplava), canakinumab (Ilaris), certolizumab pegol (Cimzia), cetuximab (Erbitux), daclizumab (Zenapax, Zinbryta), denosumab (Prolia, Xgeva), efalizumab (Raptiva), golimumab (Simponi, Simponi), and the like. Aria), Inflectra (Remicade), ipilimumab (Yervoy), ixekizumab (Taltz), natalizumab (Tysabri), nivolumab (Opdivo), olaratumab (Lartruvo), omalizumab (Xolair), palivizumab (Synagis), panitumumab (Vectibix), pembrolizumab (Keytruda), rituximab In some embodiments, the therapeutic agent may be secukinumab (Cosentyx), secukinumab (Rituxan), secukinumab (Actemra), secukinumab (Herceptin), secukinumab (Cosentyx), secukinumab (Rituxan ...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 X antibodies, anti-Ki-67 antibodies, anti-PCNA antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD5 antibodies, anti-CD7 antibodies, anti-CD8 antibodies, anti-CD9 / p24 antibodies, anti-CD1 antibodies, anti-CD11c antibodies, anti-CD13 antibodies, anti-CD14 antibodies, anti-CD15 antibodies, and anti-CD 19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD39 antibody, anti-CD41 antibody, anti-LCA / CD45 antibody, 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.

[0312] Concomitant administration does not require the therapeutic agents to be administered simultaneously, provided the timing of their administration is such that the pharmacological activities of the additional therapeutic agent and the active ingredient(s) in the pharmaceutical composition overlap in time, thereby achieving a combined therapeutic effect. Generally, each agent will be administered at a dose, for a time period, and on a schedule determined for that agent.

[0313] The pharmaceutical compositions may be administered at any dosage appropriate to achieve the desired outcome. In some embodiments, the desired outcome is a reduction in the intensity, severity, frequency, and / or delay in onset of one or more symptoms of infection. In some embodiments, the desired outcome is inhibition or prevention of infection. The required dosage will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the infection being prevented or treated, the particular composition used, and its mode of administration.

[0314] In some embodiments, the pharmaceutical compositions according to the present disclosure are administered in a single dose or in multiple doses, hi some embodiments, the pharmaceutical compositions are administered in multiple doses administered on different days.

[0315] The active ingredient (e.g., HSCs or their precursors or progeny derived from a cell line(s) or bank of expanded primary cells, or derivatives of iPSC cells or banks thereof) may also be encapsulated in microcapsules prepared, for example, by droplet formation techniques or interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are well known to those skilled in the art and are disclosed in Remington: The Science and Practice of Pharmacy, Twenty Third Edition: Elsevier (2020).

[0316] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing HSCs, their precursors, or successors, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0317] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes, as well as other techniques known to those skilled in the art.

[0318] As used herein, unless the context requires otherwise, the term "about" means ±10% of the associated numerical value.

[0319] Certain aspects and embodiments of the present disclosure are further illustrated with reference to the following examples. [Example]

[0320] Example 1: Overexpression of ETV2 increases the yield of blood endothelial cells and enhances CD34+ cell preparations during iPSC differentiation, but does not affect pluripotency. method iPSCs are known in the art and were developed from hCD34+ cells by episomal reprogramming, essentially as described in Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells, Science 318, 1917-1920, (2007), and Yu, et al. Human induced pluripotent stem cells free of vector and transgene sequences, Science 324, 797-801, (2009). Embryoid body and blood endothelial differentiation was 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).

[0321] Briefly, hiPSCs were dissociated and resuspended in medium supplemented with L-glutamine, penicillin / streptomycin, ascorbic acid, human holotransferrin, monothioglycerol, BMP4, and Y-27632. 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, IL-6, IGF-1, IL-11, SCF, and EPO. Cells were maintained in an incubator at 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.

[0322] result We transduced induced pluripotent stem cells (iPSCs) using an adenoviral vector containing both ETV2 and GFP sequences under the control of the EF1A promoter. After transduction, we observed that approximately 45% of the iPSC cultures were GFP-positive, thus confirming ETV2 overexpression (ETV2-OE). We further observed that ETV2-OE in iPSC cells maintained the pluripotent properties of iPSCs, as indicated by the stemness marker expression TRA-1-60 (Figure 3). Figure 3 shows a FACS plot depicting the transduction efficiency of iPSCs with an adenoviral vector for overexpressing ETV2 and GFP sequences.

[0323] Next, ETV2-OE-iPSCs (along with control iPSCs transduced with a vector carrying the GFP sequence without ETV2) were differentiated into embryoid bodies and then into hematopoietic endothelial cells (Strugeon et al., 2014). The results showed that overexpression of ETV2 increased the expression of CD235a - CD34 in the population + and CD31 +These results suggest that ETV2-OE promotes the formation of blood endothelial cells, as demonstrated by the expression of markers (Figure 4). Specifically, Figure 4 shows a representative flow cytometry analysis of blood endothelial cells (defined herein as CD235a-CD34+CD31+), and relative quantification demonstrates that ETV2-OE enhances the formation of blood endothelial cells compared to controls.

[0324] Furthermore, the results showed that ETV2-OE expresses CD34 + These results suggest that ETV2-OE enhances the formation of CD34+ cells (Figure 5). Figure 5 shows a representative flow cytometry analysis of CD34+ cells, and the relative quantification demonstrates that ETV2-OE enhances the formation of CD34+ cells.

[0325] Overall, these data indicate that overexpression of ETV2 in iPSCs does not affect their pluripotent properties, but promotes their ability to undergo blood endothelial and hematopoietic differentiation.

[0326] 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 for approximately 4-18 hours (by visual inspection), Yoda1 was added to the cultures for some experiments. After 4-7 days, cells were collected for analysis.

[0327] iPSCs were differentiated into embryoid bodies for 8 days. On day 8, CD34+ cells were harvested from iPSC-derived embryoid bodies and cultured for an additional 5–7 days to induce endothelial-hematopoietic (EHT) transition (with or without Yoda1). CD34+ cells were then harvested from EHT cultures between days 5–7 for further hematopoietic lineage differentiation.

[0328] CD34+ cells harvested from EHT cultures between days 5 and 7 (or between days 13 and 21 total differentiation from iPSCs) were seeded into 48-well pre-coated plates with rhDL4 and RetroNectin in medium containing AMEM, FBS, ITS-G, 2BME, ascorbic acid-2-phosphate, Glutamax, rhSCF, rhTPO, rhIL7, FLT3L, rhSDF-1a, and SB203580 for induction of T lineage differentiation.

[0329] 80% of the medium was replaced every other day between days 2 and 6. On day 7, cells were transferred to new coated plates and analyzed for the presence of pro-T cells (CD34+CD7+CD5+ / -).

[0330] 80% of the medium was changed every other day from day 8 to day 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+ / -).

[0331] 80% of the medium was changed every other day between days 15 and 20. On day 21, cells were harvested and their functional properties were assessed for CD3, CD4, CD8, CD5, CD7, and TCRab expression as surrogates for T cells via FACS and / or activation using CD3 / CD28 beads.

[0332] After 21 days of differentiation, cells were harvested and approximately 80,000 cells were replated into new 96-well culture plates in RPMI 1640 (no L-glutamine, no phenol red) with FBS, L-glutamine, and IL-2, followed by activation with 1:1 CD3 / CD28 beads. After 72 hours of activation with CD3 / CD28 beads, cells were analyzed for expression of CD3, CD69, and CD25 by FACS and IFN-γ using RT-qPCR. Supernatants were analyzed by ELISA.

[0333] result Figures 6A and 6B show that iPSC-derived HSCs induced by EHT of CD34+ cells from differentiated iPSCs (e.g., in this case, with Piezo1 activation) undergo pro-T cell differentiation similar to bone marrow (BM)-HSCs. Furthermore, Figures 7A and 7B show that iPSC-derived HSCs generated by EHT of CD34+ cells from differentiated iPSCs (in this case, with Piezo1 activation) undergo T cell differentiation and can be activated with CD3 / CD28 beads similar to BM-HSCs. Figure 8 shows that iPSC-derived HSCs (in this case, generated by Piezo1 activation) can differentiate into functional T cells, as demonstrated by INFγ expression upon stimulation with CD3 / CD28 beads. Together, these results demonstrate that iPSC-derived HSCs (i.e., induced by EHT of CD34+ cells from differentiated iPSCs) have enhanced HSC potential to further differentiate ex vivo into hematopoietic lineages, such as progenitor T cells and functional T cells.

