T cell populations derived from pluripotent stem cells and their progenitor cells

Abstract

In various aspects and embodiments, the present disclosure provides methods for generating T cell populations for cell therapy, including T lymphocytes (T cells) and progenitor T cells. In various embodiments, the present invention provides an efficient ex vivo process for developing progenitor T cells and T cell populations (including, but not limited to, CD4+CD8+ "double-positive" T cells, as well as single-positive CD4+ T helper cells and CD8+ cytotoxic T cells, and T regulatory cells) from human induced pluripotent stem cells (iPSCs). In various embodiments, cells generated according to the present disclosure are functional and / or more closely resemble corresponding lineages isolated from peripheral blood or lymphoid organs. The present invention in some aspects provides isolated cells and cell compositions produced by the methods disclosed herein, as well as methods for cell therapy.

Description

[Technical Field]

[0001] Priority This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 413,338, 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 via EFS-Web in XML format and is incorporated herein by reference in its entirety. The XML copy, created on September 25, 2023, is named GRU-016 / 121145-5016_Sequence_Listing.xml and is 30,062 bytes in size. [Background technology]

[0003] The generation of hematopoietic cells from pluripotent cells ex vivo has attracted interest from the scientific community for the promise of allocompatible cell-based therapy. Induced pluripotent stem cells (iPSCs) could potentially serve as a source for generating “off-the-shelf” therapeutic lymphocytes. Nianias, A., & Themeli, M., Induced pluripotent stem cell (iPSC)-derived lymphocytes for adoptive cell immunotherapy: recent advances and challenges. Current Hematologic Malignancy Reports, 14(4), 261-268 (2019). However, significant obstacles remain in developing methods for generating clinically relevant numbers of hematopoietic cell lineages, such as T cells, with clinically advantageous phenotypes. Thus, successful generation of T cell populations suitable for cell therapy ex vivo from iPSCs would fulfill a significant need. In various aspects and embodiments, the present invention fulfills these objectives. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Nianias, A., & Themeli, M., Induced pluripotent stem cell (iPSC)-derived lymphocytes for adoptive cell immunotherapy: recent advances and challenges. Current Hematologic Malignancy Reports, 14(4), 261-268 (2019). Summary of the Invention [Means for solving the problem]

[0005] The present disclosure, in various aspects and embodiments, provides methods for generating T cell populations for cell therapy, including T lymphocytes (T cells) and progenitor T cells. In various embodiments, the invention provides an efficient ex vivo process for developing progenitor T cells and T cell populations (including, but not limited to, precursor T cells, CD4+CD8+ "double positive" T cells, CD4+ T helper cells, CD8+ cytotoxic T cells, and T regulatory cells) from human induced pluripotent stem cells (iPSCs). In various embodiments, cells generated according to the present disclosure are functional and / or more closely resemble corresponding lineages isolated from peripheral blood or lymphoid organs. The invention in some aspects provides isolated cells and cell compositions, including cell compositions produced by the methods disclosed herein, as well as methods (and uses) for cell therapy. In one aspect, the present disclosure provides a method for preparing a T cell population or a population of T cell precursors. The method includes enriching a differentiated pluripotent stem cell population for CD34+ cells to prepare a CD34+ enriched population. Endothelial-to-hematopoietic transformation (EHT) is induced in the CD34+ enriched population for at least 2 days but not more than 12 days to prepare a population containing hematopoietic stem cells (HSCs) and / or hematopoietic stem progenitor cells (HSPCs). The resulting population containing HSCs and / or HSPCs (or a fraction thereof) is differentiated into a T cell population or a population containing progenitor T cells.

[0006] Traditionally, hematopoietic lineages are prepared by differentiating iPSCs into embryoid bodies up to day 8 and harvesting CD34+ cells. CD34 is commonly used as a marker for hemogenic endothelial cells, hematopoietic stem cells, and hematopoietic progenitor cells. According to aspects and embodiments of the present disclosure, it has been discovered that inducing endothelial-to-hematopoietic transition (EHT) of CD34+ cell populations, which may be derived from iPSC embryoid bodies, can be used for the ex vivo generation of superior T cell progenitor and T cell populations.

[0007] In a non-limiting example, to generate mature T cells, embryoid body formation is used to generate CD34+ cells from which HSCs and / or HSPCs are derived by inducing EHT. The HSC and / or HSPC population is then cultured in T cell medium supplemented with, for example, retronectin and DLL-4, for the generation of T progenitor or pre-T cells. Further culture will generate mature T cells. Foxp3 expression in α / β T cells leads to Treg generation ex vivo. Cells can optionally be harvested or recovered at certain steps, or in some embodiments, differentiation into the desired T cell population does not include a harvest / harvest step; i.e., differentiation can occur continuously in culture.

[0008] 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 methods include generating an HSC and / or HSPC population, which can include human long-term hematopoietic stem cells (LT-HSCs), from iPSCs (e.g., hiPSCs). The HSC and / or HSPC population is derived by inducing endothelial-to-hematopoietic transformation of CD34+ cells (e.g., CD34+ cells derived from embryoid bodies). The HSC and / or HSPC population (or cells isolated therefrom) is cultured with a partial or complete Notch ligand, sonic hedgehog (SHH), retronectin (or other extracellular matrix component(s)), and / or combinations thereof to produce a population comprising CD7+ progenitor T cells or a derivative cell population (e.g., a T cell population).

[0009] In various embodiments, iPSCs are prepared by reprogramming somatic cells, such as, but not limited to, CD34+ cells isolated from peripheral blood. In various embodiments, iPSCs are autologous or allogeneic to the recipient (e.g., HLA-matched at one or more loci). In various embodiments, iPSCs can be gene-edited to support HLA matching. For example, iPSCs can be gene-edited to delete one or more of HLA-A, HLA-B, and HLA-C, and to delete one or more of HLA-DP, HLA-DQ, and HLA-DR. In certain embodiments, iPSCs retain expression of at least one HLA class I and at least one HLA class II complex. In some embodiments, T cell populations are HLA-A, HLA-B, and HLA-DR. neg , homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg In some embodiments, the iPSCs are further homozygous for HLA-DRB1.

[0010] In some embodiments, processes according to the present invention can include generating CD34+ enriched cells from differentiated pluripotent stem cells (e.g., from EBs) and inducing endothelial and hematopoietic differentiation. 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 local stimuli, as well as factors such as extracellular matrix, niche factors, and cell-extrinsic factors, induction of cell-intrinsic properties, and including pharmacological and / or genetic means. In some embodiments, CD34+ enrichment and EHT are induced when the cells are at least 20% CD34+, e.g., on days 6-14 of iPSC differentiation, such as days 8, 9, 10, 11, 12, 13, or 14. Differentiation of iPSCs can be performed by known techniques.

[0011] Induction of EHT can be by any known process. In various embodiments, EHT can be induced in culture for 2 to 12 days. In some embodiments, EHT is induced in culture for about 5 to about 7 days. In some embodiments, the method includes increasing the activity or expression of DNA (cytosine-5-)-methyltransferase 3β (Dnmt3b) in a cell population, such as a CD34+ cell population comprising hemogenic endothelial cells. In some embodiments, the cells are contacted with an effective amount of an agonist of a mechanosensitive receptor or mechanosensitive channel that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezo1. An exemplary, non-limiting Piezo1 agonist is Yoda1.

[0012] In various embodiments, CD34+ cells are harvested from cultures undergoing endothelial-to-hematopoietic transition on days 10-20 of iPSC differentiation, such as days 10-20 or days 12-15 of iPSC differentiation.

[0013] In various embodiments, populations comprising HSCs and / or HSPCs or fractions thereof are differentiated into populations comprising T cell precursors or T lymphocytes. In some embodiments, the cell populations are cultured ex vivo with a partial or complete Notch ligand, SHH, extracellular matrix component(s), and / or a combination thereof to differentiate the HSCs into T cell populations (or their precursors). Differentiation into progenitor T cells can, in some embodiments, further include the presence of stem cell factor (SCF), Flt3L, and interleukin (IL)-7. For example, HSCs and / or HSPCs can be 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, the cells are cultured for 7 to 14 days to prepare progenitor or pre-T cells (e.g., CD34-, CD7+, CD5+ / -). In some embodiments, the cells are cultured for 15-28 days for the production of mature T cells (e.g., CD3+), optionally including the production of Tregs. In some embodiments, T lymphocytes and precursor T cells can be differentiated into Tregs by expression of FOXP3, which are optionally expanded in culture.

[0014] In yet other embodiments, the present invention generates T cells (e.g., CTLs, helper T cells, or Tregs) that express chimeric antigen receptors. The cells can be efficiently transduced with vectors, such as, but not limited to, retroviral or non-integrating viral vectors, non-viral vectors, and episomal or episomal hybrid vectors carrying CAR-targeted tumor antigens, including, but not limited to, CD19, CD38, CD33, CD47, and CD20. CARs can be engineered to enhance the ability of cells to recognize, bind to, and / or kill target cells. In some embodiments, the CAR enhances the ability of cells to recognize tumor cells. In some embodiments, the CAR enhances the anti-tumor activity of the cells.

[0015] In some embodiments, the present invention generates T cells that exhibit T cell activation and subsequent T cell-mediated cytotoxicity. The T cells generated herein can exhibit significantly superior performance in T cell-mediated cytotoxicity compared to CD34+-derived T cells.

[0016] In another aspect, the invention provides a cell population or a pharmaceutically acceptable composition thereof produced by the methods described herein. In some embodiments, the cell population is a progenitor T cell population capable of engraftment in the thymus, spleen, or secondary lymphoid organs upon administration to a subject in need thereof. In other embodiments, the cell population is an α / β T cell population, a CAR-T cell population, a CTL population (capable of expressing a CAR), a T helper population, or a Treg population (each as described herein).

[0017] In some embodiments, the cell population is a Treg population useful for adoptive cell therapy for a human subject having a condition selected from, for example, an autoimmune or inflammatory condition or disease, or graft-versus-host disease (GVHD). Additionally, various genetic diseases affect the immune system and can manifest as autoimmune or pro-inflammatory conditions. In some embodiments, the Treg population is a CAR-T cell that expresses a tissue-specific CAR or a cell-specific CAR.

[0018] In another aspect, the present disclosure provides an HLA-A neg , HLA-DPB1 neg , and HLA-DQB1 neg

[0010] The present invention provides a cell composition comprising a T cell population (or a progenitor thereof, such as a progenitor T cell population) that is a T cell ...

[0019] In some embodiments of this aspect, the T cell population is a T cell precursor population. In various embodiments, the T cell precursor population engrafts in the thymus or spleen. In other embodiments, the T cell population is a cytotoxic T cell (CTL) population, a helper T cell population, or a Treg population. In various embodiments, the T cell population may express a chimeric antigen receptor (CAR). Cell populations according to this aspect may be prepared according to other aspects of the disclosure.

[0020] In another aspect, the present invention provides methods for cell therapy (or uses 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).

[0021] In some embodiments, the subject has cancer, such as a hematological malignancy or a solid tumor, and in such embodiments, the subject is administered T cell precursors or T cells with anti-tumor specificity (such as CTLs that recognize tumor antigens).

[0022] In other embodiments, the present invention provides methods for cell therapy, comprising administering a Treg cell population described herein or a pharmaceutically acceptable composition thereof to a human subject in need thereof. In various embodiments, the subject has an autoimmune, alloimmune, or inflammatory disease. In some embodiments, the subject is a recipient of a tissue or organ transplant, and in some embodiments, the subject is a recipient of an allogeneic organ or tissue transplant. In some embodiments, the subject is experiencing or at risk of GVHD. For example, organs that can be transplanted include the heart, kidney, liver, lung, pancreas, intestine, and thymus, among others. Tissues for transplant may include, for example, bone, tendon (both referred to as musculoskeletal grafts), bone marrow or HSCs, cornea, skin, heart valve, nerve, and / or vein.

[0023] In some embodiments, the subject has an autoimmune condition selected from type 1 diabetes, rheumatoid arthritis (RA), psoriasis or psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, scleroderma, hemolytic anemia, pernicious anemia, and Goodpasture's syndrome.

[0024] Other aspects and embodiments of the present disclosure will be apparent from the following detailed disclosure and examples. [Brief explanation of the drawings]

[0025] [Figure 1]Figure 1 shows that ETV2 overexpression (OE) does not affect pluripotency. Figure 1 shows a FACS plot showing the transduction efficiency of iPSCs using an adenoviral vector to overexpress ETV2 and GFP sequences. ETV2 overexpression does not affect iPSC stemness, as indicated by the expression of the TRA-1-60 stemness marker. [Figure 2] ETV2 overexpression (OE) increases the yield of hemogenic endothelial cells. Representative flow cytometry analysis and relative quantification of hemogenic endothelial cells (described as CD235a-CD34+CD31+) demonstrates that ETV2-OE enhances the formation of hemogenic endothelial cells. [Figure 3] Figure 1 shows that ETV2 overexpression (OE) enhances CD34+ cell formation during iPSC differentiation. Representative flow cytometry analysis and relative quantification of CD34+ cells demonstrates that ETV2-OE enhances CD34+ cell formation. [Figure 4A] Figure 1 shows that iPSC-derived HSCs derived by Piezo1 activation undergo pre-T cell differentiation similar to bone marrow (BM)-HSCs. Figure 2 shows FACS plots of the differentiation efficiency of bone marrow (BM)-HSCs and iPSC-HSCs derived by Piezo1 activation into CD34+CD7+ pre-T cells. [Figure 4B] Figure 1 shows that iPSC-derived HSCs derived by Piezo1 activation undergo pre-T cell differentiation similar to bone marrow (BM)-HSCs. Quantification (%) of CD34+CD7+ cells derived from (1) BM-HSCs and (2) iPSC-HSCs (Piezo1-activated). Averages of three experiments are shown. [Figure 5A] Figure 1 shows 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. Figure 2 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 5B]Figure 1 shows 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 6] We demonstrate that iPSC-derived HSCs (in this example, using Piezo1 activation) can differentiate into functional T cells. IFNγ expression is a consequence of T cell activation after T cell receptor (TCR) stimulation via CD3 / CD28 beads. IFNγ expression in T cells differentiated from iPSC-derived HSCs generated upon Piezo1 activation enhances the HSC capacity to further differentiate into functional T cells. Averages of three experiments are shown. [Figure 7] We demonstrate that HSCs generated according to the present disclosure (D8+7 iPSC-CD34+ cells with or without Yoda 1 "Y") can be successfully differentiated into CD4+CD8+ ("double positive") T cells as well as TCR α / β T cells. The disclosed method substantially outperforms T cell maturation from bone marrow CD34+ cells. [Figure 8] We show that HSCs generated according to the present disclosure (D8+7iPSC-CD34+ cells with or without Y) successfully rearrange TCR and outperform bone marrow CD34+ cells. [Figure 9A]

