Pluripotent stem cell-derived t cell populations and progenitor cells thereof

Abstract

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

Description

[0001] priority

[0002] This application claims priority and the benefit of U.S. Provisional Application No. 63 / 413,338, filed on October 5, 2022, which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a Sequence Listing that has been submitted in XML format via EFS-Web and is hereby incorporated 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 of the Invention

[0005] The in vitro generation of hematopoietic cells from pluripotent cells has attracted the attention of the scientific community due to its potential application in allogeneic compatible cell therapies. 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 still exist in developing methods for preparing hematopoietic cell lineages (such as T cells) with clinically advantageous phenotypes and clinically relevant quantities. Therefore, successfully generating T cell populations suitable for cell therapy from iPSCs in vitro would meet a great need. The present invention achieves these goals in various aspects and embodiments. Summary of the Invention

[0006] The present disclosure provides, in various aspects and embodiments, 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 methods for the efficient in vitro development of progenitor T cells and T cell populations (including, but not limited to, precursor T cells, CD4+CD8+ "double positive" T cells, CD4+ helper T cells, CD8+ cytotoxic T cells, and regulatory T cells) from human induced pluripotent stem cells (iPSCs). The cells generated according to the present disclosure in various embodiments are functional and / or more closely resemble the corresponding lineages isolated from peripheral blood or lymphoid organs. The present invention provides, in some aspects, isolated cells and cell compositions, including cell compositions generated by the methods disclosed herein, as well as methods (and uses) for cell therapy.

[0007] In one aspect, the present disclosure provides a method for preparing a population of T cells or T progenitor cells. The method includes enriching CD34+ cells from a population of differentiated pluripotent stem cells to prepare a CD34+-enriched population. Endothelial-to-hematopoietic transition (EHT) is induced in the CD34+-enriched population for at least two days but no more than twelve days to prepare a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem cell progenitors (HSPCs). The resulting population comprising HSCs and / or HSPCs (or a fraction thereof) is differentiated into a population comprising a T cell population or a progenitor T cell population.

[0008] Traditionally, hematopoietic lineages have been prepared by differentiating iPSCs into embryoid bodies until day 8 to harvest CD34+ cells. CD34 is commonly used as a marker for hematopoietic endothelial cells, hematopoietic stem cells, and hematopoietic progenitor cells. According to aspects and embodiments of the present disclosure, it has been found that inducing endothelial-to-hematopoietic transition (EHT) in a population of CD34+ cells (which can be derived from iPSC-embryoid bodies) can be used to generate advanced T progenitor cells and T cell populations in vitro.

[0009] In a non-limiting example, to generate mature T cells, embryoid body formation is used to generate CD34+ cells, and HSCs and / or HSPCs are obtained therefrom by inducing EHT. The HSC and / or HSPC population is then cultured in a T cell medium supplemented with, for example, recombinant human fibrin fragment and DLL-4 to generate Tpro cells or pre-T cells. Further culture will generate mature T cells. Expression of Foxp3 in α / β T cells results in the in vitro generation of Tregs. 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 / recovery step. That is, differentiation can occur continuously in culture.

[0010] In certain aspects and embodiments, the present disclosure provides a method for generating a CD7+ progenitor T cell population or a derivative of such a population. For example, the method includes generating a population of HSCs and / or HSPCs, which can include human long-term hematopoietic stem cells (LT-HSCs) from iPSCs (e.g., hiPSCs). The HSC and / or HSPC population is obtained by inducing endothelial-to-hematopoietic transition in 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 full set of Notch ligands, Sonic Hedgehog (SHH), recombinant human fibrin fragment (or other extracellular matrix components), and / or combinations thereof to generate a population comprising CD7+ progenitor T cells or a derivative cell population (e.g., a T cell population).

[0011] 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, the iPSCs are autologous or allogeneic to the recipient (e.g., HLA-matched at one or more loci). In various embodiments, the iPSCs can be gene-edited to aid in HLA matching. For example, the 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, the iPSCs retain the expression of at least one HLA class I and at least one HLA class II complex. In some embodiments, a T cell population is derived from iPSCs that are gene-edited to be homozygous for HLA-A neg and 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.