[0334] Figure 27 shows that HSCs generated according to the present disclosure (labeled as D8+7 iPSC-CD34+) successfully differentiate into CD4+CD8+ ("double positive") T cells and TCRα / β T cells. The disclosed method substantially outperforms bone marrow CD34+ cells for T cell maturation. Figure 27 shows results with ("+Y") and without ("-Y") Yoda1 during HSC formation.

[0335] Figure 28 shows that HSCs (D8+7 iPSC-CD34+ cells (+ or -Yoda1)) generated according to the present disclosure successfully rearranged TCRs and outperformed bone marrow CD34+ cells. Shown are iPSC and EB negative controls, peripheral blood T cells as a positive control, T cells generated from BM CD34+ cells, and T cells generated according to the present disclosure, both with and without Yoda1.

[0336] Example 3: CCR5 deletion Figure 9A shows the generation of three CCR5 knockout (KO) iPSC clones. As shown in Figure 9B, CCR5-KO does not affect iPSC pluripotency. Furthermore, as shown in Figure 9C, CCR5-KO does not affect the ability of cells to undergo endothelial-hematopoietic transition.

[0337] Example 4: CD33 Deletion Figure 10A shows the generation of three CD33-KO iPSC clones. As shown in Figure 10B, CD33-KO does not affect the ability of cells to undergo endothelial-hematopoietic transition. Furthermore, CD33-KO does not affect the ability of cells to generate self-renewing HSCs (Figure 10C).

[0338] Example 5: HLA editing According to the present disclosure, cells can be HLA-modified by CRISPR-Cas9 using one or more of the following sgRNAs containing one or more spacer sequences shown in the table below. The gRNAs were designed using the following parameters: target sequences are exon 1 (to stop translation) and exon 2 (to affect the peptide groove) of each variant, a PAM motif with a 3' NGG, and a target length of 20 nucleotides. Candidate sgRNAs are evaluated for potential off-target editing. Table 2A: HLA-A 01:01 and 02:01 Table 2B: HLA-A 02:05 and 03:01 Table 2C: HLA-A 11:01 and 23:01 Table 2D: HLA-A 25:1 and 26:1 Table 2E: HLA-A 29:02 and 30:1 Table 2F: HLA-A 30:2 and 31:1 Table 2GHLA-A 33:1 Table 3A: HLA-B 07:02 and 08:01 Table 3B: HLA-B 13:02 and 14:02 Table 3C: HLA-B 15:01 and 18:01 Table 3D: HLA-B 35:01 and 38:01 Table 3E: HLA-B 40:01 and 44:02 Table 3F: HLA-B 44:03 and 50:01 Table 3G: HLA-B 52:01 and 57:01 Table 4A: HLA-DQB1 02:01 and 03:01 Table 4B: HLA-DQB1 03:02 and 03:03 Table 4C: HLA-DQB1 05:01 and 05:03 Table 4D: HLA-DQB1 06:01 and 06:03 Table 4E: HLA-DQB1 06:04 Table 5A: HLA-DRB1 01:02, 03:01, 01:01 Table 5B: HLA-DRB1 04:01 and 04:02 Table 5C: HLA-DRB1 04:04 and 07:01 Table 5E: HLA-DRB1 13:02 and 14:01 Table 5F: HLA-DRB1 15:01 and 15:02 Table 6: HLA-A 01:01, 03:01, 29:02, 33:01 Table 7: HLA-A 11:01 and 26:01 Table 8: HLA-DQB1 02:01, 06:02, 03:03, and 05:01 Table 9: HLA-DQB1 06:01 and 06:03 Table 10: HLA-A 24:02 Table 11: HLA-B 07:02, 44:03, 57:01, and 08:01 Table 12: HLA-B 14:02, 50:01, 37:01, and 52:01 Table 13: HLA-B 38:01 and 35:01 Table 14: HLA-DRB1 03:01, 15:01, 07:01, and 01:02 Table 15: HLA-DRB1 10:01, 15:02, 13:01, and 01:01 Table 16: HLA-DPB1 01:01, 02:01, 03:01, and 04:01 Table 17: HLA-DPB1 04:02, 11:01, 17:01, and 34:01

Table 2A-1

Table 2A-2

Table 2A-3

Table 2B-1

Table 2B-2

Table 2B-3

Table 2B-4

Table 2C-1

Table 2C-2

Table 2C-3

Table 2C-4

Table 2D-1

Table 2D-2

Table 2D-3

Table 2E-1

Table 2E-2

Table 2E-3

Table 2E-4

Table 2F-1

Table 2F-2

Table 2F-3

Table 2F-4

Table 2G-1

Table 2G-2

Table 3A-1

Table 3A-2

Table 3A-3

Table 3A-4

Table 3B-1

Table 3B-2

Table 3B-3

Table 3B-4

Table 3C-1

Table 3C-2

Table 3C-3

Table 3C-4

Table 3D-1

Table 3D-2

Table 3D-3

Table 3D-4

Table 3D-5

Table 3D-6

Table 3D-7

Table 3D-8

Table 3D-9

Table 3D-10

Table 3D-11

Table 3D-12

Table 3D-13

Table 3D-14

Table 3D-15

Table 3D-16

Table 3D-17

Table 3D-18

Table 3D-19

Table 3D-20

Table 3E-1

Table 3E-2

Table 3E-3

Table 3E-4

Table 3F-1

Table 3F-2

Table 3F-3

Table 3G-1

Table 3G-2

Table 3G-3

Table 3G-4

Table 4A-1

Table 4A-2

Table 4A-3

Table 4B-1

Table 4B-2

Table 4B-3

Table 4C-1

Table 4C-2

Table 4C-3

Table 4C-4

Table 4D-1

Table 4D-2

Table 4D-3

Table 4E-1

Table 4E-2

Table 4E-3

Table 5A-1

Table 5A-2

Table 5A-3

Table 5B-1

Table 5B-2

Table 5B-3

Table 5C-1

Table 5C-2

Table 5C-3

Table 5D-1

Table 5D-2

Table 5D-3

Table 5E-1

Table 5E-2

Table 5E-3

Table 5F-1

Table 5F-2

Table 5F-3

Table 6-1

Table 6-2

Table 7-1

Table 7-2

Table 8-1

Table 8-2

Table 9

Table 10-1

Table 10-2

Table 11-1

Table 11-2

Table 12

[0339] Example 6: Evaluation of off-target editing in HLA knockout HSCs Because HLA genes are located on the short arm of chromosome 6 (e.g., as shown in Figure 11), and the HLA system contains closely related genes, specific gRNA design is challenging. Sequencing of triple knockout (HLA-edited) clones was performed to confirm unwanted editing and to ensure that primary editing events (e.g., deletion(s)) did not occur within other regions of chromosome 6. Sequencing methods and other analyses were performed to assess the extent of gRNA off-target activity and select gRNAs that represented a low risk of affecting non-target HLA genes.

[0340] 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 DNA-labeled fragments eliminated all unwanted 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 DNA cleavage readouts, 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.

[0341] Table 18 below summarizes the results of the editing strategies in two representative HLA-edited clones relative to wild-type (gHSC) cells. [Table 18]

[0342] Table 19 provides non-limiting examples of gRNAs used in experiments. The gRNA sequences disclosed in Tables 2A-17 can be used to knock out expression of the indicated HLA genes. [Table 19]

[0343] 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.

[0344] These results were confirmed by phenotypic analysis of the HLA-edited clones by FACS and immunofluorescence. As shown in Figures 12A and 12B, 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 12A shows that all HLA-edited cells were positive for HLA, such as class I, to the same extent as wild-type (gHSC) cells. 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.