[0023] Figure 1 shows phenotypic analysis of HLA-edited (e.g., triple knockout) cells performed by FACS and immunofluorescence. Global expression of HLA class I molecules (HLA-A, HLA-B, and HLA-C) on the cell surface is shown, with HLA-edited cells being positive for global HLA class I expression to a similar extent as wild-type cells (gHSCs). [Figure 9B] Figure 1 shows phenotypic analysis of HLA-edited (e.g., triple knockout) cells performed by FACS and immunofluorescence. Cellular expression of HLA-A via immunofluorescence is shown, where HLA-A is not expressed in HLA-edited clones. [Figure 10]We show that the HLA-edited clones retain their pluripotency (maintain tri-lineage differentiation) as illustrated by immunofluorescence, where ectodermal differentiation is indicated by NESTIN-488 and PAX6-594 staining, mesodermal differentiation is indicated by GATA-488 staining, and endodermal differentiation is indicated by CXCR4-488 and FOX2A-594 staining. [Figure 11] Immunocompatibility of HLA-edited HSCs is shown. HLA-edited HSCs and control HSCs (WT, B2M KO, and HLA class II null) were cocultured with peripheral blood mononuclear cells (PBMCs) bearing HLA-B and HLA-C matched but mismatched HLA-A. PBMC-mediated cytotoxicity was measured by Annexin V staining assay. [Figure 12] Figure 1 shows the in vivo engraftment potential of HLA-edited HSCs. Equal proportions of mCherry HLA-edited HSCs and wild-type HSCs (gHSCs) were mixed for competitive transplantation into mice, and bone marrow (BM) and peripheral blood samples were assessed by FACS to compare the relative amounts of each cell type present in the samples. [Figure 13A] Figure 1 shows that HLA-A deletion does not affect class I peptide presentation. Schematic diagram of immunopeptidome analysis. [Figure 13B] Figure 1 shows that HLA-A deletion does not affect class I peptide presentation. Figure 1 shows the results of immunopeptidome analysis, revealing little difference in the number of peptides and representative proteins presented by class I molecules in WT and HLA-edited cells. [Figure 14A] Figure 1 shows that deletion of HLA-DP and DQ does not affect class II peptide presentation. Figure 2 shows the immunopeptidome analysis scheme. [Figure 14B] Figure 1 shows that deletion of HLA-DP and DQ does not affect class II peptide presentation. Figure 2 shows that despite deletion of HLA-DP and DQ, cells retain the ability to present a wide range of peptides through HLA class II. [Figure 15] Schematic diagram of in vivo testing of antigen-mediated immune responses: delayed-type hypersensitivity assay (DTH), sensitization phase, and elimination phase, respectively. [Figure 16A] We demonstrate that HLA-edited HSCs reconstitute a functional immune system, as evidenced by DTH responses in immunodeficient mice. Figure 16 shows a delayed-type hypersensitivity assay performed on transplanted mice, an assay involving crosstalk between different types of immune cells. Mice were sensitized by subcutaneous injection of sheep red blood cells (antigen). A functional immune system results in swelling of the left paw, as measured with a microcaliper. As can be seen in Figure 16A, non-transplanted mice, being immunodeficient, did not exhibit swelling of the left paw. Conversely, mice transplanted with umbilical cord blood CD34+ cells exhibited tissue swelling, doubling the diameter of their left paw. [Figure 16B] 16A-B are graphical evaluations of the results shown in Figure 16A, showing that HLA-edited HSCs reconstitute a functional immune system as demonstrated by DTH responses in immunodeficient mice. [Figure 17] Figure 17 shows the differentiation potential of HSCs into T cell subtypes. After a 35-day differentiation period, pre-T cells were assessed by cell sorting for the presence of CD4+, CD8+, and AB+ T cell populations. Figure 17 compares the differentiation potential of bone marrow-derived CD34+ cells, embryoid body CD34+ cells, and HSCs prepared according to the present disclosure (e.g., using Piezo1 activation) ("gHSCs"). [Figure 18]

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

[0026] The term "gHSC" is used herein to refer to the iPSC-derived hematopoietic stem cells of the present disclosure.

[0027] The terms "wild-type" (WT), "unedited," and "non-HLA edited" are used interchangeably herein to refer to the non-gene-edited cells of the present disclosure.

[0028] EB34+ cells refer to embryonic body-derived CD34+ cells, which contain hemogenic endothelial cells. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present disclosure, in various aspects and embodiments, provides methods for generating T cell populations for cell therapy, including T lymphocytes (T cells) and progenitor T cells. In various embodiments, the invention provides an efficient ex vivo process for developing progenitor T cells and T cell populations (including, but not limited to, precursor T cells, CD4+CD8+ "double positive" T cells, CD4+ T helper cells, CD8+ cytotoxic T cells, and T regulatory cells) from human induced pluripotent stem cells (iPSCs). In various embodiments, cells generated according to the present disclosure are functional and / or more closely resemble corresponding lineages isolated from peripheral blood or lymphoid organs. The invention in some aspects provides isolated cells and cell compositions, including those produced by the methods disclosed herein, and methods for cell therapy.

[0030] According to aspects and embodiments of the present disclosure, the ability of human induced pluripotent stem cells (hiPSCs) to generate essentially unlimited pluripotent stem cells (PSCs) is exploited to generate an unlimited supply of T cell populations or their progenitors. The use of primary T cells as therapeutic lymphocytes is limited by their limited availability, cell number, limited proliferation capacity, and histocompatibility issues. For example, Tregs exist in low numbers in the circulation and are difficult to isolate and expand ex vivo. Furthermore, compared to primary cells, hiPSCs are more easily genetically modified in vitro, thereby offering opportunities for improving cell targeting specificity and cell number, as well as circumventing HLA matching issues, for example. In addition, fully engineered hiPSC clones can serve as a stable and safe source compared to primary cells (Nianias and Themeli, 2019). Furthermore, unlike human embryonic stem cells (hESCs), hiPSCs are of non-embryonic origin, eliminating ethical concerns. Thus, the use of hiPSCs according to the present disclosure offers several advantages over primary cells for generating therapeutic numbers of T cells or progenitor cells, including antigen-specific or tissue-specific T cells (including Tregs).

[0031] In one aspect, the disclosure provides a method for preparing a T cell population or a population of T cell precursors. The method includes enriching a differentiated pluripotent stem cell population for CD34+ cells to prepare a CD34+ enriched population. Endothelial-to-hematopoietic transition (EHT) is induced in the CD34+ enriched population for at least 2 days but not more than 12 days to prepare a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem progenitor cells (HSPCs). In various embodiments, the HSC and / or HSPC population is a non-adherent cell population. In some embodiments, these cells are further enriched for CD34+ cells. The resulting population comprising HSCs and / or HSPCs (or a fraction thereof) is differentiated into a T cell population or a population comprising a progenitor T cell population.

[0032] Traditionally, hematopoietic lineages are prepared by differentiating iPSCs into embryoid bodies up to day 8 and harvesting CD34+ cells. CD34 is commonly used as a marker for hemogenic endothelial cells, hematopoietic stem cells, and hematopoietic progenitor cells. According to aspects and embodiments of the present disclosure, it has been discovered that inducing endothelial-to-hematopoietic transition (EHT) of CD34+ cell populations, which may be derived from iPSC embryoid bodies, can be used for the ex vivo generation of superior T progenitor and T cell populations, including, but not limited to, T regulatory cells (Tregs).

[0033] In a non-limiting example, to generate mature T cells, embryoid body formation, which takes about 8 to about 14 days (but is not limited to this), is used to generate CD34+ cells from which HSCs and / or HSPCs are derived by inducing EHT. The HSC and / or HSPC population (e.g., CD34+ cells undergoing EHT) is then cultured in T cell medium supplemented with, for example, retronectin and DLL-4 to generate T progenitor cells, which can be identified as CD34+CD7+CD5+ / -, or pre-T cells, which can be identified as CD34-CD7+CD5+. Further culture will generate mature T cells (CD3+, α / β T cells). Foxp3 expression in α / β T cells leads to ex vivo Treg generation.

[0034] For example, the earliest intrathymic progenitor cells express high levels of CD34 and CD7, do not express CD1a, and are triple negative (TN) for mature T cell markers: CD4, CD8, and CD3. Commitment to the T cell lineage is associated with expression of CD1a by CD7-expressing prothymocytes. Thus, immature stages of T cell development are typically characterized by the expression of CD34 + CD1a - (mostly immature) and CD34 + CD1a + Described as cells. CD34 by early thymocytes + CD7 + CD1a - from CD34 + CD7 + CD1a +Conversion to CD34 is associated with T cell involvement. + CD7 + CD1a + Following this stage, thymocytes progress to a CD4 immature mono-positive stage, at which point CD4 is expressed in the absence of CD8. A subset of cells then expresses CD4 + CD8 + Finally, following TCRα rearrangement, TCRαβ-expressing DP thymocytes undergo positive and negative selection and differentiate to the CD4 + CD8 - and CD4 - CD8 + Generate single positive (SP) T cells.

[0035] In various embodiments, as described in detail herein, populations comprising HSCs and / or HSPCs are differentiated into populations comprising one or more of T progenitor cells, precursor T cells, double-positive T cells, single-positive T cells (such as CD8+ or CD4+), and regulatory T cells (Tregs). Cells can optionally be harvested or recovered at certain steps, or in some embodiments, differentiation into the desired T cell population does not involve a harvest / harvest step; i.e., differentiation can occur continuously in culture.

[0036] In some embodiments, the population comprising HSCs and / or HSPCs is differentiated into a population comprising T progenitor cells. The T progenitor cells can be recovered from the culture for cell therapy or, alternatively, further differentiated in culture. In some embodiments, the T progenitor cells are further differentiated without first recovering them from the culture. For example, the T progenitor cells can be further differentiated into a population comprising one or more of precursor T cells, double-positive T cells (CD4+CD8+), single-positive T cells (CD4+CD8- or CD8+CD4-), or regulatory T cells. In some embodiments, the T progenitor cells are further differentiated into a population comprising regulatory T cells. The precursor T cells are optionally recovered from the culture prior to such differentiation.

[0037] In some embodiments, the population comprising HSCs and / or HSPCs is differentiated into a population comprising double-positive T cells (CD4+CD8+) and / or single-positive T cells (CD4+CD8-, CD8+CD4-), optionally in continuous culture (i.e., without harvesting or recovering intermediate cell populations). In various embodiments, the double-positive and / or single-positive T cells are differentiated into regulatory T cells, optionally including a step of recovering the double-positive and / or single-positive cells from the culture prior to differentiation into regulatory T cells.

[0038] In some embodiments, a population comprising HSCs and / or HSPCs is differentiated into a population comprising T regulatory cells, which can optionally occur in continuous culture (i.e., without harvesting or recovering intermediate cell populations).

[0039] 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 methods include generating an HSC and / or HSPC population, which can include human long-term hematopoietic stem cells (LT-HSCs), from iPSCs (e.g., hiPSCs). The HSC and / or HSPC population is derived by inducing endothelial-to-hematopoietic transformation of CD34+ cells (e.g., CD34+ cells derived from embryoid bodies). The HSC and / or HSPC population (or cells isolated therefrom) is cultured with a partial or complete Notch ligand, sonic hedgehog (SHH), retronectin (or other extracellular matrix component(s)), and / or combinations thereof to produce a population comprising CD7+ progenitor T cells or a derivative cell population (e.g., a T cell population).

[0040] 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 proceeds after lymphoid progenitor cells differentiate from bone marrow hematopoietic stem cells and migrate to the thymus. Specialized thymic epithelial cells guide T cells to develop along a regulated pathway. Notch signaling plays a critical role during T lineage commitment in the thymus. As lymphoid progenitor cells enter the thymus, they encounter high-density expression of Notch ligands on the thymic epithelium, which promotes thymopoiesis. The present disclosure provides HSC and / or HSPC populations generated ex vivo from iPSCs and that respond to Notch ligands, SHH, and / or extracellular matrix components by robustly producing T progenitor cells and T cell lineages ex vivo.

[0041] In various embodiments, iPSCs are prepared by reprogramming somatic cells. The term "induced pluripotent stem cells" or "iPSCs" refers to cells derived from somatic cells, such as skin or blood cells, that have been reprogrammed back to an embryonic-like pluripotent state. In some embodiments, iPSCs are generated from somatic cells, such as (but not limited to) fibroblasts or PBMCs (or cells isolated therefrom). In some embodiments, iPSCs are derived from lymphocytes (e.g., T cells, B cells, NK cells, etc.), umbilical cord blood cells (including CD3+ and / or CD4+ cells derived from umbilical cord blood), PBMCs, CD34+ cells, or other primary human tissues. In some embodiments, iPSCs are derived from CD34+ cells isolated from peripheral blood. In various embodiments, iPSCs are autologous or allogeneic (e.g., HLA-matched at one or more loci) to the recipient (the subject in need of the treatment described herein). In various embodiments, iPSCs can be gene-edited to support HLA matching (such as deletion of one or more HLA class I and / or HLA class II alleles or their master regulators, including but not limited to beta-2-microglobulin (B2M), CIITA, etc.), or to delete or express other functions. For example, iPSCs can be gene-edited to delete one or more of HLA-A, HLA-B, and HLA-C, and one or more of HLA-DP, HLA-DQ, and HLA-DR. In certain embodiments, iPSCs retain expression of at least one HLA class I and at least one HLA class II complex.

[0042] In various embodiments, the T cell population comprises: (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 - The modified cells are derived from iPSCs that have been gene-edited to be one of the following: (1) a single-stranded HLA-B gene (HLA-B), (2) a single-stranded HLA-C gene (HLA-C), and (3) a single-stranded HLA-DR gene (HLA-DQ). For the retained HLA (e.g., HLA-B, HLA-C, and HLA-DR), the cells can be homozygous or can retain only a single copy of the gene. For example, the modified cells are identified as at least (a) HLA-C+ and HLA-DR+, and optionally as one or more of (b) HLA-B-, (c) HLA-DP-, and (d) HLA-DQ-. In an exemplary embodiment, the modified cells are HLA-B+, HLA-DP-, and HLA-DQ-.

[0043] In some embodiments, the T cell population is HLA-A neg , homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg In some embodiments, the iPSCs are further homozygous for HLA-DRB1.

[0044] As used herein, the term "neg," (-), or "negative" with respect to a particular HLA class I or HLA class II molecule indicates that both copies of the gene have been disrupted in a cell line or population, and therefore the cell line or population does not exhibit significant functional expression of the gene. Such cells can be generated by complete or partial gene deletion, or alternatively, by other techniques such as siRNA. As used herein, the term "deletion" in the context of genetic modification of a targeted gene (i.e., gene editing) refers to the elimination of functional expression of the corresponding gene product (i.e., the corresponding polypeptide). Such gene editing includes complete or partial gene deletion, or deletion of a critical cis-acting expression control sequence.

[0045] In some embodiments, iPSCs are gene-edited using gRNAs that are 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more nucleotides in length. In some embodiments, the gRNAs comprise modifications at or near the 5' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5' end) and / or at or near the 3' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 3' end). In some embodiments, the modified gRNAs exhibit increased resistance to nucleases. In some embodiments, the gRNAs comprise two separate RNA molecules (i.e., "duplex gRNAs"). Duplex gRNAs comprise two separate RNA molecules, "crispr RNA" (or "crRNA") and "tracr RNA," and are well known to those of skill in the art.