[0012] In some embodiments, the method according to the aspect can include generating CD34+-enriched cells from differentiated pluripotent stem cells (e.g., from EBs) and inducing endothelial-to-hematopoietic cell differentiation. HSCs comprising a relatively high frequency of LT-HSCs can be generated from a cell population using: various stimuli or factors, including mechanical, biochemical, metabolic, and / or topographical stimuli, and factors such as induction of extracellular matrix, niche factors, extracellular extrinsic factors, cell-intrinsic properties; and including pharmacological and / or genetic means. In some embodiments, CD34+ enrichment and EHT can be induced once the cells reach at least 20% CD34+, e.g., can be induced on days 6 to 14 of iPSC differentiation, e.g., like day 8, day 9, day 10, day 11, day 12, day 13, or day 14. Differentiation of iPSCs can be performed according to known techniques.

[0013] Induction of EHT can be performed by any known method. In various embodiments, EHT can be induced in culture for 2 days to 12 days. In some embodiments, EHT is induced in culture for about 5 days 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 hematopoietic endothelial cells. In some embodiments, the cells are contacted with an effective amount of a mechanosensor or mechanosensitive channel agonist that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensor is Piezol. An exemplary, non-limiting Piezol agonist is Yoda1.

[0014] In various embodiments, CD34+ cells are harvested from a culture that undergoes an endothelial-to-hematopoietic cell transition between day 10 and day 20 of iPSC differentiation (such as day 10 to day 20 of iPSC differentiation, or day 12 to day 15).

[0015] In various embodiments, a population comprising HSCs and / or HSPCs or fractions thereof differentiates into a population comprising T progenitor cells or T lymphocytes. In some embodiments, the cell population is cultured in vitro with a Notch ligand (part or all), SHH, an extracellular matrix component, and / or a combination thereof to differentiate HSCs into a T cell population (or its precursor). In some embodiments, the differentiation into progenitor T cells may further include the presence of stem cell factor (SCF), Flt3L, and interleukin (IL)-7. For example, HSCs and / or HSPCs can be cultured in a medium comprising 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 T cells or pre-T cells (e.g., CD34-, CD7+, CD5+ / -). In some embodiments, the cells are cultured for 15 to 28 days to produce mature T cells (e.g., CD3+), including optionally producing Tregs. In some embodiments, T lymphocytes and progenitor T cells can differentiate into Tregs by the expression of FOXP3 and are optionally expanded in culture.

[0016] In still other embodiments, the present invention generates T cells expressing a chimeric antigen receptor (e.g., CTL, helper T cell, or Treg). The cells can be efficiently transduced by a vector, such as but not limited to a retroviral or non-integrating viral vector, a non-viral vector, and an episomal or episomal hybrid vector carrying a tumor antigen target (including but not limited to CD19, CD38, CD33, CD47, and CD20, etc.). The CAR can be designed to enhance the ability of the cell to recognize, bind, and / or kill target cells. In some embodiments, the CAR enhances the ability of the cell to recognize tumor cells. In some embodiments, the CAR enhances the anti-tumor activity of the cell.

[0017] In some embodiments, the present invention generates T cells that exhibit T cell activation and subsequent T cell-mediated cytotoxicity. Compared with CD34+-derived T cells, the T cells generated herein exhibit significantly superior performance in T cell-mediated cytotoxicity.

[0018] In other aspects, the present invention provides a cell population produced by the methods described herein or a pharmaceutically acceptable composition thereof. In some embodiments, the cell population is a progenitor T cell population that is capable of engrafting 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 (which may express a CAR), a helper T population, or a Treg population (each as described herein).

[0019] In some embodiments, the cell population is a Treg population that can be used for adoptive cell therapy, for example, in a human subject suffering from an autoimmune or inflammatory disorder or disease or graft-versus-host disease (GVHD) selected from the group consisting thereof. In addition, various genetic disorders can affect the immune system, presenting as an autoimmune or pro-inflammatory state. In some embodiments, the Treg population is a CAR-T cell expressing a tissue- or cell-specific CAR.