[0345] 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 12B, HLA-A was not expressed in the HLA-edited clones, indicating that the gene editing strategy was efficient in specifically deleting only the HLA-A gene. This preservation of overall class I expression with the deletion of HLA-A facilitates patient matching while avoiding NK cell-mediated rejection.

[0346] Example 7: 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 13, immunofluorescence evaluation of HLA-edited iPSC clones showed that they maintained three germ layer differentiation: ectodermal differentiation indicated by NESTIN-488 and PAX6-594 staining, mesodermal differentiation indicated by GATA-488 staining, and endodermal differentiation indicated by CXCR4-488 and FOX2A-594 staining.

[0347] 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 (GVHD). 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 promote patient matching while preventing 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 HLA-edited HSC grafts.

[0348] Wild-type (gHSC) and HLA-edited HSCs were cocultured with PBMCs matched for HLA-B and HLA-C markers but mismatched for 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 HLA-mismatched HSCs, respectively. Figure 14 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. HSCs cocultured with CD8+ T cells sorted from the same PBMC donor protected HLA-edited and B2M KO HSCs from CD8+ T cell cytotoxicity. Conversely, HSCs co-cultured with sorted NK cells alone protected WT (gHSC) and HLA-edited cells from NK cell-mediated cytotoxicity.

[0349] In summary, the immunocompetence results indicate that CD8+ T cells present in PBMC samples were involved in killing cells with mismatched HLA molecules (WT) and CIITA KO, whereas NK cells present in PBMC were involved in killing HLA-null cells (B2M KO). However, HLA-edited HSCs were protected from CD8+ T cell-mediated cytotoxicity (because the mismatched HLA-A was knocked out) and from NK cell-mediated cytotoxicity (because HLA class I molecule expression was largely preserved).

[0350] Example 8: Evaluation of the 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 WT HSCs. Equal proportions of mCherry HLA-edited and unedited HSCs were mixed and transplanted into mice, from which bone marrow (BM) and peripheral blood samples were collected and assessed by FACS to compare the relative amounts of each cell type present in the samples. As shown in Figure 15, both HLA-edited and WT HSCs contributed to approximately equal engraftment in BM and peripheral blood samples. These results confirm that HLA-edited HSCs (prepared according to the present disclosure) are comparable to WT HSCs in their engraftment and reconstitution potential. Therefore, the characteristics of WT HSCs are expected to be consistent with those of the HLA-edited HSCs of the present disclosure.

[0351] Example 9: Differentiation of HLA-edited HSCs into hematopoietic lineages Experiments were performed to determine whether HLA deletion affects the ability of HSCs to differentiate into different types of immune cells. HLA-edited HSCs were differentiated into pro-T cells using a process essentially as described in Example 2. It was found that the HLA-edited HSCs were capable of differentiating into pro-T cells, which was comparable to wild-type (non-HLA-edited) HSCs as measured by their CD34+-CD7+ expression (Figures 16A and 16B). Furthermore, upon differentiation into NK cells, it was shown that the HLA-edited HSCs were capable of differentiating into NK cells, which was comparable to wild-type HSCs as measured by their CD3-CD56+ expression (Figure 17). Similarly, it was determined that the HLA-edited HSCs were capable of differentiating into the monocyte / macrophage lineage, which was comparable to wild-type HSCs as measured by their CD11b+-CD14+ expression (Figure 18A). Furthermore, the CD11b+-CD14+ gated population showed equivalent HLA-I and HLA-II expression, indicating that HLA-edited HSCs preserved the overall expression of both class I and class II molecules (Figure 18B).

[0352] The global expression of HLA-DQB1 and HLA-DPB1 supported by edited HSCs and other class II molecules was assessed by evaluating their expression in macrophages differentiated from HSCs. The study design is shown schematically in Figure 19A. It was found that deletion of HLA-DQB1 and HLA-DPB1 did not affect the expression of other HLA class II molecules (Figure 19B). For example, HLA-DR is equally expressed in both WT and HLA-edited cells (Figure 19C). In Figures 19B and 19C, CIITA-KO is used as a positive control.

[0353] Antigen-presenting cells (APCs) bind to helper CD4 receptors via HLA-II molecules. + Presents antigens to T cells. Helper CD4 + Activation of T cells results in antigen-specific CD8 T cells that further develop into antigen-specific CTLs. +HLA class I molecules are expressed on the surface of all nucleated cells and display peptide fragments of proteins intracellularly to CD8+ CTLs. CTLs induce cytotoxic killing of target (infected) cells upon recognition of HLA-I-peptide complexes expressed on the cell surface. Therefore, we performed studies to determine whether deletion of HLA-A affects class I peptide presentation by edited HSCs. As shown in Figures 20A and 20B, immunopeptidome analysis indicates that deletion of HLA-A does not affect overall class I peptide presentation. HLA-A-edited cells showed equivalent peptide and protein presentation when compared to wild-type (gHSC) HSCs. Furthermore, as shown in Figures 21A and 21B, deletion of HLA-DQB1 and HLA-DPB1 does not affect overall class II peptide presentation by macrophages differentiated from HSCs. Taken together, these data suggest that despite the loss of HLA-A, HLA-DQ, and HLA-DP molecules, cells preserve the ability to present a wide range of class I and class II peptides.

[0354] Example 10: In vivo testing of antigen-mediated immune responses. Figure 22 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 challenge, CD4+ helper T cells are activated and cytokines recruit macrophages and other immune cells, which induce tissue swelling at the site of antigen exposure.

[0355] A delayed-type hypersensitivity assay was performed in transplanted mice. Specifically, mice were sensitized by subcutaneous injection of sheep red blood cells as antigen. If the mice had a functional immune system, APCs would process the antigen and present the peptide antigen to CD4+ T cells. The mice were then challenged by subcutaneous injection of the same antigen into the left paw. At this point, T cells were activated and secreted cytokines that recruit macrophages and other immune cells at the antigen injection site, causing tissue swelling. In this assay, a functional immune system could cause swelling of the left paw, as measured with a microcaliper.

[0356] As can be seen in Figures 23A and 23B, control (non-transplanted) mice, due to their immunodeficiency, did not show swelling of the left paw. Conversely, mice transplanted with cord blood CD34+ cells showed tissue swelling, with the diameter of the left paw doubling. Similar immune system responses were observed in both WT (non-edited HSCs) and HLA-edited HSCs (triple KO) transplanted mice.

[0357] Example 11: In vivo testing of HSC-derived T cells In vitro activation of HSC-derived T cells was measured, and the results are shown in Figure 24. The top panel of Figure 24 shows FACS analysis of activated T cells from different sources, including HSCs prepared using Piezo1 activation. T cells prepared from HSCs demonstrated comparable or superior activation, as measured by increased CD107 expression. The bottom panel shows Dynabeads activation, where activated T cells express proinflammatory cytokines. HSC-derived T cells prepared using Piezo1 activation expressed higher levels of proinflammatory cytokines, as exemplified by TNF-α and interferon-gamma expression levels.

[0358] Example 12: Evaluation of the properties of CCR5 knockout HSCs to develop into pro-T cells To determine whether CCR5 knockout (CCR5-KO) HSCs could differentiate equally well into pro-T cells as their wild-type counterparts from which they were derived, we measured CD34, CD7, and CD5 expression in HSCs and CCR5-KO. As can be seen in Figure 25, HSCs differentiated equally well into CD34+CD7+CD5+ pro-T cells as bone marrow-derived CD34+ cells. Similarly, CCR5-KOs differentiated equally well into CD34+CD7+CD5+ pro-T cells as their gHSC counterparts.

[0359] Next, the properties of CCR5 knockout HSCs for differentiation into double-positive (CD4+CD8+) T cells were evaluated. As can be seen in Figure 26, CCR5 knockout HSCs differentiated equally well into double-positive (CD4+CD8+) T cells compared to their gHSC counterparts (i.e., HSCs of the present disclosure) from which they were derived.