[0046] Generally, various gene editing technologies are known that can be applied in accordance with various embodiments of the present disclosure, including, but not limited to, zinc finger (ZF), transcription activator-like effector (TALE), etc. Fusion proteins comprising one or more of these DNA binding domains and the cleavage domain of a Fokl endonuclease can be used to generate double-stranded breaks in desired regions of DNA within a cell (see, e.g., U.S. Patent Application Publication No. 2012 / 0064620, U.S. Patent Application Publication No. 2011 / 0239315, U.S. Patent No. 8,470,973, U.S. Patent Application Publication No. 2013 / 0217119, U.S. Patent No. 8,420,782, U.S. Patent Application Publication No. 2011 / 0301073, U.S. Patent Application Publication No. 2011 / 0145940, U.S. Patent No. 8,450,471, U.S. Patent No. 8,440,431, U.S. Patent No. 8,440,432, and U.S. Patent Application Publication No. 2013 / 0122581, the entire contents of which are incorporated herein by reference). In some embodiments, gene editing is performed using a CRISPR-associated Cas system (e.g., CRISPR-Cas9) known in the art. See, for example, US8,697,359, US8,906,616, and US8,999,641, each of which is incorporated herein by reference in its entirety. In various embodiments, gene editing employs a type II Cas endonuclease (such as Cas9) or a type V Cas endonuclease (such as Cas12a). Type II and type V Cas endonucleases are guide RNA dependent. Design of gRNAs to guide desired gene editing (while limiting or avoiding off-target editing) is known in the art. See, for example, Mohr SE, et al., CRISPR guide RNA design for research applications, FEBSJ. 2016 Sep; 283(17):3232-3238.In still other embodiments, non-canonical Type II or Type V Cas endonucleases with homology (albeit low primary sequence homology) to S. pyogenes Cas9 or Prevotella and Francisella 1 (Cpf1 or Cas12a) can be employed. Many such non-canonical Cas endonucleases are known in the art. Nidhi S, et al. Novel CRISPR-Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives, Int J Mol Sci. 2021 Apr;22(7):3327. In still other embodiments, gene editing employs base editing or prime editing to incorporate mutations without creating double-strand breaks. See, for example, Antoniou P, et al., Base and Prime Editing Technologies for Blood Disorders, Front. Genome Ed., 28 January 2021; Matsuokas IG, Prime Editing: Genome Editing for Rare Genetic Diseases Without Double-Strand Breaks or Donor DNA, Front. Genet., 9 June 2020. Various other gene editing processes are known, including the use of dead Cas (dCas) systems (e.g., Cas fusion proteins) to target DNA-modifying enzymes to desired targets using dCas as a guide RNA-dependent system. Brezgin S, Dead Cas Systems: Types, Principles, and Applications, Int J Mol Sci. 2019 Dec;20(23):6041.

[0047] Base editors that can introduce precise genome modifications without generating double-stranded DNA breaks can also be used for gene editing in cells (e.g., iPSCs) (e.g., designing gene therapy vectors). Base editors inherently cannot create DSBs and contain a catalytically inactive nuclease, such as Cas9 nickase (nCas9), fused to a nucleic acid base deaminase enzyme, possibly a DNA glycosylase inhibitor. Currently, there are two major categories of base editors: cytidine base editors (CBEs) and adenine base editors (ABEs), which catalyze C>T and A>G transversions. Base editors can be delivered, for example, via HDAd5 / 35++ vectors, to efficiently edit promoters and enhancers to activate or inactivate genes. Exemplary methods are described in U.S. Patent Nos. 9,840,699, 10,167,457, 10,113,163, 11,306,324, 11,268,082, 11,319,532, and 11,155,803. Also contemplated are prime editors comprising a reverse transcriptase conjugated to (e.g., fused with) a Cas endonuclease and a polynucleotide useful as a DNA synthesis template conjugated to (e.g., fused with) a guide RNA, as described in WO2020 / 191153.

[0048] Exemplary vectors that can be used for genome editing applications include plasmids, retroviral vectors, lentiviral vectors, adenoviral vectors (e.g., Ad5 / 35, Ad5, Ad26, Ad34, Ad35, Ad48), parvoviruses (e.g., adeno-associated virus (AAV) vectors), herpes simplex virus vectors, baculovirus vectors, negative-stranded RNA viruses such as coronaviruses, orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive-stranded RNA viruses such as picornaviruses and alphaviruses, and herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses. Examples of vectors that can be used include, but are not limited to, double-stranded DNA viruses, including canarypox, vaccinia, or modified vaccinia viruses. Vectors containing a nucleic acid molecule of interest can be delivered to cells (e.g., iPS cells, endothelial cells, hemogenic endothelial cells, HSCs (ST-HSCs or LT-HSCs)) via any method known in the art, including, but not limited to, transduction, transfection, infection, and electroporation. Any of these vectors may contain a transposable element (such as a piggyBac transposon or a Sleeping Beauty transposon). Transposons insert specific sequences of DNA into the vertebrate genome. A gene of interest can be integrated into the genome of a mammalian cell by transposase-catalyzed cleavage of a similar excision site present in the cell's nuclear genome.

[0049] For increased efficiency, in some embodiments, Cas and gRNA can be combined before delivery into cells. The Cas-gRNA complex is known as a ribonucleoprotein (RNP). Several methods have been developed for direct delivery of RNP into cells. For example, RNP can be delivered to cells in culture by lipofection or electroporation. Electroporation using a nucleofection protocol can be employed, which allows RNP to rapidly enter the cell's nucleus and immediately begin cleaving the genome. See, for example, Zhang S, Shen J, Li D, Cheng Y. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR / Cas9 genome editing. Theranostics. 2021 Jan 1;11(2):614-648, incorporated herein by reference in its entirety. In some embodiments, Cas9 and gRNA are electroporated into donor iPSCs and / or HSCs as RNP.

[0050] Generally, a protospacer adjacent motif (PAM) is required for Cas nuclease cleavage and is typically found 3-4 nucleotides downstream of the cleavage site. The PAM is a short DNA sequence (usually 2-6 base pairs in length) following the DNA region targeted for cleavage by a CRISPR system such as CRISPR-Cas9. In some embodiments, the PAM sequence, sgRNA, or base editing tool targeting a haplotype or polymorphism at an HLA locus does not contain four Gs, four Cs, a GC repeat, or a combination thereof.

[0051] In some embodiments, a CRISPR / Cas9 system specific for a unique HLA haplotype can be developed by using the gRNAs described herein to design a single gRNA that targets each of the donor-specific HLA-A, HLA-DPB1, and HLA-DQB1 genes (for example). To perform gene knockout, the gRNA targets the Cas9 protein to the appropriate site for editing. The Cas9 protein can then perform a double-strand break (DSB), which repairs the DNA through a non-homologous end joining (NHEJ) mechanism, generating an indel that results in a frameshift mutation and terminates the function of the resulting protein. However, off-target gene recombination can occur, altering the function of an otherwise intact gene. For example, the Cas9 endonuclease can generate DSBs at undesired off-target locations, even in the presence of some degree of mismatch. This off-target activity can result in genomic instability events such as point mutations and genomic structural perturbations. In various embodiments, an sgRNA targeting HLA-A can target a region of chromosome 6 defined as 29942532-29942626. In various embodiments, an sgRNA targeting HLA-DQB1 can target a region of chromosome 6 defined as 32665067-32664798. In various embodiments, an sgRNA targeting HLA-DPB1 can target a region of chromosome 6 defined as 33080672-33080935.

[0052] gRNAs can be used to develop clonal iPSCs. Such iPSC lines can be assessed for (i) on-target editing, (ii) off-target editing, and (iii) translocation editing, for example, using sequencing as described herein. Specifically, such assays can be performed by multiplex PCR using primers designed to target and enrich for the region of interest, followed by next-generation sequencing (e.g., Amplicon sequencing, AMP-seq). On-target and translocation panels can amplify the intended edited region and allow for the selection of iPSC clones with the expected edits that do not contain chromosomal translocations resulting from unintended DSB cleavage site fusions. Off-target panels can enrich for any potential off-target regions identified through sequencing, allowing for the selection of iPSC clones with negligible off-target mutations. Together, these assays enable the screening of iPSC clones to select clones with the desired edits while ruling out potential CRISPR / Cas9-related genome integrity issues.

[0053] In some embodiments, to further ensure the genomic stability and integrity of the reprogrammed and edited iPSCs, genetic and genomic assays can be performed to select clones that have not undergone translocation and mutation events and have not integrated the episomal vector. For example, whole genome sequencing (WGS) can be performed on CD34+ cells and iPSC clones after reprogramming, and the genomes can be compared for differences resulting from editing. These analyses provide an assessment of which iPSC clone genomes differ from the CD34+ starting material, allowing for the informed selection of iPSC clones that have not undergone mutations during reprogramming.

[0054] In some embodiments, karyotyping using a system such as the KARYOSTAT assay is used to select iPSC clones that did not develop indels or translocations during reprogramming, as described, for example, in Ramme AP, et al., "Supporting dataset of two integration-free induced pluripotent stem cell lines from related human donors," Data Brief. 2021 May 15;37:107140, incorporated herein by reference in its entirety. The KARYOSTAT assay allows visualization of chromosomal abnormalities with a resolution similar to G-banding karyotyping. The size of structural abnormalities that can be detected is >2 Mb for chromosome gains and >1 Mb for chromosome losses. The KARYOSTAT array is functionalized for balanced whole-genome coverage with low-resolution DNA copy number analysis, and the array covers all 36,000 RefSeq genes, including 14,000 OMIM targets. The assay allows for the detection of aneuploidy, submicroscopic abnormalities, and mosaic events.

[0055] In some embodiments, array comparative genomic hybridization (aCGH) analysis is used to select iPSC clones that have not developed copy number aberrations (CNAs) during reprogramming, as described, for example, in Wiesner et al. "Molecular Techniques," Editor(s): Klaus J. Busam, Pedram Gerami, Richard A. Scolyer, "Pathology of Melanocytic Tumors," Elsevier, 2019, pp. 364-373, ISBN 9780323374576, and Hussein SM, et al. "Copy number variation and selection during reprogramming to pluripotency," Nature. 2011 Mar 3;471(7336):58-62, which are incorporated herein by reference in their entireties. aCGH is a technique that analyzes the entire genome for CNAs by comparing sample DNA with reference DNA.

[0056] In some embodiments, a targeted hematologic malignancy NGS panel analysis is used to select iPSC clones that did not develop hematologic malignancy mutations during reprogramming. For example, a targeted hematologic malignancy NGS panel focuses on genes associated with myeloid leukemia, lymphoma, and / or other hematologic malignancies, generating smaller, more manageable datasets than more broad methods. A targeted hematologic malignancy NGS panel analysis involves the use of highly multiplexed PCR to amplify regions associated with hematologic malignancies, followed by next-generation sequencing.

[0057] In some embodiments, droplet digital PCR (ddPCR) is used to select iPSC clones that have not integrated the episomal vector and have been passaged sufficiently for episomal vector clearance. As discussed herein, iPSC reprogramming of CD34+ cells can be achieved by delivering episomal vectors encoding reprogramming factors. However, episomal vectors can randomly integrate into the cellular genome, although this is rare, which can disrupt developmental processes, homeostasis, and the like. Therefore, ddPCR can be used to detect residual episomal vectors in iPSC cultures, allowing for the selection of iPSC clones that have not integrated the episomal vector.

[0058] In some embodiments, after assessing that selected clones do not contain editing-related genomic abnormalities, the clones can be further tested for spontaneous mutations that may arise during expansion, such as mutations affecting hematologic malignancies, indels, translocations, and numerical abnormalities, as described for pre-edited reprogrammed clones. Analysis of spontaneous mutations can include whole genome sequencing (WGS), KARYOSTAT analysis, array comparative genomic hybridization (aCGH) analysis, targeted hematologic malignancies NGS panel AMP-Seq analysis, and / or droplet digital PCR (ddPCR).

[0059] In certain embodiments, the iPSCs are homozygous for at least one retained class I and class II locus. In some embodiments, the iPSCs are derived from T cells, e.g., with known or unknown TCR specificity. In some embodiments, the T cells have TCRs with specificity for one or more autoantigens or other antigens of interest. Exemplary autoantigens are described herein. In yet other embodiments, the iPSCs can be gene-edited to express a chimeric antigen receptor (CAR) to direct the resulting T cells to a tissue or organ of interest.

[0060] Somatic cells can be reprogrammed by expression of reprogramming factors selected from Sox2, Oct3 / 4, c-Myc, Nanog, Lin28, and klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc, Nanog, Lin28, and klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc, and klf4. Methods for preparing iPSCs are described, for example, in U.S. Pat. Nos. 10,676,165, 9,580,689, and 9,376,664, which are incorporated by reference in their entireties. In various embodiments, reprogramming factors are expressed using well-known viral vector systems, such as lentivirus, Sendai, or measles virus systems. Alternatively, reprogramming factors can be expressed by introducing mRNA(s) encoding the reprogramming factors into somatic cells. Furthermore, iPSCs can be generated by introducing non-integrating episomal plasmids expressing reprogramming factors, i.e., for the generation of transgene-free and virus-free iPSCs. Known episomal plasmids that have limited replicative capacity and are therefore lost over several cell generations can be employed.

[0061] In some embodiments, human pluripotent stem cells (e.g., iPSCs) are gene-edited. Gene editing can include, but is not limited to, modification of HLA genes (e.g., deletion of one or more HLA class I and / or HLA class II genes), deletion of β2 microglobulin (β2M), deletion of CIITA, deletion or addition of T cell receptor (TCR) genes, or addition of chimeric antigen receptor (CAR) genes. Exemplary CARs can target antigens specific to any desired organ or tissue, and in some embodiments, are specific for antigens unique to the donor organ. For example, iPSCs can be T cell receptor (TCR)-transduced iPSCs. Exemplary TCRs can be specific for autoantigens of interest. Such embodiments enable the production of large-scale regenerative Tregs with desired antigen specificity. Alternatively, engineered iPSCs with one or more HLA knockouts and TCR knockouts can be placed in a bioreactor for feeder-free and serum-free differentiation under GMP-grade conditions to generate fully functional histocompatible T cells.

[0062] In some embodiments, the iPSCs are CD3 + T-iPSCs are prepared from T cells, or in some embodiments, from T lymphocytes (T-iPSCs). For example, T lymphocytes can be isolated with a desired antigen specificity (e.g., using cell sorting with HLA peptide ligands) and reprogrammed into T-iPSCs. These T-iPSCs are then redifferentiated into populations containing the desired T cell or T progenitor cell populations according to the present disclosure. When T-iPSCs are generated from antigen-specific T cells, they inherit rearranged T cell receptor (TCR) genes. In these embodiments, T cells redifferentiated from T-iPSCs exhibit the same antigen specificity as the original T cells.

[0063] In various embodiments, iPSCs are prepared and expanded using a culture system. The expanded iPSCs can be recovered from the culture for differentiation into embryoid bodies (EBs). EBs generated by differentiation of iPSCs are three-dimensional aggregates of iPSCs and contain three (or alternatively, two or one) embryonic germ cell layers depending on the differentiation method(s). EB preparation is described, for example, in US2019 / 0177695, which is incorporated herein by reference in its entirety. In some embodiments, EBs prepared by differentiation of iPSCs are expanded in a bioreactor, as described, for example, in Abecasis B. et al., "Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches." J. of Biotechnol. 246 (2017) 81-93. EBs can be used to generate any desired cell type. Other methods involving 3D suspension culture for the growth or differentiation of EBs are described in WO2020 / 086889, which is incorporated herein by reference in its entirety.