[0020] In another aspect, the present disclosure provides a cell composition comprising a T cell population (or its progenitor, such as a progenitor T cell population) that is HLA-A neg , HLA-DPB1 neg and HLA-DQB1 neg . Despite such gene deletions and / or gene editing, the T cell composition retains antigen presentation function comparable to that of unedited cells and the ability to differentiate from precursors (as described herein) into a more mature T cell phenotype. The cell composition of this aspect provides an advantage in HLA matching of the recipient to avoid, for example, GVHD. In various embodiments, the T cell population is homozygous for both HLA-B and HLA-C. In some embodiments, the T cell population is homozygous for HLA-DRB1.

[0021] In some embodiments of this aspect, the T cell population is a T progenitor cell population. In various embodiments, the T progenitor cell 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). The cell population according to this aspect can be prepared according to other aspects of the present disclosure.

[0022] In other aspects, the present invention provides a method for cell therapy (or the use of a cell composition for cell therapy), comprising administering to a human subject in need thereof the cell population described herein or a pharmaceutically acceptable composition thereof. In various embodiments, the methods described herein are used to treat blood (malignant and non-malignant), bone marrow, and immune diseases. In various embodiments, the human subject has a condition comprising one or more of the following: lymphopenia, cancer, infectious diseases (e.g., viral diseases such as HPV or HIV), immunodeficiency, autoimmune diseases, skeletal dysplasia, hemoglobinopathies, anemia, bone marrow failure syndromes, and genetic disorders (e.g., genetic disorders affecting the immune system).

[0023] In some embodiments, the subject has cancer, such as a hematological malignancy or a solid tumor. In such embodiments, T progenitor cells or T cells with anti-tumor specificity (such as CTLs that recognize tumor antigens) are administered to the subject.

[0024] In other embodiments, the present invention provides a method for cell therapy, comprising administering to a human subject in need thereof a population of Treg cells described herein or a pharmaceutically acceptable composition thereof. In various embodiments, the subject has an autoimmune disease, an alloimmune disease, or an inflammatory disease. In some embodiments, the subject is a tissue or organ transplant recipient, 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 developing GVHD. Organs that can be transplanted include, for example, the heart, kidney, liver, lung, pancreas, intestine, and thymus, etc. Tissues for transplantation can include, for example, bone, tendon (both referred to as musculoskeletal grafts), bone marrow or HSC, cornea, skin, heart valve, nerve, and / or vein.

[0025] In some embodiments, the subject has an autoimmune disorder, and in some embodiments, the autoimmune disorder 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.

[0026] Other aspects and embodiments of the present disclosure will become apparent from the following detailed disclosure and working examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Showing that ETV2 overexpression (OE) does not affect pluripotency. Figure 1FACS plots showing the transduction efficiency of overexpressing ETV2 and GFP sequences in iPSCs using an adenovirus vector. As shown by the expression of the TRA-1-60 stem cell marker, overexpression of ETV2 does not affect the stem cell characteristics of iPSCs.

[0028] Figure 2 Showing that overexpression (OE) of ETV2 increases the yield of hematopoietic endothelial cells. Representative flow cytometry analysis and relative quantification of hematopoietic endothelial cells (described as CD235a-CD34+CD31+) indicate that ETV2-OE enhances the formation of hematopoietic endothelial cells.

[0029] Figure 3 Showing that overexpression (OE) of ETV2 enhances the formation of CD34+ cells during iPSC differentiation. Representative flow cytometry analysis of CD34+ cells, and relative quantification indicate that ETV2-OE enhances the formation of CD34+ cells.

[0030] Figure 4A and Figure 4B Showing that iPSC-derived HSCs activated by Piezo1 undergo pro-T cell differentiation similar to that of bone marrow (BM)-HSCs. Figure 4A FACS plots of the differentiation efficiency of bone marrow (BM) HSCs and iPSC-HSCs activated by Piezo1 into CD34+CD7+ pro-T cells. Figure 4B Quantification of CD34+CD7+ cells (%) derived from (1) BM-HSCs and (2) iPSC-HSCs (Piezo1-activated). Figure 4B Showing the mean of three experiments.