[0360] Example 13 - Evaluation of differentiation and maturation of HSC-derived T cells (pro-T cells) Next, we tested the ability of HSC-derived T cells (pro-T cells) to differentiate into mature T cells. After a 35-day differentiation period, pro-T cells were assessed by cell sorting for the presence of CD4+, CD8+, and AB+ T cell populations. As shown in Figure 29, pro-T cells differentiate into CD4+, CD8+, and αβ+ T cells more efficiently than bone marrow (BM)-derived CD34+ cells and embryonic body (EB)-derived CD34+ cells.

[0361] Next, to test their functional properties, each of the T cell populations 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 triggering T cell activation. The degree of activation was assessed by measuring the subsequent T cell-mediated cytotoxicity compared to a pan T cell control. As shown in Figure 30, pro T cells demonstrated statistically significant superior cytotoxicity compared to both BM CD34+ T cells and EB CD34+ T cells.

[0362] Because the overall differentiation process of pro T cells is 35 days long, transduction experiments were performed to test whether the time required to differentiate HSCs could be shortened. Pro 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 the 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 31, HSC-derived pro T cells could be transduced with high efficiency, with over 80% of the cells expressing the anti-CD19 CAR, as evidenced by cell sorting.

[0363] Next, we evaluated the ability of pro-T cells to effectively mature into CD4+ / CD8+ T cells via CAR transduction. Pro-T cells were transduced with LVs by anti-CD19 CAR, along with bone marrow (BM)-derived CD34+ cells and embryonic body (EB)-derived CD34+ cells (and 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 32, the results indicated that CAR transduction promoted T cell maturation, and an increased degree of T cell maturation was observed in pro-T cells compared to bone marrow (BM)-derived CD34+ cells and embryonic body (EB)-derived CD34+ cells.

[0364] The ability of LV-transduced pro 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. Untransduced cells and pan T cells served as negative and positive controls, respectively. As shown in Figure 33, CAR pro T cells functioned effectively via T cell-mediated lysis and exhibited a similar degree of cytotoxicity to CAR pro T cells derived from BM CD34+ cells. Conversely, CAR pro T cells derived from EB CD34+ cells did not exhibit the ability to kill target cells.

[0365] Example 14 - Evaluation of HSC properties that develop into pro-T cells. The ability of HSCs to develop into pro-T cells was assessed by measuring the CD34-CD7+ marker on pro-T cells. As shown in Figure 34, FACS analysis demonstrated that HSCs produced according to the present disclosure successfully differentiated into CD34-CD7+ pro-T cells compared to bone marrow-derived CD34+ cells or EB-derived CD34+ cells.

[0366] Next, we measured the expression of T cell-specific transcription factors and thymic engraftment molecules. Figure 35A shows increased TCF7 expression, and Figure 35B shows increased CCR7 expression in the HSC-derived pro T cells of the present disclosure. Figure 36A shows that HSC-derived pro T cells engraft and differentiate in the thymus. Figure 36B 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 pro T cells in the thymus. The pro T cells in this example were prepared from HSCs using Piezo1 activation, as previously described.

Claims

1. HLA-A ｎｅｇ and (1) HLA-DPA1 ｎｅｇ and / or HLA-DPB1 ｎｅｇ and / or (2) HLA-DQA1 ｎｅｇ and / or HLA-DQB1 ｎｅｇ wherein said cell population is homozygous for or comprises single genes for HLA-C, HLA-DRB1, and optionally HLA-B.

2. The cell population is HLA-DPB1 ｎｅｇ and / or HLA-DQB1 ｎｅｇ The cell population of claim 1 ,

3. The cell population is HLA-DPB1 ｎｅｇ and HLA-DQB1 ｎｅｇ The cell population of claim 2 ,

4. The cell population of any one of claims 1 to 3, wherein the cell population comprises a deletion or inactivation of both DPB1 genes.

5. 3. The cell population of claim 1 or 2, wherein one or both DQB1 genes are retained, and optionally one or both DQA1 genes are retained.

6. 3. The cell population of claim 1 or 2, wherein the cell population comprises a deletion or inactivation of both DQB1 genes and, optionally, both DQA1 genes.

7. The cell population of claim 6, wherein one or both DPB1 genes are retained and / or one or both DPA1 genes are retained.

8. The cell population of claim 6 or 7, further comprising a deletion of one or both of the genes for DQB2 and / or DQB3.

9. The cell population of claim 3 , wherein the cell population comprises deletion or inactivation of both DPB1 genes and both DQB1 genes.

10. The cell population of any one of claims 1 to 9, wherein the cell population is homozygous or contains a single copy of HLA-B.

11. The cell population of any one of claims 1 to 9, wherein the cell population is homozygous or comprises a deletion or inactivation of both HLA-B genes.

12. The cell population according to any one of claims 1 to 11, wherein the cell population is homozygous for HLA-DRB1 or retains one copy of HLA-DRB1 and the other copy of HLA-DRB1 is deleted or inactivated.

13. 13. The cell population of claim 12, wherein the cell population is homozygous or heterozygous or has only a single copy of one, two, three, or four of DRB2, DRB3, DRB4, and DRB5.

14. The cell population of claim 12, wherein one or more of DRB2, DRB3, DRB4, and DRB5 are deleted or inactivated.

15. The cell population of any one of claims 1 to 14, wherein the cell population is homozygous for HLA-E or has one HLA-E gene deleted or inactivated.

16. The cell population according to any one of claims 1 to 14, wherein the cell population is heterozygous for HLA-E.

17. 17. The cell population of any one of claims 1 to 16, wherein the cell population is homozygous for HLA-F or one or both HLA-F genes are deleted or inactivated.

18. The cell population according to any one of claims 1 to 16, wherein the cell population is heterozygous for HLA-F.

19. 19. The cell population of any one of claims 1 to 18, wherein the cell population is homozygous for HLA-G or one or both HLA-G genes are deleted or inactivated.

20. The cell population according to any one of claims 1 to 18, wherein the cell population is heterozygous for HLA-G.

21. The cell population of any one of claims 1 to 20, wherein the cells are human stem cells or human progenitor cells.

22. 22. The cell population of claim 21, wherein the stem cells are pluripotent stem cells, which are optionally human induced pluripotent stem cells (hiPSCs).

23. 23. The cell population of claim 22, wherein the iPSCs are derived from umbilical cord blood, bone marrow biopsy of human HSCs, hCD34+ cells from mobilized peripheral blood, human CD34+ cells, immune cells, immune progenitor cells, hematopoietic cells, non-hematopoietic cells, and cells from banked organs.

24. The cell population according to any one of claims 21 to 23, wherein the stem cells are hematopoietic stem cells (HSCs) or a cell population derived therefrom.

25. 25. The cell population of any one of claims 1 to 24, wherein the cells are of a hematopoietic cell lineage, optionally wherein the hematopoietic lineage is selected from common lymphoid progenitor (CLP) cells, granulocyte-monocyte progenitor (GMP) cells, precursor T cells, T lymphocytes, B lymphocytes, natural killer cells, neutrophils, monocytes, macrophages, erythrocytes, megakaryocytes, and platelets.

26. The cell population of any one of claims 1 to 20, wherein the cell population is derived directly from a human donor or patient.

27. 27. The cell population of claim 26, wherein the cell population is human stem cells, human progenitor cells (including, but not limited to, CMP, CLP, GMP, B cells, macrophages, T cells, and subtypes thereof).

28. 24. The cell population of any one of claims 1 to 23, wherein the cells are non-hematopoietic stem cells, which are optionally mesenchymal stem cells, neural stem cells, or epithelial stem cells, or the cells are non-hematopoietic cells, optionally selected from neurons, astrocytes, oligodendrocytes, cardiomyocytes, skeletal muscle cells, hepatocytes, pancreatic beta cells, and lung epithelial cells, or precursors thereof.