[0064] In some embodiments, processes according to each aspect can include generating CD34+ enriched cells from pluripotent stem cells (e.g., EBs) and inducing endothelial and hematopoietic differentiation. 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 local stimuli, as well as factors such as extracellular matrix, niche factors, and cell-extrinsic factors, induction of cell-intrinsic properties, and including pharmacological and / or genetic means.

[0065] In some embodiments, the method includes preparing hematopoietic endothelial cells from pluripotent stem cells prior to induction of EHT. In some embodiments, overexpression of GATA2 / ETV2, GATA2 / TAL1, or ER71 / GATA2 / SCL can lead to the formation of hematopoietic endothelial cells from PSC sources. In some embodiments, the method includes overexpression of the E26 transformation-specific variant 2 (ETV2) transcription factor in iPSCs. After CD34+ enrichment, HSCs are then generated from the endothelial cells using mechanical, biochemical, pharmacological, and / or genetic stimulation or modification. ETV2 can be expressed by introduction of an encoding non-integrating episomal plasmid for constitutive or inducible expression of ETV2 and for the production of transgene-free hematopoietic ECs. In some embodiments, ETV2 is expressed from mRNA introduced into iPSCs. The mRNA can be introduced using any available method, including electroporation or lipofection. Differentiation of ETV2-expressing cells can include the addition of VEGF-A. See Wang K, et al., Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with mRNA. Sci. Adv. Vol. 6 (2020). Cells generated in this manner can be used for robust generation of CD34+ cells and subsequent EHT induction according to embodiments of the present disclosure.

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

[0067] Induction of EHT can be by any known process. In some embodiments, induction of EHT generates an HSC population including LT-HSCs. In some embodiments, EHT generates a cell population including HSPCs. In some embodiments, EHT generates HSCs and / or HSPCs through endothelial or hemogenic endothelial cell (HEC) progenitor cells using mechanical, biochemical, pharmacological, and / or genetic means (e.g., via stimulation, inhibition, and / or genetic modification). In some embodiments, EHT generates a stem cell population including one or more of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs), and HSPCs. In various embodiments, EHT can be induced in culture for 2 to 12 days, such as about 4 to about 8 days (e.g., about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days). In some embodiments, EHT is induced in culture for about 5 to about 7 days.

[0068] In some embodiments, HSC and / or HSPC populations or fractions thereof are differentiated into T cells or their progenitor or derivative cells, with or without the use of an agonist of a mechanosensitive receptor or mechanosensitive channel, such as Yoda1. In some embodiments, the use of an agonist of a mechanosensitive receptor or mechanosensitive channel (e.g., Yoda1) is optional. Thus, in some embodiments, CD34+ cells are enriched from a differentiated pluripotent stem cell population to prepare a CD34+ enriched population. Endothelial-hematopoietic transformation of the CD34+ enriched cell population is induced for at least 2 days but not more than 12 days, optionally with the use of an agonist of a mechanosensitive receptor or mechanosensitive channel, such as Yoda1, jedi1, jedi2, or ssRNA40. HSC and / or HSPC are differentiated (e.g., as described herein) into a progenitor T cell population or a T cell population. In some embodiments, endothelial-to-hematopoietic transformation of the CD34+ enriched cell population is induced for at least 2 days, 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. Full EHT differentiation proceeds over 12 days or less.

[0069] In some embodiments, the method comprises increasing the expression or activity of dnmt3b in PSCs, embryoid bodies, CD34+ 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 3β (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, inducing EHT comprises increasing the expression or activity of dnmt3b.

[0070] In some embodiments, the cells are contacted with an effective amount of an agonist of a mechanosensitive receptor or mechanosensitive channel that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezo1. An exemplary Piezo1 agonist is Yoda1. In some embodiments, the mechanosensitive receptor is Trpv4. An exemplary Trpv4 agonist is GSK1016790A. Yoda1 (2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine) is a small molecule agonist developed for the mechanosensitive ion channel Piezo1. Syeda R, Chemical activation of the mechanotransduction channel Piezo1. eLife (2015). Yoda1 has the following structure: [ka]

[0071] Derivatives of Yodal can be employed in various embodiments. For example, derivatives containing a 2,6-dichlorophenyl core are employed in some embodiments. Exemplary agonists are disclosed in Evans EL, et al., "Yoda1 analogue (Dooku1) which antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation," British Journal of Pharmacology 175(1744-1759):2018. Still other Piezo1 agonists include Jedi1, Jedi2, single-stranded (ss) RNA (e.g., ssRNA40), and derivatives and analogs thereof. See Wang Y., et al., "A lever-like transduction pathway for long-distance chemical- and mechano-gating of the mechanosensitive Piezo1 channel," Nature Communications (2018) 9:1300; and Sugisawa, et al., "RNA Sensing by Gut Piezo1 Is Essential for Systemic Serotonin Synthesis," Cell, Volume 182, Issue 3, 2020, Pages 609-624, which are incorporated herein by reference in their entireties. These Piezo1 agonists are commercially available. In various embodiments, the effective amount of Piezo1 agonist or derivative is in the range of about 1 μM to about 500 μM, or about 5 μM to about 200 μM, or about 5 μM to about 100 μM, or in some embodiments, about 25 μM to about 150 μM, or about 25 μM to about 100 μM, or about 25 μM to about 50 μM. Alternatively, single-stranded (ss) RNA (e.g., ssRNA) and derivatives and analogs thereof can be used for Piezo1 activation.

[0072] 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, hemogenic endothelial cells (HECs), HSCs, hematopoietic progenitor cells, and hematopoietic lineage(s). In certain embodiments, Piezo1 activation is applied to at least EBs generated from iPSCs, CD34+ cells isolated from EBs, and / or combinations thereof, which, according to various embodiments, enables superior generation of T progenitor cells and derived T cell lineages (e.g., Tregs and mature α / β T cells) compared to other methods for inducing EHT. Advantageously, progenitor T cells generated by Piezo1 activation during EHT have superior engraftment potential than progenitor T cells prepared during EHT without Piezo1 activation.

[0073] In some embodiments, a pharmacological agent, such as, but not limited to, retinoic acid, a retinoid acid receptor (RAR) agonist, dibutyl cyclic AMP, a protein kinase inhibitor, ascorbic acid, dexamethasone, forskolin (FSK), baicalin, or 2-methyl-5-hydroxytryptamine, or a combination thereof, is applied to CD34+ cells (i.e., CD34+ enriched cells). In certain embodiments, pharmacological agent activation may further be applied to iPSCs, embryoid bodies, ECs, hemogenic endothelial cells (HECs), HSCs, hematopoietic progenitor cells, and hematopoietic lineage(s) (e.g., to expand the cell population). In certain embodiments, an agent, such as retinoic acid or a retinoid acid receptor (RAR) agonist activation, is applied to at least EBs generated from iPSCs, CD34+ cells isolated from EBs, and / or combinations thereof.

[0074] Alternatively, or in addition, Dnmt3b activity or expression can be increased directly in cells, for example, in CD34+ enriched cells. For example, Dnmt3b mRNA expression can be increased by, but not limited to, delivering a transcript encoding Dnmt3b to cells, or by introducing a transgene encoding Dnmt3b, or by transgene-free methods such as introducing a non-integrating episome into cells. In some embodiments, gene editing is employed to introduce genetic modifications into Dnmt3b-expressing elements in cells, such as, but not limited to, increasing promoter strength, ribosome binding, RNA stability, and / or affecting RNA splicing.

[0075] In some embodiments, the methods involve increasing the activity or expression of Gimap6 in a cell, alone or in combination with Dnmt3b and / or other genes that are up- or down-regulated upon cyclic strain or Piezo1 activation. To increase Gimap6 activity or expression, an mRNA transcript encoding Gimap6 can be introduced into the cell; a transgene-free approach can also be employed, including, but not limited to, introducing an episome into the cell; or, alternatively, a transgene encoding Gimap6 can be introduced. In some embodiments, gene editing is employed to introduce genetic modifications (such as one or more modifications to increase promoter strength, ribosome binding, RNA stability, or to affect RNA splicing) into a Gimap6-expressing element in the cell.

[0076] In embodiments of the present disclosure employing mRNA delivery to cells, known chemical modifications can be used to circumvent the innate immune response in cells. For example, synthetic RNAs containing only canonical nucleotides can bind to pattern recognition receptors and induce a strong immune response in cells. This response can result in translation block, secretion of inflammatory cytokines, and cell death. RNAs containing certain non-canonical nucleotides can avoid detection by the innate immune system and can be translated into proteins with high efficiency. See US Pat. No. 9,181,319, incorporated herein by reference, particularly for nucleotide modifications to circumvent the innate immune response.

[0077] In some embodiments, expression of Dnmt3b and / or Gimap6 is increased by introducing a transgene into cells, which can direct the desired level of overexpression (with varying promoter strength or other selection of expression control elements). Various viral vectors or transfection reagents (including lipid nanoparticles) known in the art can be used to introduce the transgene. In some embodiments, expression of Dnmt3b and / or Gimap6 is increased by transgene-free methods (e.g., episomal delivery). In some embodiments, expression or activity of Dnmt3b and / or Gimap6, or other genes disclosed herein, is increased using gene editing techniques, for example, to introduce one or more modifications to increase promoter strength, ribosome binding, or RNA stability.

[0078] In some embodiments, the method includes applying cyclic 2D, 3D, or 4D stretch to cells. In various embodiments, the cells subjected to cyclic 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, as described in WO 2017 / 096215, which is incorporated herein by reference in its entirety. The 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 stretching 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 (e.g., the FlexCell™ Tension System, CytoStretcher System) attached to a flexible, biocompatible and / or biomimetic surface. For example, the applied cyclic stretching can be about 1% to about 20% cyclic strain (e.g., about 6% cyclic strain) for several hours or days (e.g., about 7 days). In various embodiments, the cyclic strain is applied for at least about 1 hour, at least about 2 hours, at least about 6 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 120 hours, at least about 144 hours, or at least about 168 hours.

[0079] Alternatively, or in addition, EHT is stimulated by Trpv4 activation, which can be by contacting cells (e.g., CD34+ enriched cells, ECs, or HECs) with one or more Trpv4 agonists, optionally selected from GSK1016790A, 4α-PDD, or analogs and / or derivatives thereof.

[0080] When a cell population is described herein as having a particular phenotype, it is understood that the phenotype represents a substantial portion of the cell population, such as at least 25%, at least 40%, or at least about 50%, or at least about 60%, or at least about 75%, or at least about 80%, or at least about 90% of the cell population. Furthermore, in various steps, the cell population can be enriched for cells of the desired phenotype and / or depleted of cells of an undesired phenotype, such that the cell population contains at least about 75%, or at least about 80%, or at least about 90% of the desired phenotype. Such positive and negative selection methods are known in the art. For example, cells can be sorted based on cell surface antigens (including those described herein) using a fluorescence-activated cell sorter or magnetic beads that bind to certain cell surface antigens. Negative selection columns can be used to remove cells expressing undesirable cell surface markers. In some embodiments, cells are enriched for CD34+ cells (before and / or after undergoing EHT). In some embodiments, the cell population is cultured under conditions that promote the proliferation of CD34+ cells, thereby producing an expanded population of stem cells. In various embodiments, the cells are enriched for T cell precursors (using cell surface markers described herein, such as CD7+), which can optionally be further differentiated in culture. Additionally, after T cell precursor or T cell differentiation, the cells can be enriched for markers such as CD3, CD4, and / or CD8. Tregs can be further enriched in the resulting population, for example, by CD25+ enrichment.

[0081] In various embodiments, CD34+ cells (e.g., suspension and / or adherent cells) are harvested from cultures undergoing endothelial-hematopoietic transition between days 10 and 20 of iPSC differentiation, such as between days 10 and 17 or between days 12 and 15 of iPSC differentiation.

[0082] In various embodiments, the HSC and / or HSPC population (e.g., CD34+ enriched cells isolated therefrom) is further expanded. For example, the cells can be expanded according to the methods disclosed in US8,168,428, US9,028,811, US10,272,110, and US10,278,990, which are incorporated by reference in their entireties. In some embodiments, the ex vivo expansion of HSC or CD34+ enriched cells employs prostaglandin E2 (PGE2) or a PGE2 derivative. In some embodiments of the present disclosure, the HSCs comprise at least about 0.01% LT-HSCs, or at least about 0.05% LT-HSCs, or at least about 0.1% LT-HSCs, or at least about 0.5% LT-HSCs, or at least about 1% LT-HSCs.

[0083] Hematopoietic stem cells (HSCs), which give rise to erythroid, myeloid, and lymphoid lineages, can be identified based on the expression of CD34 and the absence of lineage-specific markers (referred to as Lin-). In some embodiments, a population of stem cells containing HSCs is enriched, for example, as described in US9,834,754, which is incorporated herein by reference in its entirety. For example, this process can include sorting a cell population based on the expression of one or more of CD34, CD90, CD38, and CD43. CD34 + , CD90 + , CD38 - , and CD43 - In some embodiments, a stem cell population for differentiation into hematopoietic lineages is selected for further differentiation from a fraction that is at least about 80% CD34 + , or at least about 90% CD34 + , or at least about 95% CD34 + is.

[0084] In some embodiments, HSC / HSPC populations, or CD34+ enriched cell or fraction thereof, or derivative populations, are expanded as described in US 2020 / 0308540, which is incorporated herein by reference in its entirety. For example, the cells are expanded by exposing them to an aryl hydrocarbon receptor antagonist, including, for example, SR1 or an SR1 derivative. See also Wagner et al., Cell Stem Cell 2016;18(1):144-55, and Boitano A., et al., Aryl Hydrocarbon Receptor Antagonists Promote the Expansion of Human Hematopoietic Stem Cells. Science 2010 Sep 10;329(5997):1345-1348.

[0085] In some embodiments, CD34 + Compounds that promote cell proliferation include pyrimidoindole derivatives, including, for example, UM171 or UM729 (see US2020 / 0308540, incorporated herein by reference).

[0086] In some embodiments, the stem cell population or CD34+ enriched cells are further enriched for or modified to express periostin and / or platelet-derived growth factor receptor alpha (pdgfra), as described in WO2020 / 205969 (incorporated herein by reference in its entirety). Such expression can be by transgene-free methods, including but not limited to, by delivering an encoding transcript to the cells, by introducing an encoding transgene, or by introducing a non-integrating episome into the cells. In some embodiments, gene editing is employed to introduce genetic modifications into expression elements in the cells, such as to modify promoter activity or strength, ribosome binding, RNA stability, or affect RNA splicing.

[0087] 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 in the stem cell population, or transiently silenced (e.g., by siRNA). Inhibition of EZH1 can direct myeloid progenitor cells (e.g., CD34+CD45+) to the lymphoid lineage. See WO2018 / 048828, which is incorporated herein by reference in its entirety. In yet another embodiment, EZH1 is overexpressed in the stem cell population.

[0088] In various embodiments, the population comprising HSCs and / or HSPCs or fractions thereof is differentiated into a population comprising T progenitor cells or T lymphocytes.

[0089] In some embodiments, the cell population is cultured ex vivo with a partial or complete Notch ligand, SHH, extracellular matrix component(s), and / or a combination thereof to differentiate the HSCs into a T cell population (or its progenitor cells). Furthermore, according to known processes, heterologous OP9-DL1 cells are often employed for T cell differentiation. 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 use. There is a need for a feeder-free system capable of generating T lymphocytes from hiPSCs for clinical use, and in some embodiments, the present invention fulfills this goal.

[0090] The term "Notch ligand" as used herein refers to a ligand capable of binding to a Notch receptor polypeptide present on the membrane of a hematopoietic stem cell or progenitor T cell. Notch receptors include Notch-1, Notch-2, Notch-3, and Notch-4. Notch ligands typically have a DSL domain (D-Delta, S-Serrate, and L-Lag2) containing 20-22 amino acids at the amino terminus and 3-8 EGF repeats on the extracellular surface. In various embodiments, Notch ligands include Delta-like-1 (DLL1), Delta-like-4 (DLL4), SFIP3, Delta-like-5 (DLL5), SFIP4, SFIP5, SFIP6, SFIP7, SFIP8, SFIP9, SFIP10, SFIP11, SFIP12, SFIP13, SFIP14, SFIP15, SFIP16, SFIP17, SFIP18, SFIP19, SFIP20, SFIP21, SFIP22, SFIP23, SFIP24, SFIP25, SFIP26, SFIP27, SFIP28, SFIP29 ...9, SFIP29, SFIP29, SFIP29, SFIP29, SFIP29, SFIP29, SFIP29, SFIP29, SFIP29, SFIP29, SFIP29, SFIP29, SFIP2 Max (disclosed in PCT / US2020 / 041765 and PCT / US2020 / 030977, which are incorporated by reference in their entireties), or at least one functional portion thereof. A key signal delivered by thymic stromal cells to invading lymphoid progenitor cells in vivo is mediated by DL4, which is expressed by cortical thymic epithelial cells.

[0091] The earliest intrathymic progenitor cells express high levels of CD34 and CD7, do not express CD1a, and are triple negative (TN) for mature T cell markers: CD4, CD8, and CD3. Commitment to the T cell lineage is associated with expression of CD1a by CD7-expressing prothymocytes. Thus, immature stages of T cell development are typically characterized by CD34 + CD1a - (mostly immature) and CD34 + CD1a + Described as cells. CD34 by early thymocytes + CD7 + CD1a - from CD34 + CD7 + CD1a + Conversion to CD34 is associated with T cell involvement. + CD7 + CD1a +Following this stage, thymocytes progress to a CD4 immature mono-positive stage, at which point CD4 is expressed in the absence of CD8. A subset of cells then expresses CD4 + CD8 + Finally, following TCRα rearrangement, TCRαβ-expressing DP thymocytes undergo positive and negative selection and differentiate to the CD4 + CD8 - and CD4 - CD8 + Generate single positive (SP) T cells.

[0092] In some embodiments, progenitor T cells are isolated by enrichment for CD7 expression. In some embodiments, progenitor T cells are expanded as described in US2020 / 0308540, which is incorporated herein by reference in its entirety. For example, cells can be expanded by exposing them to an aryl hydrocarbon receptor antagonist, including, for example, SR1 or an SR1 derivative. See also Wagner et al., Cell Stem Cell 2016;18(1):144-55. In some embodiments, the proliferation-promoting compound comprises a pyrimidoindole derivative, including, for example, UM171 or UM729 (see US2020 / 0308540, which is incorporated herein by reference).

[0093] 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 precursor T cells include CD7 + CD1a + CD5 + cells, and optionally CD34 + Includes.

[0094] In some embodiments, the progenitor T cells exhibit reduced levels, minimal, or no CD34 expression (compared to the HSC population), 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.

[0095] In some embodiments, the Notch ligand is an anti-Notch (agonist) antibody that can bind to and participate in Notch signaling. In some embodiments, the antibody is a monoclonal antibody (including a human or humanized antibody), a single-chain antibody (scFv), a nanobody, or other antibody fragment or antigen-binding molecule that can activate the Notch signaling pathway.

[0096] In some embodiments, the Notch ligand is a Delta family Notch ligand. In some embodiments, the Delta family ligand is Delta-1 (Genbank Accession No. AF003522, Homo sapiens), Delta-like 1 (DLL1, Genbank Accession Nos. NM_005618 and NP_005609, Homo sapiens; Genbank Accession Nos. X80903, 148324, M. musculus), Delta-4 (Genbank Accession No. AF273454, BAB18580, Mus musculus; Genbank Accession Nos. AF279305, AAF81912, Homo sapiens), and / or Delta-like 4 (DLL4, Genbank Accession Nos. Q9NR61, AAF76427, AF253468, NM_019074, Homo sapiens; Genbank Accession No. NM 019454, Mus musculus). Notch ligands are commercially available or can be produced, for example, by recombinant DNA techniques.

[0097] 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. A functional derivative of a Notch ligand (including a fragment or portion thereof) will be capable of binding to and activating a Notch receptor. Binding to a Notch receptor can be determined by a variety of methods known in the art, including in vitro binding assays and receptor activation / cell signaling assays.

[0098] In some embodiments, the Notch ligand is DLL4 with one or more affinity-enhancing mutations, such as one or more (or all) of the following: for hDLL4, G28S, F107L, I143F, H194Y, L206P, N257P, T271L, F280Y, S301R, and Q305P. See Gonzalez-Perez, et al., Affinity-matured DLL4 ligands as broad-spectrum modulators of Notch signaling, Nature Chemical Biology (2022).

[0099] In various embodiments, the Notch ligand is soluble and optionally immobilized on microparticles or nanoparticles that are optionally paramagnetic to enable magnetic enrichment or concentration processes. In yet other embodiments, the Notch ligand is immobilized on a 2D or 3D culture surface, optionally using other adhesion molecules such as VCAM-1. See US 2020 / 0399599, incorporated herein by reference in its entirety. In other embodiments, the beads or particles are constructed of biological materials, such as polymers (e.g., polystyrene or PLGA), gold, iron dextran, or particles formed from lipids and / or proteins. In various embodiments, the particles have a diameter or largest dimension of about 0.01 μm (10 nm) to about 500 μm (e.g., about 1 μm to about 7 μm). In still other embodiments, polymer scaffolds with conjugated ligands can be employed, as described in WO 2020 / 131582, incorporated herein by reference in its entirety. For example, the scaffold can be constructed from polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivatives, gelatin, collagen, agarose, hyaluronic acid, poly(lysine), polyhydroxybutyrate, poly-epsilon-caprolactone, polyphosphazine, poly(vinyl alcohol), poly(alkylene oxide), poly(ethylene oxide), poly(allylamine), poly(acrylate), poly(4-aminomethylstyrene), Pluronic® polyol, poloxamer, poly(uronic acid), poly(anhydride), poly(vinylpyrrolidone), and any combination thereof. In some embodiments, the scaffold comprises pores having diameters of about 1 pm to 100 pm.

[0100] In some embodiments, the C-terminus of the Notch ligand is conjugated to a support of choice. In some embodiments, this can involve adding a sequence to the C-terminus of the Notch ligand that can be enzymatically conjugated to the support, for example, via a biotin molecule. In another embodiment, the Notch ligand-Fc fusion is prepared such that the Fc segment can be immobilized by binding to Protein A or Protein G that has been conjugated to a support. Of course, any of the known protein conjugation methods can be employed.

[0101] Thus, in various embodiments, the Notch ligand can be immobilized, functionalized, and / or embedded in a 2D or 3D culture system. The Notch ligand can be incorporated with components of an extracellular matrix, such as one or more selected from fibronectin, retronectin, and laminin. In some embodiments, the Notch ligand and / or components of the extracellular matrix are embedded in an inert material that provides 3D culture conditions. Exemplary materials include, but are not limited to, cellulose, alginate, and combinations thereof. In some embodiments, the Notch ligand, components of the extracellular matrix, or combinations thereof, are in contact with culture conditions that provide cells with a topographical pattern and / or texture (e.g., roughness) that promotes differentiation and / or proliferation.

[0102] In some embodiments, HSCs are differentiated into precursor T cells by culturing them in medium containing TNF-α and / or an antagonist of the aryl hydrocarbon / dioxin receptor (SR1) and in the presence of a Notch ligand. See US2020 / 0390817, US2021 / 0169934, and US2021 / 0169935, which are incorporated by reference in their entireties. In some embodiments, HSCs are cultured in medium containing TNF-α, IL-7, thrombopoietin (TPO), Flt3L, and stem cell factor (SCF), and optionally SR1, in the presence of immobilized delta-like-4 ligand and fibronectin fragments. In some embodiments, cells are cultured with retronectin, a recombinant human fibronectin containing three functional domains: a human fibronectin cell-binding domain (C domain), a heparin-binding domain (H domain), and a CS-1 sequence domain. In some embodiments, cells are cultured in the presence of immobilized delta-like-4 ligand and retronectin. In some embodiments, cells are cultured in the presence of immobilized delta-like-4 ligand, TNF-α, and retronectin. In some embodiments, cells are cultured in the presence of immobilized delta-like-1 ligand and retronectin. In some embodiments, cells are cultured in the presence of SFIP3 and retronectin. In some embodiments, cells are cultured in the presence of immobilized delta-like-4 ligand and an SHH molecule and / or a functional derivative thereof. Exemplary fibronectin fragments include one or more of RGDS, CS-1, and heparin-binding motifs. The fibronectin fragments can be free in solution or immobilized on a culture surface or particle. In some embodiments, cells are cultured for 5 to 7 days to prepare CD7+ progenitor T cells. In some embodiments, cells are cultured for 8 to 13 days to prepare pre-T cells (e.g., CD34-, CD7+, CD5+ / -). In some embodiments, the cells are cultured for 15-21 days for the production of mature T cells (e.g., CD3+). In some embodiments, the cells are cultured for 21 days or longer for the production of Tregs.

[0103] In various embodiments, the method produces a Treg population by culturing a population comprising HSCs and / or HSPCs with a Notch ligand (including any of the embodiments described above), with or without component(s) of the extracellular matrix, and optionally adding TNF-α to the culture at a particular stage of differentiation. Thus, in some embodiments, the cells produced are precursor or progenitor cells committed to the T cell lineage ("progenitor T cells"). In some embodiments, the cells are CD7+ precursor T cells. 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) or CD4+CD8- single positive cells. In some embodiments, the cells are CD4+CD8- single positive (SP) cells, which in some embodiments may be TCRhi. In some embodiments, the cells are TCRαβ+. In various embodiments, the cells are CD3+.

[0104] In various embodiments, the precursor T cells are further cultured under suitable conditions to generate cells of a desired T cell population (e.g., Tregs) comprising one or more Notch ligands. For example, the cells can be cultured in the presence of one or more Notch ligands as described for a period of time sufficient to form cells of a desired T cell population. In some embodiments, HSC / HSPC 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, the precursor T cell or HSC / HSPC population is cultured in suspension or with soluble or conjugated Notch ligand in suspension in an adherent form in a bioreactor, optionally a closed or automated closed bioreactor. One or more cytokines, extracellular matrix component(s), and thymic niche factor(s) that promote commitment and differentiation into a desired T cell population may also be added to the culture or reactor. In various embodiments, the HSC and / or HSPC 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 progenitor 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 the Treg lineage. In some embodiments, the stem cell population is cultured for less than about 28 days, or less than about 21 days, or less than about 15 days to produce a Treg population.

[0105] In various embodiments, the HSC / HSPC population is cultured in artificial thymic organoids (ATOs). See Hagen, M. et al. (2019). ATOs involve culturing HSCs (or aggregates of HSCs) with Notch ligand-expressing stromal cell lines under serum-free conditions. Artificial thymic organoids are 3D systems that induce the differentiation of hematopoietic progenitor cells into naive CD3+CD8+ and CD3+CD4+ T cells. In some embodiments, the artificial thymic organoids contain DLL4 and BMP2, or functional fragments thereof.

[0106] In various embodiments, the method includes generating Tregs. In certain embodiments, the Tregs express CD3 and T cell receptors. In some embodiments, the Tregs express CD4 T cells, which are optionally expanded in culture. + In some embodiments, the iPSCs, CD34+ cells, or derivatives thereof are modified to express a chimeric antigen receptor (CAR).

[0107] Regulatory T cells (Tregs) have the potential to be useful in a wide variety of clinical applications. For example, they can be used to control harmful immune responses seen in patients with autoimmune diseases such as childhood (type 1) diabetes, rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease, and to suppress transplant rejection in patients who have received heart, liver, or kidney transplants. However, their clinical application is limited due to the low frequency of Tregs in peripheral blood (approximately 1-2% in humans). Therefore, successful generation of Tregs from iPSCs ex vivo would fill a significant need.

[0108] T lymphocytes and precursor T cells can be differentiated into Tregs by expression of FOXP3. Tregs can optionally be further isolated or enriched by positive and / or negative selection. In various embodiments, the present disclosure provides cell populations comprising at least about 40%, or at least about 50%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90% Tregs, which can be antigen- or tissue-specific. Tregs can be defined as CD3+CD4+CD25+ and FoxP3+ cells. Additional cell surface markers in some embodiments include CTLA-4, CD39, CD73, GITR, and / or LAG-3. In exemplary embodiments, at least about 50%, or at least about 75%, or at least about 80% of the Tregs in the population express CTLA-4. In various embodiments, at least about 50%, or at least about 75%, or at least about 80% of the Tregs in the population express CD39 and / or CD73.

[0109] In some embodiments, pluripotent stem cells are cultured under conditions that allow the formation of embryoid bodies. The embryoid bodies are dissociated, and CD34+ cells are isolated and used to induce EHT, followed by differentiation of T cells or progenitor T cells by culturing the cells undergoing EHT with at least one Notch ligand (as further described herein). A vector containing a nucleic acid sequence encoding Foxp3 is introduced during or after T cell differentiation, thereby promoting the formation of Tregs. In some embodiments, a population of T cells also expresses an exogenous gene to provide tissue-targeting functionality, such as a tissue-specific T cell receptor (TCR). For example, engineered Tregs are also engineered to express a pancreatic islet-specific T cell receptor (TCR), targeting the engineered Tregs to sites of pancreatic-related disease. Optionally, the engineered Tregs include insertion of an IL-2 signaling complex, which provides a growth and functional advantage to the engineered Tregs.

[0110] Tregs are CD4 + CD25 +Tregs can be defined as Tregs. Tregs control immune responses to self and foreign antigens and help prevent autoimmune diseases. In some embodiments, differentiation of cells into Tregs involves modifying Treg precursor cells (e.g., CD4+ αβ T cells or their precursor cells) to constitutively express FOXP3. The FOXP3 gene provides instructions for producing the forkhead box P3 (FOXP3) protein. The FOXP3 protein is a transcription factor involved in regulating the immune system and is involved in the production of regulatory T cells. In some embodiments, iPSCs, CD34+ cells (e.g., isolated before or after EHT), precursor T cells, CD25+ T cells, CD4+CD8+ cells, or CD4+ cells (e.g., αβ T cells) are gene-edited to provide FOXP3 expression, which can be constitutive and stable. In some embodiments, a regulatory sequence comprising a strong enhancer and / or promoter is inserted to operably control expression of the FOXP3 gene in a constitutive and stable manner. In some embodiments, an enhancer-binding domain is placed upstream of the FOXP3 promoter to activate the promoter and increase transcription of the FOXP3 gene. In some embodiments, a transcription activation domain containing a specific DNA sequence that can be bound by a transcription factor is inserted, thereby allowing the transcription factor to control the transcription rate. Specific transcription factors may include, but are not limited to, SP1, AP1, C / EBP, heat shock factor, ATF / CREB, c-Myc, Oct-1, and / or NF-1. In some embodiments, the activation domain is used to silence inhibitory mechanisms that prevent transcription of the FOXP3 gene. In some embodiments, the FOXP3 gene (including a coding sequence with a constitutive expression control sequence) is inserted to provide constitutive FOXP3 expression. Various modes of introducing donor templates, gene-editing proteins, and gRNAs are known, including the use of viral vectors such as AAV and lipid nanoparticles. See US2021 / 0253652, incorporated herein by reference in its entirety.In some embodiments, a FOXP3 donor gene with constitutive expression control sequences is inserted using CRISPR / Cas9.