29. The cell population is selected from the group consisting of DRB1*03:01, DRB1*15:01, DRB1*01.02, DRB1*07.01, DRB1*04:01, DRB1*07:01, DRB1*01:01, DRB1*01:02, DRB1*04:04, DRB1*13:02, DRB1*13:01, DRB1*11:04, DR The cell population of any one of claims 1 to 28, having a DRB1 haplotype selected from DRB1*15:02, DRB1*03:02, DRB1*11:01, DRB1*15:03, DRB1*04:07, DRB1*08:04, DRB1*04:02, DRB1*14:01, and DRB1*10:

01.

30. 30. The cell population of any one of claims 1 to 29, wherein the cell population has an HLA-C haplotype selected from C*07:01, C*04:01, C*07:02, C*06:02, C*03:04, C*05:01, C*02:02, C*12:03, C*03:03, and C*16:

01.

31. the cell population 【Table 20-1】 【Table 20-2】 The cell population according to any one of claims 1 to 30, having an HLA-C and HLA-DRBI haplotype selected from:

32. The cell population according to any one of claims 1 to 31, wherein the cell population has an HLA-B haplotype selected from B*08:01 and B*07:

02.

33. 32. The cell population of any one of claims 1 to 31, wherein the cell population has an HLA-B haplotype selected from one or more of B*08:01, B*07:02, B*44:02, B*44:03, B*35:01, B*57:01, B*15:01, B*14:02, B*40:01, B*53:01, B*49:01, B*51:01, B*13:02, and B*38:

01.

34. the cell population C*07:01~B*08:01~DRB1*03:01、C*07:02~B*07:02~DRB1*15:01、C*05:01~B*44:02~DRB1*04:01、C*16:01~B*44:03~DRB1*07:01、C*04:01~B*35:01~DRB1*01:01、C*06:02~B*57:01~DRB1*07:01、C*06:02~B*13:02~DRB1*07:01、C*08:02~B*14:02~DRB1*01:02、C*3:04~B*40:01~DRB1*04:04、C*04:01~B*44:03~DRB1*07:01、C*03:04~B*40:01~DRB1*13:02、C*03:04~B*15:01~DRB1*04:01、C*05:01~B*18:01~DRB1*03:01、C*05:01~B*44:02~DRB1*13:01、C*07:02~B*07:02~DRB1*01:01、C*04:01~B*35:02~DRB1*11:04、C*12:02~B52:01~DRB1*15:02、C*03:03~B*15:01~DRB1*13:01、C*07:02~B*07:02~DRB1*07:01、C*05:01~B*44:02~DRB1*15:01、C*12:03~B*38:01~DRB1*13:01、C*17:01~B*42:01~DRB1*03:02、C*08:02~B*14:01~DRB1*07:01、C*01:02~B*27:05~DRB1*01:01、C*04:01~B*35:01~DRB1*11:01、C*06:02~B*50:01~DRB1*07:01、C*07:01~B*18:01~DRB1*11:04、C*04:01~B*53:01~DRB1~*13:02、C*12:03~B*18:01~DRB1*15:01、C*07:02~B*07:02~DRB1*11:01、C*05:01~B*44:02~DRB1*01:01、C*04:01~B*53:01~DRB1*15:03、C*07:02~B*07:02~DRB1*04:01、C*08:02~B*14:02~DRB1*13:02、C*04:01~B*35:01~DRB1*07:01、C*07:01~B*08:01~DRB1*15:01、C*07:02~B*39:05~DRB1*04:07、C*04:01~B*53:01~DRB1*08:04, C*07:02~B*07:02~DRB1*13:01, C*12:03~B*38:01~DRB1*04:02, C*03:03~B*15:0 1~DRB1*04:01, C*04:01~B*35:01~DRB1*14:01, C*06:02~B*37:01~DRB1*10:01, C*07:01~B*49:01~DRB1*11:01, C The cell population of any one of claims 1 to 31, comprising an HLA-C to HLA-B to DRB1 haplotype selected from C*03:04 to B*40:01 to DRB1*04:01, C*15:02 to B*51:01 to DRB1*11:01, C*03:02 to B*58:01 to DRB1*03:01, C*03:03 to B*55:01 to DRB1*14:01, and C*04:01 to B*35:01 to DRB1*13:

01.

35. 35. A collection of cell populations according to any one of claims 1 to 34, wherein said cell populations in said collection represent at least two different HLA-C haplotypes.

36. 36. The collection of claim 35, wherein the cell populations in the collection represent at least four, or at least six, or at least eight, or at least ten different HLA-C haplotypes.

37. 36. The collection of claim 35, wherein the cell populations in the collection represent at least 12 different HLA-C haplotypes.

38. The collection of any one of claims 35 to 37, wherein the cell population comprises at least the following HLA-C haplotypes: C*07:01 and C*04:

01.

39. 39. The collection of claim 38, wherein the cell population comprises at least the following HLA-C haplotypes: C*07:01, C*04:01, C*07:02, C*06:02, C*03:04, and C*05:

01.

40. 40. The collection of claim 39, wherein the cell population comprises at least the following HLA-C haplotypes: C*07:01, C*04:01, C*07:02, C*06:02, C*03:04, C*05:01, C*02:02, C*12:03, C*03:03, and C*16:

01.

41. 41. The collection of any one of claims 35 to 40, wherein the cell population in the collection is homozygous for the HLA-B gene or comprises a deletion or inactivation of one or both of the HLA-B genes.

42. The collection of any one of claims 35 to 40, wherein the cell populations in the collection represent at least two different HLA-B haplotypes.

43. 43. The collection of claim 42, wherein the cell populations in the collection represent at least four, or at least six, or at least eight, or at least ten, or at least twelve different HLA-B haplotypes.

44. 44. The collection of claim 43, wherein the collection comprises a cell population having at least the following HLA-B haplotypes: B*08:01 and B*07:

02.

45. 45. The collection of claim 44, wherein the collection comprises a cell population having at least the following HLA-B haplotypes: B*08:01, B*07:02, B*44:02, B*35:01, B*14:02, and B*40:

01.

46. 46. ​​The collection of claim 45, wherein the collection comprises a cell population having at least the following HLA-B haplotypes: B*08:01, B*07:02, B*44:02, B*44:03, B*35:01, B*57:01, B*15:01, B*14:02, B*40:01, and B*53:

01.

47. 46. ​​The collection of claim 45, wherein the collection comprises a cell population having at least the following HLA-B haplotypes: B*08:01, B*07:02, B*44:02, B*44:03, B*35:01, B*57:01, B*15:01, B*14:02, B*40:01, B*53:01, B*49:01, B*51:01, B*13:02, and B*38:

01.

48. 48. The collection of any one of claims 35 to 47, wherein the cell populations in the collection represent at least two, at least four, at least six, at least eight, or at least ten different HLA-C to HLA-B haplotypes.

49. 49. The collection of claim 48, wherein the cell populations in the collection represent at least 12 different HLA-C to HLA-B haplotypes.

50. 50. The collection of claim 49, wherein the cell populations in the collection represent at least 20 different HLA-C to HLA-B haplotypes.

51. The collection of any one of claims 48 to 50, wherein the collection comprises a cell population having the following haplotypes: C*07:01 to B*08:01 and C*07:02 to B*07:

02.

52. 52. The collection of claim 51, wherein the collection comprises a cell population having the following haplotypes: C*07:01 to B*08:01, C*07:02 to B*7:02, C*05:01 to B*44:02, C*16:01 to B*44:03, C*04:01 to B*35:01, C*03:04 to B*40:01, and C*08:02 to B*14:

02.

53. 52. The collection of claim 51, wherein the collection comprises a cell population having the following haplotypes: C*07:01 to B*08:01, C*07:02 to B*7:02, C*05:01 to B*44:02, C*16:01 to B*44:03, C*04:01 to B*35:01, C*03:04 to B*40:01, C*08:02 to B*14:02, C*06:02 to B*57:01, C*3:03 to B*15:01, and C*04:01 to B*53:

01.

54. 52. The collection of claim 51, wherein the collection comprises a cell population having the following haplotypes: C*07:01 to B*08:01, C*07:02 to B*7:02, C*05:01 to B*44:02, C*16:01 to B*44:03, C*04:01 to B*35:01, C*03:04 to B*40:01, C*08:02 to B*14:02, C*06:02 to B*57:01, C*3:03 to B*15:01, C*04:01 to B*53:01, C*07:01 to B*49:01, C*15:02 to B*51:01, C*06:02 to B*13:02, and C*12:03 to B*38:

01.

55. 55. The collection of any one of claims 35 to 54, wherein the cell populations in the collection represent at least two, or at least four, or at least six, or at least eight, or at least ten different DRB1 haplotypes.

56. 56. The collection of claim 55, wherein the cell populations in the collection represent at least 12 different DRB1 haplotypes.

57. 57. The collection of claim 56, wherein the collection comprises a cell population having at least the following DRB1 haplotypes: DRB1*03:01 and DRB1*15:

01.

58. 58. The collection of claim 57, wherein the collection comprises a cell population having at least the following DRB1 haplotypes: DRB1*03:01, DRB1*15:01, DRB1*04:01, DRB1*07:01, and DRB1*01:

01.

59. 58. The collection of claim 57, wherein the collection comprises a cell population having at least the following DRB1 haplotypes: DRB1*03:01, DRB1*15:01, DRB1*04:01, DRB1*07:01, DRB1*01:01, DRB1*01:02, DRB1*04:04, DRB1*13:02, DRB1*13:01, and DRB1*11:

04.

60. 58. The collection of claim 57, wherein the collection comprises a cell population having at least the following DRB1 haplotypes: DRB1*03:01, DRB1*15:01, DRB1*04:01, DRB1*07:01, DRB1*01:01, DRB1*01:02, DRB1*04:04, DRB1*13:02, DRB1*13:01, DRB1*11:04, DRB1*15:02, DRB1*03:02, and DRB1*11:

01.

61. 58. The collection of claim 57, wherein the collection comprises a cell population having at least the following DRBI haplotypes: DRB1*03:01, DRB1*15:01, DRB1*04:01, DRB1*07:01, DRB1*01:01, DRB1*01:02, DRB1*04:04, DRB1*13:02, DRB1*13:01, DRB1*11:04, DRB1*15:02, DRB1*03:02, DRB1*11:01, DRB1*15:03, DRB1*04:07, DRB1*08:04, DRB1*04:02, DRB1*14:01, and DRB1*10:

01.

62. The collection comprises the following HLA-C, HLA-DRB1 haplotypes: 【Table 21-1】 【Table 21-2】 36. The collection of claim 35, comprising a cell population having one or more of:

63. 63. The collection of any one of claims 35 to 62, wherein the cell populations in the collection represent at least two, or at least four, or at least six, or at least eight, or at least ten different HLA-C to HLA-B to DRB1 haplotypes.

64. 64. The collection of claim 63, wherein the cell populations in the collection represent at least 12 different, or at least 20 different HLA-C to HLA-B to DRB1 haplotypes.

65. The collection of claim 63 or 64, wherein the collection comprises a cell population having at least the following haplotypes: C*07:01 to B*08:01 to DRB1*03:01 and C*07:02 to B*07:02 to DRB1*15:

01.

66. 66. The collection of claim 65, wherein the collection comprises a cell population having at least the following haplotypes: C*07:01 to B*08:01 to DRB1*03:01, C*07:02 to B*07:02 to DRB1*15:01, C*05:01 to B*44:02 to DRB1*04:01, C*16:01 to B*44:03 to DRB1*07:01, C*04:01 to B*35:01 to DRB1*01:01, C*06:02 to B*57:01 to DRB1*07:01, C*06:02 to B*13:02 to DRB1*07:01, and C*08:02 to B*14:02 to DRB1*01:

02.

67. The collection comprises at least the following haplotypes: C*07:01 to B*08:01 to DRB1*03:01, C*07:02 to B*07:02 to DRB1*15:01, C*05:01 to B*44:02 to DRB1*04:01, C*16:01 to B*44:03 to DRB1*07:01, C*04:01 to B*35:01 to DRB1*01:01, C*06:02 to B*57:01 to DRB1*07:01, C*06:02 to B*13:02 to DRB1*07:01, C*08:02 to B*14:02 to DRB1*01:02 ...

67. The collection of claim 66, comprising a cell population having C*03:04 to B*40:01 to DRB1*04:04, C*04:01 to B*44:03 to DRB1*07:01, C*03:04 to B*40:01 to DRB1*13:02, C*03:04 to B*15:01 to DRB1*04:01, C*05:01 to B*18:01 to DRB1*03:01, C*05:01 to B*44:02 to DRB1*13:01, C*07:02 to B*07:02 to DRB1*01:01, and C*04:01 to B*35:02 to DRB1*11:

04.

68. The collection comprises at least the following haplotypes: C*07:01 to B*08:01 to DRB1*03:01, C*07:02 to B*07:02 to DRB1*15:01, C*05:01 to B*44:02 to DRB1*04:01, C*16:01 to B*44:03 to DRB1*07:01, C*04:01 to B*35:01 to DRB1*01:01, C*06:02 to B*57:01 to DRB1*07:01, C*06:02 to B*13:02 ~DRB1*07:01, C*08:02~B*14:02~DRB1*01:02, C*3:04~B*40:01~DRB1*04:04, C*04:01~B*44:03~DRB1*07:01, C*03:04~B *40:01~DRB1*13:02, C*03:04~B*15:01~DRB1*04:01, C*05:01~B*18:01~DRB1*03:01, C*05:01~B*44:02~DRB1*13:01, C* 07:02~B*07:02~DRB1*01:01, C*04:01~B*35:02~DRB1*11:04, C*12:02~B52:01~DRB1*15:02, C*03:03~B*15:01~DRB1*13 :01, C*07:02~B*07:02~DRB1*07:01, C*05:01~B*44:02~DRB1*15:01, C*12:03~B*38:01~DRB1*13:01, C*17:01~B*42:01~ 68. The collection of claim 67, comprising a cell population having DRB1*03:02, C*08:02 to B*14:01 to DRB1*07:01, C*01:02 to B*27:05 to DRB1*01:01, C*04:01 to B*35:01 to DRB1*11:01, C*06:02 to B*50:01 to DRB1*07:01, C*07:01 to B*18:01 to DRB1*11:04, and C*04:01 to B*53:01 to DRB1*13:

02.

69. The collection includes at least the following haplotypes: C*07:01~B*08:01~DRB1*03:01, C*07:02~B*07:02~DRB1*15:01, C*05:01~B*44:02~DRB1*04:01, C*16:01~B*44:03~DRB1*07:01, C*04:01~B*35:01~DRB1*01:01, C*06:02~B*57:01~DRB1*07:01, C*06:02~B*13:02~DRB1*07:01, C*08:02~B*14:02~DRB1*01:02, C*3:04~B*40:01~DRB1*04:04, C*04:01~B*44:03~DRB1*07:01, C*3:04~B*40:01~DRB1*13:02, C*3:04~B*15:01~DRB1*04:01, C*05:01~B*18:01~DRB1*03:01, C*05:01~B*44:02~DRB1*13:01, C*07:02~B*07:02~DRB1*01:01, C*04:01~B*35:02~DRB1*11:04, C*12:02~B52:01~DRB1*?15:02, C*3:03~B*15:01~DRB1*13:01, C*07:02~B*07:02~DRB1*07:01, C*05:01~B*44:02~DRB1*15:01, C*12:03~B*38:01~DRB1*13:01, C*17:01~B*42:01~DRB1*03:02, C*08:02~B*14:01~DRB1*07:01, C*01:02~B*27:05~DRB1*01:01, C*04:01~B*35:01~DRB1*11:01, C*06:02~B*50:01~DRB1*07:01, C*07:01~B*18:01~DRB1*11:04, C*04:01~B*53:01~DRB1~*13:02, C*12:03~B*18:01~DRB1*15:01, C*07:02~B*07:02~DRB1*11:01, C*05:01~B*44:02~DRB*01:01, C*04:01~B*53:01~DRB1*15:03, C*07:02~B*07:02~DRB1*04:01, C*08:02~B*14:02~DRB1*13:02, C*04:01~B*35:01~DRB1*07:01, C*07:01~B*08:01~DRB1*15:01,C*07:02~B*39:05~DRB1*04:07, C*04:01~B*53:01~DRB1*08:04, C*07:02~B*07:02~DRB1*13:01, C*12:03~B*38:0 1~DRB1*04:02, C*03:03~B*15:01~DRB1*04:01, C*04:01~B*35:01~DRB1*14:01, C*06:02~B*37:01~DRB1*10:01, C 67. The collection of claim 66, comprising a cell population having C*07:01 to B*49:01 to DRB1*11:01, C*03:04 to B*40:01 to DRB1*04:01, C*15:02 to B*51:01 to DRB1*11:01, C*03:02 to B*58:01 to DRB1*03:01, C*03:03 to B*55:01 to DRB1*14:01, and C*04:01 to B*35:01 to DRB1*13:

01.