[0111] In some embodiments, Tregs are expanded in the presence of growth factors such as IL-2. Additionally, expansion protocols may include the use of anti-CD3 and agonistic anti-CD28 antibodies, which may be conjugated to a substrate surface (including beads) or provided in soluble form. In some embodiments, expansion of Tregs is not desired (or expansion is minimal) to avoid loss of desired function through further culture. Furthermore, Tregs may have limited expansion capacity. In some embodiments, Tregs are expanded in culture for 7 days or less, or for about 4 days or less, or for 2 days or less. In some embodiments, Tregs are engineered to have an expansion advantage, e.g., by expressing a signaling complex, e.g., as described in US 2021 / 0253652, which is incorporated herein by reference in its entirety. In some embodiments, the signaling complex involves an interleukin-2 receptor component, optionally involving a receptor signaling subunit shared by IL-2 and IL-15. In some embodiments, the signaling complex is as described in US 2020 / 0123224, the disclosure of which is incorporated herein by reference in its entirety. For example, each chimeric protein component of the complex can have half of a rapamycin-binding complex as an extracellular domain fused to half of an intracellular signaling complex (e.g., an IL-2 signaling complex). Delivery of a nucleic acid encoding the signaling complex into a host cell enables intracellular signaling in the cell that can be controlled by the presence of rapamycin or a rapamycin-related compound.

[0112] In yet other embodiments, the present invention generates T cells (e.g., CTLs, helper T cells, or Tregs) that express chimeric antigen receptors. The cells can be efficiently transduced with vectors, such as, but not limited to, retroviral or non-integrating viral vectors (e.g., adenovirus, adeno-associated virus, integration-deficient retrolentivirus, poxvirus), or non-viral vectors (e.g., plasmid vectors, artificial chromosomes), or episomal or episomal hybrid vectors carrying CAR-targeted tumor antigens (e.g., CD19, CD38, CD33, CD47, CD20, etc.). CARs are designed to enhance the ability of cells to recognize, bind to, and kill target cells (e.g., cancer cells or tumor cells). In some embodiments, the CAR enhances the ability of cells to recognize tumor cells. In some embodiments, the CAR enhances the anti-tumor activity of the cells. In some embodiments, the CAR is a G-protein coupled receptor 87 (GPR87) CAR, and solute carrier family 7 member 11 (SLC7A11(xCT)) CAR, TNF receptor superfamily member 17 (BCMA) CAR, CD30 CAR, CD19 CAR, CD22-CAR, CD33 CAR, CD133-CAR, mesothelin-CAR, CD70 CAR, CD73 CAR, which target the following tumors or tumor antigens, including but not limited to: (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) Disialoganglioside 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 antigen 133 (CD133) - glioblastoma, cholangiocarcinoma, adenocarcinoma, (xv) Cancer / Testis Antigen 1B (CTAG1B) - melanoma and ovarian cancer; (xvi) Mucin 1 - seminal vesicle cancer, (xvii) folate receptor-a (FR-a)-ovarian cancer; (xviii) a growth factor receptor selected from one or more of ErbB1, ErbB2, ErbB3, or ErbB4, IGF1R, IGF2R, TβR I-II, VEGFR1, VEGFR2, VEGFR3, PDGFR(α / β), or FGFR1-4. (xix)EGFRvIII-Glioblastoma. (xx) Claudin 18.2 - solid tumors, advanced gastric adenocarcinoma, pancreatic adenocarcinoma. (xxi) Mesothelin - mesothelioma, metastatic pancreas, ovary, cervix, lung.

[0113] 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; Zhang C, et al., Chimeric Antigen Receptor T-Cell Therapy. In: StatPearls [Internet]. Treasure Island, FL: StatPearls Publishing, each of which is incorporated herein by reference.

[0114] In some embodiments, the CAR comprises the intracellular domain from Fcε receptor gamma (FcεRIγ). However, in further contemplated embodiments, the CAR may also comprise the T cell receptor (TCR) CD3ζ (CD3ζ) intracellular domain, alone or in combination with additional components from second or third generation CAR constructs (e.g., CD28, CD134, CD137, and / or ICOS).

[0115] In some embodiments, the present invention generates T cells that exhibit T cell activation and subsequent T cell-mediated cytotoxicity to a similar extent as non-immunocompatible T cells (e.g., pan-T cell lines, among other T cells used as experimental controls to assess T cell-mediated cytotoxicity). The T cells generated herein can exhibit significantly superior performance in T cell-mediated cytotoxicity compared to CD34+-derived T cells.

[0116] In another aspect, the present disclosure provides an HLA-A neg , HLA-DPB1 neg , and HLA-DQB1 neg

[0010] The present invention provides a cell composition comprising a T cell population (or progenitor cells thereof, such as a precursor T cell population) that is a T cell ...

[0117] In some embodiments of this aspect, the T cell population is a T cell precursor population. In various embodiments, the T cell precursor population engrafts in the thymus, spleen, or secondary lymphoid organs upon administration. In other embodiments, the T cell population is a cytotoxic T cell (CTL) population, a helper T cell population, or a Treg population. In various embodiments, the T cell population may express a chimeric antigen receptor (CAR). Cell populations according to this aspect may be prepared according to other aspects of the present disclosure.

[0118] In some aspects, the present invention provides a cell population or a pharmaceutically acceptable composition thereof described herein or produced by a method described herein. In some embodiments, the cell population is a progenitor T cell population capable of engraftment in the thymus, spleen, or secondary lymphoid organs upon administration to a subject in need thereof. In other embodiments, the cell population is an α / β T cell population, a CAR-T cell population, a CTL population (capable of expressing a CAR), a T helper population, or a Treg population (each described herein). In various embodiments, a composition for cell therapy is prepared comprising the cell population and a pharmaceutically acceptable vehicle. The pharmaceutical composition is administered in a concentration of 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 7or at least about 10 8 cells, or at least about 10 9 cells, or at least about 10 10 cells, or at least about 10 11 cells, or at least about 10 12 cells, or at least about 10 13 cells, or at least about 10 14 For example, in some embodiments, a pharmaceutical composition containing about 100,000 to about 400,000 T cell precursors per kilogram (e.g., about 200,000 cells / kg) is administered. In other embodiments, the cells are administered at a concentration of about 10 5 ~Approx. 5×10 5 cells (e.g., approximately 2.5 x 10 5 cells / kg), or approximately 10 per kilogram 6 ~Approx. 5×10 6 cells (e.g., approximately 2.5 x 10 6 cells / kg), or approximately 5 x 10 cells per kilogram 6 ~about 10 7 cells (e.g., approximately 5 x 10 6 cells / kg), or approximately 10 per kilogram 7 ~about 10 8 cells (e.g., approximately 5 x 10 7 cells / kg), or approximately 10 per kilogram 8 ~about 10 9 cells (e.g., approximately 5 x 10 8 cells / kg), or approximately 10 per kilogram 9 ~about 10 10 cells, or approximately 10 per kilogram 10 ~about 10 11 cells, or approximately 10 per kilogram 11 ~about 10 12 cells, or approximately 10 per kilogram 12 ~about 10 13 cells, or approximately 10 per kilogram 13 ~about 10 14 It is administered in individual cells.

[0119] The 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 may be provided in unit vials or bags and stored frozen until use. In certain embodiments, the volume of the composition is about 1 fluid ounce to 1 pint.

[0120] In some embodiments, the cell population is a Treg population useful for adoptive cell therapy for a human subject having a condition selected from, for example, an autoimmune or inflammatory condition or disease, or graft-versus-host disease (GVHD). Additionally, various genetic disorders affect the immune system and can manifest as autoimmune or pro-inflammatory conditions. In some embodiments, the Treg population is a CAR-T cell. In various embodiments, the regulatory T cells can express a tissue- or cell-specific CAR. That is, the cells can express a CAR specific for an organ or tissue of interest, such as the pancreas, liver, skin, muscle, bone, joint, thyroid, nerve, etc. In some embodiments, the Tregs comprise a TCR or CAR that targets the cells to pancreatic islets, for example, for the treatment of type 1 diabetes or prediabetes.

[0121] In some embodiments, the cell population is derived from autologous cells, or universally matched donor cells, or HLA-modified or HLA-null cells (e.g., as described herein), i.e., the cell population is generated from iPSCs prepared from the recipient subject's cells or prepared from donor cells (e.g., universal donor cells, HLA-matched cells, HLA-modified cells, or HLA-null cells).

[0122] In another aspect, the present invention provides methods 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).

[0123] In some embodiments, the subject has cancer, such as a hematological malignancy or a solid tumor, and in such embodiments, the subject is administered T cell precursors or T cells with anti-tumor specificity (such as CTLs that recognize tumor antigens).

[0124] In some embodiments, the subject has a condition selected from acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, 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 anemia, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, hemophagocytic lymphohistopathy, congenital metabolic disorders, severe congenital neutropenia, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, and leukocyte adhesion deficiency. T cells 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 a myeloablative, non-myeloablative, or immunotoxin-based (eg, anti-c-Kit, anti-CD45, etc.) conditioning regime.

[0125] In other embodiments, the present invention provides methods for cell therapy, comprising administering a Treg cell population described herein or a pharmaceutically acceptable composition thereof to a human subject in need thereof. In various embodiments, the subject has an autoimmune, alloimmune, or inflammatory disease. In some embodiments, the subject is a recipient of a tissue or organ transplant, and in some embodiments, the subject is a recipient of an allogeneic organ or tissue transplant. In some embodiments, the subject is experiencing or at risk of GVHD. For example, organs that can be transplanted include the heart, kidney, liver, lung, pancreas, intestine, and / or thymus. 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. Kidneys, livers, and hearts are the most commonly transplanted organs. Corneas and musculoskeletal grafts are the most commonly transplanted tissues.

[0126] In some embodiments, the subject has an autoimmune condition selected from type 1 diabetes, rheumatoid arthritis (RA), psoriasis or psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, scleroderma, hemolytic anemia, pernicious anemia, and Goodpasture's syndrome.

[0127] In some embodiments, the subject has an immune condition such as celiac disease, hyperimmunoglobulin E syndrome, and IPEX syndrome.

[0128] As used herein, the term "about" means ±10% of the associated numerical value.

[0129] Certain aspects and embodiments of the present disclosure are further illustrated with reference to the following examples. [Example]

[0130] Example 1 - ETV2 overexpression increases the yield of hemogenic endothelial cells and enhances CD34+ cell formation during iPSC differentiation, but does not affect pluripotency. method iPSCs were developed from hCD34+ cells by episomal reprogramming, as known in the art and essentially as described in Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells, Science 318, 1917-1920, (2007), and J. Yu, et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801, (2009). Embryoid bodies and hemogenic endothelial differentiation were performed essentially as described in R. Sugimura, et al., Hematopoietic stem and progenitor cells from human pluripotent stem cells. Nature 545, 432-438, (2017); C. M. Sturgeon, et al., Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat Biotechnol 32, 554-561, (2014); J. Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920, (2007); and J. Yu, et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801, (2009).

[0131] Briefly, hiPSCs were dissociated and resuspended in medium supplemented with L-glutamine, penicillin / streptomycin, ascorbic acid, human holo-transferrin, monothioglycerol, BMP4, and Y-27632. The cells were then seeded onto 10 cm dishes (EZSPHERE or low-attachment plates) for EB formation. On day 1, bFGF and BMP4 were added to the medium. On day 2, the medium was replaced with medium containing SB431542, CHIR99021, bFGF, and BMP4. On day 4, the cell medium was replaced with medium supplemented with VEGF and bFGF. On day 6, the cell medium was replaced with medium supplemented with bFGF, VEGF, interleukin (IL)-6, IGF-1, IL-11, SCF, and EPO. Cells were maintained in an incubator with 5% CO2, 5% O2, and 95% humidity. To harvest CD34+ cells, EBs were dissociated on day 8, cells were filtered through a 70 μm strainer, and CD34+ cells were isolated by CD34 magnetic bead staining.

[0132] result Induced pluripotent stem cells (iPSCs) were transduced using an adenoviral vector containing both ETV2 and GFP sequences under the control of the EF1A promoter. After transduction, approximately 45% of the iPSC cultures were observed to be GFP-positive, confirming ETV2 overexpression (ETV2-OE). It was further observed that ETV2-OE in iPSC cells retained the pluripotent properties of iPSCs, as indicated by the stemness marker expression TRA-1-60 (Figure 1). Figure 1 shows a FACS plot demonstrating the transduction efficiency of iPSCs using an adenoviral vector for overexpressing ETV2 and GFP sequences.

[0133] Next, ETV2-OE-iPSCs (along with control iPSCs transduced with a vector carrying the GFP sequence but without ETV2) were differentiated into embryoid bodies and then into hemogenic endothelial cells (Strugeon et al., 2014). The results showed that overexpression of ETV2 significantly increased the expression of CD235a. - CD34 in the population + and CD31 +These results suggest that ETV2-OE enhances the formation of hemogenic endothelial cells, as evidenced by the expression of markers (Figure 2). Specifically, Figure 2 shows representative flow cytometry analysis and relative quantification of hemogenic endothelial cells (defined here as CD235a-CD34+CD31+), demonstrating that ETV2-OE enhances the formation of hemogenic endothelial cells compared to controls.

[0134] Furthermore, the results showed that ETV2-OE expresses CD34 + This suggests that ETV2-OE enhances CD34+ cell formation (Figure 3). Figure 3 shows representative flow cytometry analysis and relative quantification of CD34+ cells demonstrating that ETV2-OE enhances CD34+ cell formation.

[0135] Overall, these data indicate that ETV2 overexpression in iPSCs does not affect their pluripotent properties, but promotes their ability to undergo hemogenic endothelial and hematopoietic differentiation.

[0136] Example 2 - iPSC-derived HSCs generated by Piezo1 activation undergo T cell differentiation similar to bone marrow-derived HSCs. method To analyze EHT, EB-derived CD34+ cells were suspended in medium containing Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3. After the cells had adhered to the bottom of the well (by visual inspection) for approximately 4-18 hours, Yoda1 was added to the cultures for some experiments. After 4-7 days, cells were collected for analysis.

[0137] iPSCs were differentiated into embryoid bodies for 8 days. On day 8, CD34+ cells from iPSC-derived embryoid bodies were harvested 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 on days 5–7 for further hematopoietic lineage differentiation and analysis.