70. 70. The collection of any one of claims 35 to 69, wherein the cell populations are each contained within a separate container suitable for cryopreservation, thawing, and / or maintaining the viability of the cell line.

71. 71. The collection of claim 70, wherein the cell lines further comprise a cryoprotectant.

72. 72. A method for cell therapy comprising administering to a recipient in need thereof a cell population or tissue derived from a cell population according to any one of claims 1 to 34, or a cell population derived from a cell population in a collection according to any one of claims 35 to 71.

73. 73. The method of claim 72, wherein the administered cell population or tissue is matched to the recipient for at least HLA-C and HLA-DRB1.

74. 74. The method of claim 73, wherein the administered cell population or tissue is further matched to the recipient for HLA-B.

75. 75. The method of any one of claims 72 to 74, wherein the administered cell population or tissue is further matched to the recipient for HLA-DQB1.

76. 76. The method of any one of claims 72 to 75, wherein a population of HSCs is administered to the recipient.

77. 76. The method of any one of claims 72 to 75, wherein an immune cell line is administered to the recipient.

78. 78. The method of claim 77, wherein the immune cell lineage is a T cell, a precursor T cell, an NK cell, a B cell, a monocyte, a macrophage, a neutrophil, a monocyte, an erythrocyte, a megakaryocyte, or a platelet.

79. 79. The method of any one of claims 76-78, wherein the subject has a condition selected from hematological malignancies, aplastic anemia, hemoglobinopathies, inborn errors of metabolism, and severe immunodeficiencies.

80. 80. The method of claim 79, wherein the subject has a condition selected from acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, a myeloproliferative disorder, myelodysplastic syndrome, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, aplastic anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, thalassemia major, sickle cell disease, severe combined immunodeficiency disease (SCID), acquired immunodeficiency syndrome (AIDS), Wiskott-Aldrich syndrome, hemophagocytic lymphohistiocytosis, inborn errors of metabolism, epidermolysis bullosa, severe congenital neutropenia, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, and leukocyte adhesion deficiency.

81. 81. The method of claim 80, wherein the T cell lineage is a T regulatory cell or a cytotoxic T cell.

82. 82. The method of claim 80 or 81, wherein the cell expresses a chimeric antigen receptor (CAR).

83. 83. The method of claim 81 or 82, wherein the recipient has a condition selected from one or more of lymphopenia, cancer, immunodeficiency, autoimmune disease, skeletal dysplasia, bone marrow failure syndrome, and a genetic disorder affecting the immune system.

84. 84. The method of any one of claims 79-83, wherein the recipient has undergone lymphocyte-depleting, cytoreductive, or immunomodulatory therapy prior to administration of the cell therapy.

85. 76. The method of any one of claims 72 to 75, wherein the cell population is iPSCs and a cell population or tissue derived from the cell line is administered to the recipient.

86. 86. The method of claim 85, wherein the cell population or tissue is selected from mesenchymal stem cells, neural stem cells, and epithelial stem cells.

87. 87. The method of claim 86, wherein the cell population or tissue is selected from neurons, astrocytes, oligodendrocytes, cardiac myocytes, skeletal muscle cells, hepatocytes, pancreatic beta cells, and lung epithelial cells, or precursors thereof.

88. 25. A method for producing the cell population of claim 23 or 24, comprising: Providing iPSCs; The iPSCs are modified to express HLA-A ｎｅｇ and (1) HLA-DPA1 ｎｅｇ and / or HLA-DPB1 ｎｅｇ and / or (2) HLA-DQA1 ｎｅｇ and / or HLA-DQB1 ｎｅｇ preparing an HLA-modified iPSC population, wherein the iPSC population is homozygous for or contains single genes for HLA-C, HLA-DRB1, and optionally HLA-B; preparing embryoid bodies (EBs) from the iPSC population; dissociating the EBs and enriching for CD34+ cells to prepare a CD34+ enriched cell population; inducing endothelial hematopoietic transition (EHT) of the CD34+ enriched cell population to produce a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem progenitor cells (HSPCs); Optionally, harvesting CD34+ cells from the HSC and / or HSPC-containing population to enrich for the population undergoing EHT; Optionally, differentiating the cell population undergoing EHT into a hematopoietic lineage.

89. 89. The method of claim 88, wherein EHT is induced for at least 2 days and not more than 12 days.

90. 90. The method of claim 88 or 89, wherein the iPSCs are HLA-modified using CRISPR-Cas9, CRISPR-Casl2, STAR-CRISPR, CRISPR-CasX, a CRISPR-associated transposase, a zinc finger nuclease, an RNA editor, an isolated genomic domain platform editing, or a combination thereof.

91. 91. The method of any one of claims 88 to 90, wherein the iPSCs are HLA-modified by electroporation using CRISPR-Cas9 endonuclease and one or more guide RNAs (gRNAs) as ribonucleoproteins.

92. 90. The method of claim 88 or 89, wherein the iPSCs are HLA-modified using one or more of siRNA(s) and antisense oligonucleotide(s).

93. 93. The method of any one of claims 88 to 92, wherein the iPSC population is a human iPSC population derived from lymphocytes, umbilical cord blood cells, peripheral blood mononuclear cells, CD34+ cells, or primary human tissue.

94. 94. The method of any one of claims 88 to 93, wherein CD34+ enrichment and endothelial-hematopoietic conversion are induced between days 7 and 15 of iPSC differentiation.

95. 95. The method of claim 94, wherein the CD34+ cells are harvested from a culture undergoing EHT, and the method comprises harvesting CD34+ floating and / or adherent cells.

96. The method of claim 94 or 95, wherein the induction of endothelial hematopoietic transition comprises increasing the expression or activity of dnmt3b.

97. 97. The method of claim 96, wherein the induction of endothelial hematopoietic conversion comprises applying repeated stretch to the CD34+ enriched population, wherein the repeated stretch is, optionally, 2D, 3D, or 4D repeated stretch.

98. 98. The method of claim 97, wherein said induction of endothelial-hematopoietic transition comprises Piezo1 activation.

99. 99. The method of claim 98, wherein the Piezo1 activation is by contacting the CD34+ enriched cells or a fraction thereof with one or more Piezo1 agonists optionally selected from Yoda1, Jedi1, Jedi2, or analogs, derivatives, ssRNA agonists, and combinations thereof.

100. 100. The method of claim 98 or 99, wherein said induction of endothelial-hematopoietic conversion comprises Trpv4 activation, optionally by contacting said CD34-enriched cells with one or more Trpv4 agonists, optionally selected from one or more of GSK1016790A, 4α-PDD, and analogs or derivatives thereof.

101. 101. The method of any one of claims 88-100, wherein the CD34+ enriched cells undergoing EHT are differentiated into one or more of common lymphoid progenitor (CLP) cells, granulocyte-monocyte progenitor (GMP) cells, precursor T cells, T lymphocytes, B lymphocytes, natural killer cells, neutrophils, monocytes, macrophages, erythrocytes, megakaryocytes, and platelets.

102. 102. The method of claim 101, wherein the CD34+ enriched cells that undergo EHT are differentiated ex vivo into precursor T cells, T cells, or NK cells.

103. 103. The method of claim 102, wherein the CD34+ enriched cells that undergo EHT are cultured with a partial or complete Notch ligand to produce a population comprising a CD7+ progenitor T cell or derivative cell population.

104. A cell population produced by the method of any one of claims 88 to 103.

105. A method for producing an HLA-modified cell according to any one of claims 1 to 34, comprising: A method comprising contacting a cell with a Cas endonuclease and one or more guide (gRNA) that targets the Cas endonuclease to one or more HLA-specific or HLA allele-specific regions.

106. 106. The method of Claim 105, wherein the Cas endonuclease comprises Cas9.

107. 107. The method of claim 105 or 106, wherein each gRNA is a single guide RNA (sgRNA).

108. 108. The method of any one of Claims 105-107, wherein contacting comprises electroporating said Cas endonuclease and said one or more gRNAs as ribonucleoproteins.

109. 109. The method of any one of Claims 105-108, wherein the cell is homozygous or heterozygous for HLA-A*01:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2A or Table 6.

110. 109. The method of any one of claims 105-108, wherein the cell is homozygous or heterozygous for HLA-A*02:05, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2B.

111. 109. The method of any one of claims 105-108, wherein the cell is homozygous or heterozygous for HLA-A*03:01, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 2B or Table 6.

112. 109. The method of any one of claims 105-108, wherein the cell is homozygous or heterozygous for HLA-A*23:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2C.

113. 109. The method of any one of claims 105-108, wherein the cell is homozygous or heterozygous for HLA-A*29:02, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 2E or Table 6.

114. 109. The method of any one of Claims 105-108, wherein the cell is homozygous or heterozygous for HLA-A*25:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2D.

115. 109. The method of any one of Claims 105-108, wherein the cell is homozygous or heterozygous for HLA-A*33:01, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 2G or Table 6.

116. 109. The method of any one of claims 105-108, wherein the cell is homozygous or heterozygous for HLA-A*11:01, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 2C or Table 7.

117. 109. The method of any one of claims 105-108, wherein the cell is homozygous or heterozygous for HLA-A*26:01, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 2D or Table 7.

118. 109. The method of any one of claims 105-108, wherein the cell is homozygous or heterozygous for HLA-A*30:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2E.

119. 109. The method of any one of claims 105-108, wherein the cell is homozygous or heterozygous for HLA-A*30:02, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2F.

120. 109. The method of any one of claims 105-108, wherein the cell is homozygous or heterozygous for HLA-A*31:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 2F.

121. 109. The method of any one of claims 105-108, wherein the cell is homozygous or heterozygous for HLA-A*24:02, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 10.

122. 122. The method of any one of claims 105-121, wherein the cell is homozygous or heterozygous for DQB1*02:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 4A and Table 8.

123. 122. The method of any one of claims 105-121, wherein the cell is homozygous or heterozygous for DQB1*06:02, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 8.

124. 122. The method of any one of claims 105-121, wherein the cell is homozygous or heterozygous for DQB1*03:03, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 4B or Table 8.

125. 122. The method of any one of claims 105-121, wherein the cell is homozygous or heterozygous for DQB1*05:01, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 4C or Table 8.

126. 122. The method of any one of claims 105-121, wherein the cell is homozygous or heterozygous for DQB1*06:01, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 4D or Table 9.

127. 122. The method of any one of claims 105-121, wherein the cell is homozygous or heterozygous for DQB1*06:03, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 9.

128. 122. The method of any one of claims 105-121, wherein the cell is homozygous or heterozygous for DQB1*02:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 4A.

129. 122. The method of any one of claims 105-121, wherein the cell is homozygous or heterozygous for DQB1*03:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 4A.

130. 122. The method of any one of claims 105-121, wherein the cell is homozygous or heterozygous for DQB1*03:02, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 4B.

131. 122. The method of any one of claims 105-121, wherein the cell is homozygous or heterozygous for DQB1*05:03, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 4C.

132. 122. The method of any one of claims 105-121, wherein the cell is homozygous or heterozygous for DQB1*06:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 4D.

133. 122. The method of any one of claims 105-121, wherein the cell is homozygous or heterozygous for DQB1*06:04, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 4E.

134. 134. The method of any one of claims 105-133, wherein the cell is homozygous or heterozygous for HLA-B*07:02, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 3A or Table 11.

135. 134. The method of any one of claims 105-133, wherein the cell is homozygous or heterozygous for HLA-B*13:02, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 3B.

136. 134. The method of any one of claims 105-133, wherein the cell is homozygous or heterozygous for HLA-B*44:03, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 3F or Table 11.

137. 134. The method of any one of claims 105-133, wherein the cell is homozygous or heterozygous for HLA-B*18:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 3C.

138. 134. The method of any one of claims 105-133, wherein the cell is homozygous or heterozygous for HLA-B*57:01, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 3G or Table 11.

139. 134. The method of any one of claims 105-133, wherein the cell is homozygous or heterozygous for HLA-B*08:01, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 3A or Table 11.

140. 134. The method of any one of claims 105-133, wherein the cell is homozygous or heterozygous for HLA-B*40:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 3E.

141. 134. The method of any one of claims 105-133, wherein the cell is homozygous or heterozygous for HLA-B*14:02, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 3B or Table 12.

142. 134. The method of any one of Claims 105-133, wherein the cell is homozygous or heterozygous for HLA-B*50:01, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 3F or Table 12.

143. 134. The method of any one of Claims 105-133, wherein the cell is homozygous or heterozygous for HLA-B*37:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 12.

144. 134. The method of any one of Claims 105-133, wherein the cell is homozygous or heterozygous for HLA-B*52:01, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 3G or Table 12.

145. 134. The method of any one of Claims 105-133, wherein the cell is homozygous or heterozygous for HLA-B*38:01, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 3D or Table 13.

146. 134. The method of any one of Claims 105-133, wherein the cell is homozygous or heterozygous for HLA-B*35:01, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 3D or Table 13.

147. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*03:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5A or Table 14.

148. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*15:01, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 5F or Table 14.

149. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*07:01, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 5C or Table 14.

150. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*01:02, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5A or Table 14.

151. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*10:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 15.

152. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*15:02, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 5F or Table 15.

153. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*13:01, and wherein the cell is contacted with one or two sgRNAs comprising a nucleotide sequence independently selected from Table 5D or Table 15.

154. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*01:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5A or Table 15.

155. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*04:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5B.

156. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*04:02, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5B.

157. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*04:04, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5C.

158. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*11:04, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5D.

159. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*13:02, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5E.

160. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*14:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 5E.

161. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*03:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 14.

162. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*15:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 14.

163. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*07:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 14.

164. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*01:02, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 14.

165. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*10:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 15.

166. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*15:02, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 15.

167. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*13:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 15.

168. 147. The method of any one of claims 105-146, wherein the cell is homozygous or heterozygous for DRB1*01:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 15.

169. 169. The method of any one of claims 105-168, wherein the cell is homozygous or heterozygous for DPB1*01:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 16.

170. 169. The method of any one of claims 105-168, wherein the cell is homozygous or heterozygous for DPB1*02:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 16.

171. 169. The method of any one of claims 105-168, wherein the cell is homozygous or heterozygous for DPB1*03:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 16.

172. 169. The method of any one of claims 105-168, wherein the cell is homozygous or heterozygous for DPB1*04:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 16.

173. 169. The method of any one of claims 105-168, wherein the cell is homozygous or heterozygous for DPB1*04:02, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 17.

174. 169. The method of any one of claims 105-168, wherein the cell is homozygous or heterozygous for DPB1*11:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 17.

175. 169. The method of any one of claims 105-168, wherein the cell is homozygous or heterozygous for DPB1*17:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 17.

176. 169. The method of any one of claims 105-168, wherein the cell is homozygous or heterozygous for DPB1*34:01, and wherein the cell is contacted with one or two sgRNAs comprising nucleotide sequences independently selected from Table 17.

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