[0138] CD34+ cells harvested from EHT cultures on days 5–7 (or days 13–21 total of differentiation from iPSCs) were seeded onto 48-well plates pre-coated with rhDL4 and retronectin. T lineage differentiation was induced in medium containing aMEM, FBS, ITS-G, 2BME, ascorbic acid-2-phosphate, Glutamax, rhSCF, rhTPO, rhIL7, FLT3L, rhSDF-1α, and SB203580.

[0139] 80% of the medium was changed every other day from days 2 to 6. On day 7, cells were transferred to new coated plates and analyzed for the presence of pre-T cells (CD34+CD7+CD5+ / -).

[0140] 80% of the medium was changed every other day from days 8 to 13. On day 14, 100,000 cells / well were transferred to new coated plates, and cells were analyzed for the presence of pre-T cells (CD34-CD7+CD5+ / -).

[0141] 80% of the medium was changed every other day from days 15 to 20. Cells were harvested on day 21 and analyzed via FACS for CD3, CD4, CD8, CD5, CD7, TCRab expression on behalf of T cells, and / or activated using CD3 / CD28 beads to assess their functional properties.

[0142] After 21 days of differentiation, cells were harvested and approximately 80,000 cells were replated into new 96-well culture plates in RPMI 1640 (without L-glutamine, without phenol red) with FBS, L-glutamine, and IL-2, and then activated with 1:1 CD3 / CD28 beads. After 72 hours of activation with CD3 / CD28 beads, cells were analyzed for CD3, CD69, and CD25 expression by FACS and for IFN-γ expression using RT-qPCR. Supernatants were analyzed by ELISA.

[0143] result Figures 4A and 4B show that iPSC-derived HSCs derived from differentiated iPSCs (e.g., in this case, involving Piezo1 activation) undergo pre-T cell differentiation, similar to bone marrow (BM)-HSCs. Furthermore, Figures 5A and 5B show that iPSC-derived HSCs generated from differentiated iPSCs (e.g., in this case, involving Piezo1 activation) undergo CD34+ EHT, similar to BM-HSCs, and can be activated with CD3 / CD28 beads. Figure 6 shows that iPSC-derived HSCs (generated with 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., derived from differentiated iPSCs in CD34+ EHT) have enhanced HSC capacity to further differentiate into progenitor and functional T cells ex vivo. The experiment shown in Figure 6 involves Piezo1 activation during HSC formation.

[0144] Figure 7 shows that HSCs generated according to the present disclosure (labeled as D8+7 iPSC-CD34+) successfully differentiate into CD4+CD8+ ("double positive") T cells as well as TCR α / β T cells. The disclosed method substantially outperforms bone marrow CD34+ cells for T cell maturation. Figure 7 shows results with ("+Y") and without ("-Y") Yoda1 during HSC formation.

[0145] FIG. 8 shows that HSCs (D8+7 iPSC-CD34+ cells (+ or - Yoda1) generated in accordance with 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 in accordance with the present disclosure with or without Yoda1.

[0146] Example 3 - Evaluation of off-target editing in HLA knockout HSCs HLA typing of triple knockout (HLA edited) HSC clones was performed to check for unwanted editing and ensure that major editing events, such as deletion(s), did not occur within other regions of chromosome 6. Sequencing methods and analyses were performed to assess the extent of gRNA off-target activity and select gRNAs that represent a low risk of affecting non-target HLA genes.

[0147] Sequencing was performed using in situ cleavage labeling in fixed and permeabilized cells by ligating full-length P5 sequencing adapters to end-prepared DSBs. Genomic DNA was extracted, fragmented, end-prepared, and ligated using chemically modified semi-functional P7 adapters. The resulting DNA library contained a mixture of functional DSB-labeled fragments (P5:P7) and non-functional genomic DNA fragments (P7:P7). Subsequent DNA sequencing of the DNA library enriched for the DNA-labeled fragments, eliminating all irrelevant non-functional DNA. Because the library preparation was PCR-free, each resulting sequencing read was equivalent to a single labeled DSB end from the cell. This generated a readout of DNA cleavage, enabling direct detection and quantification of genomic DSBs by sequencing without the need for error correction, and allowed for the unambiguous mapping of off-target mutations.

[0148] Table 1 below summarizes the results of the editing strategy in two representative HLA-edited clones relative to wild-type cells (gHSC). [Table 1]

[0149] Table 2 provides non-limiting examples of gRNAs used in experiments that can be used to knock out expression of the indicated HLA genes. [Table 2-1] [Table 2-2]

[0150] 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.

[0151] These results were confirmed by phenotypic analysis of the HLA-edited clones by FACS and immunofluorescence. As shown in Figures 9A and 9B, the HLA-edited cells tested positive for overall expression of HLA class I molecules, comparable to that of wild-type cells (gHSCs). Specific expression of HLA-A via immunofluorescence confirmed that HLA-A was not expressed in the HLA-edited cells, supporting the finding that the gene editing strategy successfully deleted only the HLA-A gene. Specifically, Figure 9A shows that all HLA-edited cells were positive for HLA-like class I molecules to the same extent as wild-type cells (i.e., gHSCs). This result indicates that despite the deletion of HLA-A, other class I molecules, such as HLA-B and HLA-C, were expressed and were not affected by the gene editing strategy.

[0152] To confirm that the HLA-A gene had been deleted, the specific expression of HLA-A was analyzed by immunofluorescence. As can be seen in Figure 9B, HLA-A was not expressed in the HLA-edited clones, indicating that the gene editing strategy was effective in specifically deleting only the HLA-A gene. Such preservation of overall class I expression with deletion of HLA-A would facilitate patient matching while avoiding NK cell-mediated rejection.

[0153] Example 4 - Evaluation of pluripotency and immune compatibility of HLA-edited HSCs The ability of HLA-edited cells to preserve pluripotency was assessed. As shown in Figure 10, immunofluorescence evaluation of HLA-edited iPSC clones showed that they maintained tri-lineage differentiation, with ectoderm differentiation indicated by NESTIN-488 and PAX6-594 staining, mesoderm differentiation indicated by GATA-488 staining, and endoderm differentiation indicated by CXCR4-488 and FOX2A-594 staining.

[0154] 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 facilitate patient matching while avoiding NK cell-mediated rejection. Therefore, the immune compatibility of HLA-edited HSCs was tested by coculture with peripheral blood mononuclear cells (PBMCs) to assess whether immune cells would reject grafts of HLA-edited and wild-type HSCs (gHSCs).

[0155] Wild-type (gHSC) and HLA-edited HSCs were cocultured with PBMCs bearing HLA-B and HLA-C markers matched but mismatched HLA-A. B2M KO HSCs, which lack expression of HLA class I molecules, and CIITA KO HSCs, which lack expression of class II molecules, were used as controls to compare the degree of PBMC-mediated cytotoxicity for HLA-null and HLA-mismatched HSCs, respectively. Figure 11 shows the results of a PBMC-mediated cytotoxicity assay in the coculture, measured by Annexin V staining. The results show that deletion of HLA-A in HLA-edited HSCs protected the cells from PBMC-mediated cytotoxicity, while WT, B2M KO, and CIITA KO HSCs were susceptible to PBMC-mediated cytotoxicity. Coculture of HSCs with sorted CD8+ T cells from the same PBMC donor protected HLA-edited and B2M KO HSCs from CD8+ T cytotoxicity. Conversely, HSCs co-cultured with sorted NK cells protected only WT and HLA-edited cells from NK cell-mediated cytotoxicity.

[0156] In summary, the immunocompatibility results indicate that CD8+ T cells present in the PBMC samples were involved in killing cells bearing mismatched HLA molecules (WT) and CIITA KO, while NK cells present in the PBMCs were involved in killing HLA-null cells (B2M KO). However, HLA-edited HSCs were protected from CD8+ T cell-mediated cytotoxicity (because mismatched HLA-A had been knocked out) and from NK cell-mediated cytotoxicity (because HLA class I molecule expression was largely preserved).

[0157] Example 5 - Evaluation of in vivo engraftment potential of HLA-edited HSCs To evaluate the engraftment potential of HLA-edited HSCs, the ability of the cells to engraft in vivo was assessed by competitive transplantation against wild-type HSCs. Equal proportions of mCherry HLA-edited HSCs and wild-type HSCs (gHSCs) were mixed and transplanted into mice, from which bone marrow (BM) and peripheral blood samples were collected and evaluated by FACS to compare the relative amounts of each cell type present in the samples. As shown in Figure 12, both HLA-edited HSCs and wild-type HSCs contributed to approximately equal engraftment in the BM and peripheral blood samples. These results confirm that HLA-edited HSCs (prepared according to the present disclosure) are comparable to wild-type HSCs in their engraftment and reconstitution potential. Therefore, the properties of wild-type HSCs are expected to match those of the HLA-edited HSCs of the present disclosure for generating T cell lineages.

[0158] Example 6 - Differentiation of HLA-edited HSCs into CD4+ / CD8+ T cells Antigen-presenting cells (APCs) present antigens to helper CD4+ T cells through HLA-II molecules. Activation of helper CD4+ T cells promotes the generation of antigen-specific CD8+ T cells, which further evolve into antigen-specific CTLs. Similarly, HLA class I molecules are expressed on the surface of all nucleated cells and present peptide fragments of proteins to CD8+ CTLs from within the cell. CTLs induce cytotoxic killing of target (infected) cells upon recognition of HLA-I-peptide complexes expressed on the cell surface. Therefore, studies were performed to determine whether deletion of HLA-A affects class I peptide presentation of edited HSCs. As shown in Figures 13A and 13B, immunopeptidome analysis indicates that deletion of HLA-A does not affect overall class I peptide presentation. HLA-A-edited cells exhibited comparable peptide and protein presentation compared to wild-type HSCs (gHSCs). Furthermore, as shown in Figures 14A and 14B, deletion of HLA-DQB1 and HLA-DPB1 does not affect overall class II peptide presentation by macrophages differentiated from HSCs. Together, these data suggest that despite the deletion of HLA-A, HLA-DQ, and HLA-DP molecules, the cells (and their derived lineages) retain the ability to present a wide range of class I and II peptides.

[0159] Example 7 - In vivo testing of antigen-mediated immune responses. Figure 15 is a schematic diagram of the delayed-type hypersensitivity reaction, showing the sensitization and elicitation stages of antigen presentation. Briefly, upon antigen injection, the antigen is processed by antigen-presenting cells (APCs) and presented by MHC class II molecules on the surface of the APCs. CD4+ T cells recognize peptide-MHC on the antigen-presenting cells (APCs). Upon antigen administration, CD4+ helper T cells are activated, and cytokines recruit macrophages and other immune cells that induce tissue swelling.

[0160] A delayed-type hypersensitivity assay was performed on transplanted mice. Specifically, mice were sensitized by subcutaneous injection of sheep red blood cells as an antigen. If the mouse has a functional immune system, APCs process the antigen and present the peptide antigen to CD4+ T cells. The mouse was then stimulated by subcutaneous injection of the same antigen in the left paw. At this point, T cells are activated and secrete cytokines that recruit macrophages and other immune cells to the antigen injection site, resulting in tissue swelling. In this assay, a functional immune system resulted in swelling of the left paw, as measured with a microcaliper.

[0161] As can be seen in Figures 16A and 16B, control (non-transplanted) mice, due to their immunodeficiency, did not show swelling of their left paws. Conversely, mice transplanted with cord blood CD34+ cells showed tissue swelling, doubling the diameter of their left paws. Similar immune system responses were found in both WT (non-edited HSCs) and HLA-edited HSCs (HLA-edited) transplanted mice.

[0162] Example 8 - Evaluation of different types of T cells and pre-T cells derived from HSCs Next, we tested the ability of various types of T cells and pre-T cells derived from HSCs to differentiate into mature T cells. After a 35-day differentiation period, T cell progenitors and their derivatives were assessed by cell sorting for the presence of CD4+, CD8+, and AB+ T cell populations. As shown in Figure 17, pre-T cells differentiated more efficiently into CD4+, CD8+, and αβ+ T cells than bone marrow (BM)-derived CD34+ cells and embryonic body (EB)-derived CD34+ cells.

[0163] Next, to test their functional properties, each of the T cell populations was cocultured with a CD19+ lymphoma cell line and an anti-CD3 / CD19 bispecific antibody. In this experimental model, the bispecific antibody engaged both the CD3 receptor on T cells and the CD19 cell surface receptor on lymphoma cells, thus inducing T cell activation. The degree of activation was assessed by measuring subsequent T cell-mediated cytotoxicity compared to a pan-T cell positive control. As shown in Figure 18, the T cells demonstrated statistically significant superiority in cytotoxicity compared to both BM CD34+ and EB CD34+ T cells.

[0164] Because the overall differentiation process of T cells is 35 days long, transduction experiments were performed to test whether the time required to differentiate HSCs could be shortened. Pre-T cells were cultured in activation medium (approximately 7 days) to increase the transduction efficiency of the cells. Next, the cells were transduced with lentiviral (LV) particles encoding an anti-CD19 CAR transgene. The cells were cultured for an additional 4-5 days (12 days total) to assess their maturation and killing capacity. As shown in Figure 19, HSC-derived pre-T cells could be transduced with high efficiency, with more than 80% of the cells expressing the anti-CD19 CAR, as evidenced by cell sorting.

[0165] Next, we evaluated the ability of pre-T cells to effectively mature into CD4+ / CD8+ T cells via CAR transduction. Pre-T cells were transduced with LVs using an anti-CD19 CAR, along with bone marrow (BM)-derived CD34+ cells and embryonic body (EB)-derived CD34+ cells (as well as pan-T cells as a positive control). T cell subsets were screened by cell sorting for the presence of CD4 or CD8 cell surface marker expression. As shown in Figure 20, the results indicated that CAR transduction promoted T cell maturation, and an increased degree of T cell maturation was observed in pre-T cells compared to bone marrow (BM)-derived CD34+ cells and embryonic body (EB)-derived CD34+ cells.

[0166] The ability of LV pre-transduced T cells to function via anti-CD19 receptor-mediated cytotoxicity was assessed. T cell subsets were co-cultured with a CD19+ leukemia cell line (NALM6) expressing a luciferase reporter gene (Luc+) to measure the extent of T cell-mediated cytolysis, using untransduced cells and pan-T cells as negative and positive controls, respectively. As shown in Figure 21, CAR T cells functioned effectively via T cell-mediated lysis and exhibited a similar degree of cytotoxicity to pre-CAR T cells derived from BM CD34+ cells. Conversely, CAR T cells derived from EB CD34+ cells did not exhibit the ability to kill target cells.

[0167] Example 9 - Evaluation of HSC properties evolving into pre-T cells. The ability of HSCs to evolve into pre-T cells was assessed by measuring the CD34-CD7+ marker on pre-T cells. As shown in Figure 22, FACS analysis demonstrated that HSCs produced according to the present disclosure successfully differentiated into CD34-CD7+ pre-T cells compared to bone marrow-derived CD34+ cells or EB-derived CD34+ cells.

[0168] Next, the expression of T cell-specific transcription factors and thymic engraftment molecules was measured. Figure 23A shows increased TCF7 expression in pre-T cells derived from HSCs of the present disclosure, and Figure 23B shows increased CCR7 expression. Figure 24A shows that pre-T cells derived from HSCs engraft and differentiate in the thymus. Figure 24B shows FACS analysis of the CD3+ cell population of cells gated on the CD45+ cell population, demonstrating the superior engraftment and differentiation potential of pre-T cells derived from HSCs in the thymus. Pre-T cells in this example were prepared from HSCs using Piezo1 activation, as previously described.

[0169] In vitro activation of HSC-derived T cells was also measured, as illustrated in Figure 25. The top panel of Figure 25 shows FACS analysis of activated T cells from different sources, including HSCs of the present disclosure (e.g., prepared using Piezol activation). T cells prepared from HSCs of the present disclosure showed comparable or superior activation, as measured by increased CD107 expression. The bottom panel shows Dynabeads activation, where activated T cells express inflammatory cytokines. T cells derived from HSCs according to the present disclosure (e.g., prepared using Piezol activation) expressed higher levels of inflammatory cytokines, as exemplified by TNF-α and interferon-gamma expression levels.

[0170] Example 10 - Evaluation of the properties of CCR5 knockout HSCs that evolve into pre-T cells. To determine whether CCR5 knockout (CCR5-KO) HSCs could differentiate into pre-T cells comparable to their wild-type counterparts from which they were derived (i.e., HSCs of the present disclosure), studies were performed in which CD34, CD7, and CD5 expression of HSCs and CCR5-KOs was measured. As can be seen in Figure 26, HSCs were successfully differentiated into CD34+CD7+CD5+ pre-T cells comparable to bone marrow-derived CD34+ cells. Similarly, CCR5-KOs, like their wild-type counterparts, were successfully differentiated into CD34+CD7+CD5+ pre-T cells.

[0171] Next, the properties of CCR5 knockout HSCs that differentiate into double-positive (CD4+CD8+) T cells were evaluated, which were comparable to the HSCs from which they were derived. As can be seen in Figure 27, CCR5 knockout HSCs differentiated equally well into double-positive (CD4+CD8+) T cells compared to their wild-type counterparts from which they were derived (i.e., HSCs of the present disclosure).

[0172] References 1.Nianias,A.& Themeli,M.Induced Pluripotent Stem Cell(iPSC)-Derived Lymphocytes for Adoptive Cell Immunotherapy:Recent Advances and Challenges.Curr Hematol Malig Rep 14,261-268(2019). 2.Brauer,P.M.,Singh,J.,Xhiku,S.& Zuniga-Pfluecker,J.C.T Cell Genesis:In Vitro Veritas Est?Trends Immunol 37,889-901(2016). 3.Kennedy,M.et al.T Lymphocyte Potential Marks the Emergence of Definitive Hematopoietic Progenitors in Human Pluripotent Stem Cell Differentiation Cultures.Cell Reports 2,1722-1735(2012). 4.Sturgeon,C.M.,Ditadi,A.,Awong,G.,Kennedy,M.& Keller,G.Wnt Signaling Controls the Specification of Definitive and Primitive Hematopoiesis From Human Pluripotent Stem Cells.Nat Biotechnol 32,554-561(2014). 5.Chang,C.-W.,Lai,Y.-S.,Lamb,L.S.& Townes,T.M.Broad T-Cell Receptor Repertoire in T-Lymphocytes Derived from Human Induced Pluripotent Stem Cells.PLoS One 9,(2014). 6.Nishimura,T.et al.Generation of Rejuvenated Antigen-Specific T Cells by Reprogramming to Pluripotency and Redifferentiation.Cell Stem Cell 12,114-126(2013). 7.Themeli,M.et al.Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy.Nat Biotechnol 31,928-933(2013). 8.Vizcardo,R.et al.Regeneration of Human Tumor Antigen-Specific T Cells from iPSCs Derived from Mature CD8+T Cells.Cell Stem Cell 12,31-36(2013). 9.Montel-Hagen,A.et al.Organoid-induced differentiation of conventional T cells from human pluripotent stem cells.Cell Stem Cell 24,376-389.e8(2019). 10.Guo,R.et al.Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors.Cell Research 30,21-33(2020). 11.Nagano,S.et al.High Frequency Production of T Cell-Derived iPSC Clones Capable of Generating Potent Cytotoxic T Cells.Molecular Therapy-Methods & Clinical Development 16,126-135(2020). 12.Iriguchi,S.et al.A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy.Nature Communications 12,430(2021).

Claims

1. 1. A method for preparing a T cell population or a progenitor thereof, comprising: enriching the differentiated pluripotent stem cell population for CD34+ cells to prepare a CD34+ enriched population; inducing endothelial-to-hematopoietic transformation of the CD34+ enriched cell population for at least 2 days but not more than 12 days to produce a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem progenitor cells (HSPCs); differentiating said population comprising HSCs and / or HSPCs into a progenitor T cell population or a T cell population.

2. The method of claim 1 , wherein the population comprising HSCs and / or HSPCs comprises a non-adherent cell population.

3. 3. The method of claim 1 or 2, wherein the population comprising HSCs and / or HSPCs is differentiated into a population comprising one or more of T progenitor cells, precursor T cells, double positive T cells, single positive T cells, and regulatory T cells.

4. The method of claim 3, wherein the population comprising HSCs and / or HSPCs is differentiated into a population comprising T cell precursors.

5. The method of claim 4, wherein the T cell precursors are recovered from the culture.

6. 6. The method of claim 4 or 5, wherein the T cell precursors are further differentiated into a population comprising one or more of precursor T cells, double-positive T cells, single-positive T cells, or regulatory T cells.

7. The method of claim 6, wherein the T cell precursors are further differentiated into a population comprising double-positive T cells.

8. The method of claim 6, wherein the T cell precursors are further differentiated into a population comprising regulatory T cells.

9. 4. The method of claim 3, wherein the population comprising HSCs and / or HSPCs is differentiated into a population comprising double-positive T cells and / or single-positive T cells.

10. 10. The method of claim 9, wherein the double positive and / or single positive T cells are differentiated into regulatory T cells, optionally comprising a step of recovering the double positive and / or single positive cells from the culture prior to differentiation into regulatory T cells.

11. The method of claim 3, wherein the population comprising HSCs and / or HSPCs is differentiated into a population comprising T regulatory cells.

12. 12. The method of any one of claims 1 to 11, wherein the PSC population is a human iPSC population derived from lymphocytes, umbilical cord blood cells, peripheral blood mononuclear cells, CD34+ cells, or primary human tissue.

13. 13. The method of claim 12, wherein the PSC population is derived from CD34+ enriched cells isolated from peripheral blood.

14. 14. The method of claim 12 or 13, wherein the iPSCs are homozygous for one or more HLA class I and / or HLA class II genes.

15. 15. The method of claim 14, wherein the iPSCs are homozygous for HLA-DRB1.

16. 14. The method of claim 12 or 13, wherein the iPSCs have been gene-edited to delete one or more HLA class I genes, one or more class II genes, and / or one or more genes that govern HLA or MHC expression or presentation capacity.

17. 17. The method of claim 16, wherein the iPSCs comprise a deletion of HLA-A.

18. 18. The method of claim 16 or 17, wherein the iPSCs comprise a deletion of HLA-DPB1 and / or HLA-DQB1.

19. The method according to claim 16, wherein the one or more genes controlling the ability to express or present HLA or MHC are β2-microglobulin and / or CIITA.

20. 19. The method of any one of claims 14 to 18, wherein the iPSCs comprise a deletion of HLA-A and are homozygous for both HLA-B and HLA-C, and comprise a deletion of HLA-DPB1 and HLA-DQB1 and are homozygous for HLA-DRB1.

21. 21. The method of any one of claims 1 to 20, wherein CD34+ cell enrichment and endothelial-hematopoietic transition are induced between days 8 and 15 of iPSC differentiation.

22. 22. The method of claim 21, wherein the endothelial-to-hematopoietic conversion generates a HSC and / or HSPC population comprising long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs), and HSPCs.

23. 23. The method of claim 21 or 22, wherein the CD34+ cells are harvested from a culture undergoing EHT, including harvesting of CD34+ non-adherent cells.

24. 24. The method of claim 23, wherein EHT is induced in culture for about 4 to about 8 days.

25. 25. The method of claim 24, wherein EHT is induced in culture for about 5 to about 7 days.

26. The method of any one of claims 1 to 25, wherein the induction of EHT comprises increasing the expression or activity of dnmt3b.

27. 27. The method of claim 26, wherein the inducing EHT comprises applying cyclic stretch to the CD34+ enriched cells.

28. 28. The method of claim 27, wherein the cyclic stretching is 2D, 3D, or 4D cyclic stretching.

29. The method of claim 26, wherein the induction of endothelial-hematopoietic transition comprises Piezo1 activation.

30. 30. The method of claim 29, wherein the Piezo1 activation is by contacting the CD34+ enriched cells or a fraction thereof, optionally with one or more Piezo1 agonists selected from Yoda1, Jedi1, Jedi2, ssRNA40, or analogs or derivatives thereof, or by RNA activation.

31. The method of any one of claims 1 to 25, wherein the induction of endothelial-hematopoietic transition comprises Trpv4 activation.

32. 32. The method of claim 31, wherein said Trpv4 activation is by contacting said CD34+ enriched cells, optionally with one or more Trpv4 agonists selected from GSK1016790A, 4α-PDD, or analogs or derivatives thereof.

33. 33. The method of any one of claims 1 to 32, wherein the HSC and / or HSPC 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.

34. 34. The method of claim 33, wherein the CD7+ progenitor T cells do not express CD34 or express reduced levels of CD34 compared to the HSC population.

35. 35. The method of claim 33 or 34, wherein the CD7+ precursor T cells express CD5.

36. 36. The method of any one of claims 33 to 35, wherein the Notch ligand comprises at least one of DLL1, DLL4, SFIP3, or a functional portion thereof.

37. 37. The method of claim 36, wherein the Notch ligand comprises DLL4 with one or more affinity-enhancing mutations.

38. 36. The method of any one of claims 33 to 35, wherein the Notch ligand is immobilized, functionalized and / or embedded in a 2D or 3D culture system.

39. 39. The method of any one of claims 33 to 38, wherein the Notch ligand is optionally incorporated with a component of the extracellular matrix selected from fibronectin, retronectin, and laminin, derivatives or analogues thereof, and / or combinations thereof.

40. 40. The method of claim 39, wherein the Notch ligand and / or extracellular matrix components are optionally embedded in an inert material that provides 3D culture conditions, selected from cellulose, alginate, and combinations thereof.

41. 41. The method of claim 39 or 40, wherein the Notch ligand, extracellular matrix component, or a combination thereof is in contact with culture conditions that provide a topographical pattern and / or roughness to the cells.

42. 42. The method of any one of claims 33 to 41, wherein the Notch ligand, extracellular matrix component, topographical pattern and / or roughness, or a combination thereof, is optionally cultured with a cytokine and / or growth factor selected from one or more of TNF-α and SHH.

43. 43. The method of any one of claims 33 to 42, wherein the HSC population or a fraction thereof is cultured in an artificial thymus organoid, optionally comprising DLL4 and BMP2 or a functional fragment thereof.

44. 44. The method of claim 43, wherein the T cells express at least one of CD3 and a T cell receptor.

45. 45. The method of any one of claims 33 to 44, comprising generating regulatory T cells from the precursor T cells or αβ CD3+ and / or CD4+ T cells generated therefrom.

46. 46. ​​The method of claim 45, wherein the regulatory T cells express CD8 and / or CD4.

47. 47. The method of claims 45 and 46, wherein differentiation into Tregs comprises modifying Treg progenitor cells to express FOXP3.

48. 48. The method of claim 47, wherein differentiation into Tregs comprises modifying Treg progenitor cells to constitutively express FOXP3.

49. 49. The method of claim 47 or 48, wherein the cells modified to express FOXP3 are the iPSCs, the CD34+ cells pre-EHT, the CD34+ cells post-EHT, progenitor T cells, CD4+ / CD8+ T cells, CD8+ αβ T cells, or CD4+ αβ T cells.

50. 50. The method of any one of claims 1 to 49, wherein the T cell population expresses a chimeric antigen receptor (CAR) or a T cell receptor that is optionally cell or tissue specific.

51. HLA-A ｎｅｇ , HLA-DPB1 ｎｅｇ , and HLA-DQB1 ｎｅｇ A cell composition comprising a T cell population or a progenitor T cell population,

52. 52. The cell composition of claim 51, wherein the T cell population is homozygous for both HLA-B and HLA-C.

53. 53. The cell composition of claim 51 or 52, wherein the T cell population is homozygous for HLA-DRB1.

54. The cell composition of any one of claims 51 to 53, wherein the T cell population is a T cell precursor population.

55. The cell composition of any one of claims 51 to 53, wherein the T cell population is a cytotoxic T cell (CTL) population.

56. The cell composition of any one of claims 51 to 53, wherein the T cell population is a helper T cell population.

57. The cell composition of any one of claims 51 to 54, wherein the T cell population is a Treg population.

58. The cell composition of any one of claims 51 to 57, wherein the T cell population expresses a CAR.

59. At least about 10 ４ The cell composition of any one of claims 51 to 58, comprising cells.

60. At least about 10 ５ cells, or at least about 10 ６ cells, or at least about 10 ７ cells, 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 The cell composition of any one of claims 51 to 58, comprising cells.

61. 61. A method for cell therapy comprising administering to a human subject in need thereof a T cell population or a progenitor T cell population according to any one of claims 51 to 60, or a T cell population or progenitor cells thereof according to any one of claims 1 to 50.

62. 62. The method of claim 61, wherein the human subject has a condition comprising one or more of lymphopenia, cancer, immunodeficiency, autoimmune disease, viral infection, skeletal dysplasia, and bone marrow failure syndrome.

63. 62. The method of claim 61, wherein the human subject has cancer, optionally a hematological malignancy or a solid tumor, and the T cell population comprises precursor T cells, CD8+ cytotoxic T cells, or CD4+ helper T cells.

64. 64. The method of any one of claims 61 to 63, wherein the T cell population or T cell precursor comprises a CAR that targets a tumor-associated antigen.

65. 62. The method of claim 61, wherein the human subject is a recipient of a tissue or organ transplant and the T cells are regulatory T cells.

66. 66. The method of claim 65, wherein the human subject is a recipient of an allogeneic organ or tissue transplant.

67. 67. The method of claim 65 or 66, wherein the human subject is experiencing or at risk of GVHD.

68. 62. The method of claim 61, wherein the human subject has an autoimmune, alloimmune, or inflammatory disease state and the T cell population comprises regulatory T cells.

69. 69. The method of claim 68, wherein the autoimmune condition is selected from type 1 diabetes, rheumatoid arthritis (RA), psoriasis or psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, scleroderma, hemolytic anemia, pernicious anemia, and Goodpasture's syndrome.

70. 62. The method of claim 61, wherein the human subject has an immune condition selected from celiac disease, hyperimmunoglobulin E syndrome, and IPEX syndrome, and the T cell population comprises regulatory T cells.

71. 71. The method of any one of claims 65 to 70, wherein the cell population is modified to express a tissue-specific T cell receptor (TCR).

72. 72. The method of any one of claims 65-71, wherein the cell population is modified to insert an IL-2 signaling complex, optionally a chimeric IL-2 signaling complex, that provides a growth advantage to FOXP3-expressing cells.

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