[0031] Figure 5A and Figure 5B Showing that iPSC-derived HSCs generated by Piezo1 activation undergo T cell differentiation, and such T cells can be activated by CD3 / CD28 beads similar to T cells derived from BM-HSCs. Figure 5A FACS plots of the activation efficiency (CD3+CD69+ expression) of T cells differentiated from BM-HSCs and iPSC-HSCs generated by Piezo1 activation. Figure 5B Quantification of CD3+CD69+ cells (%) derived from (1) BM-HSCs and (2) iPSC-HSCs (Piezo1-activated). Figure 5B Showing the mean of three experiments.

[0032] Figure 6It is shown that iPSC-derived HSCs (activated by Piezo1 in this example) can differentiate into functional T cells. IFNγ expression is a result of T cell activation following stimulation of the T cell receptor (TCR) via CD3 / CD28 beads. The enhanced expression of IFNγ in T cells differentiated from iPSC-derived HSCs after Piezo1 activation increases the ability of HSCs to further differentiate into functional T cells. Figure 6 The average values of three experiments are shown.

[0033] Figure 7 It is shown that HSCs generated according to the present disclosure (D8+7iPSC-CD34+ cells, with or without Yoda 1, "Y") successfully differentiate into CD4+CD8+ ("double positive") T cells as well as TCRα / β T cells. The method of the present disclosure is significantly superior to T cell maturation from bone marrow CD34+ cells.

[0034] Figure 8 It is shown that HSCs generated according to the present disclosure (D8+7iPSC-CD34+ cells, with or without Y) successfully rearrange the TCR and are superior to bone marrow CD34+ cells.

[0035] Fig. 9A and Fig. 9B It shows the phenotypic analysis of HLA-edited (e.g., triple knockout) cells by FACS and immunofluorescence. Fig. 9A It shows the overall expression of HLA class I molecules (HLA-A, HLA-B, and HLA-C) on the cell surface, where HLA-edited cells are positive for overall HLA class I expression to a degree similar to wild-type cells (gHSCs). Fig. 9B It shows the cell expression of HLA-A via immunofluorescence, where HLA-A is not expressed in HLA-edited clones.

[0036] Fig.10 It is shown that HLA-edited clones retain their pluripotency (maintaining tri-lineage differentiation), as indicated 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.

[0037] Fig.11 It shows the immunocompatibility of HLA-edited HSCs. HLA-edited HSCs and control HSCs (WT, B2M KO, and HLA class II null) are co-cultured with peripheral blood mononuclear cells (PBMCs) that are HLA-B and HLA-C matched but HLA-A mismatched. PBMC-mediated cytotoxicity is measured by annexin V staining assay.

[0038] Fig.12 Demonstrate 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, where bone marrow (BM) and peripheral blood samples were evaluated by FACS to compare the relative amounts of each cell type present in the samples.

[0039] Fig.13A and 13B Show that the deletion of HLA-A does not affect the presentation of class I peptides. Fig.13A Show a schematic diagram of immunopeptidome analysis. Fig. 13B Show the results of immunopeptidome analysis, which show minimal differences in the number of peptides and representative proteins presented by class I molecules in WT and HLA-edited cells.

[0040] Fig.14A and 14B Show that the deletion of HLA-DP and DQ does not affect the presentation of class II peptides. Fig.14A Show a graph of immunopeptidome analysis. Fig. 14B Show that, despite the deletion of HLA-DP and DQ, the cells still retained their ability to present a broad spectrum of peptides via HLA class II.

[0041] Fig.15 Is a schematic diagram of the in vivo testing of antigen-mediated immune responses: the delayed-type hypersensitivity assay (DTH), the sensitization phase, and the elimination phase, respectively.

[0042] Fig.16A and 16B Show that HLA-edited HSCs reconstituted a functional immune system, as confirmed by the DTH response of immunodeficient mice. Fig.16A Show the delayed-type hypersensitivity assay performed on transplanted mice, which involves the interaction of different types of immune cells. Mice were sensitized by subcutaneous injection of sheep red blood cells (antigen). A functional immune system led to swelling of the left paw, which was measured with a micrometer caliper. As Fig.16A can be seen, non-transplanted mice did not show any left paw swelling due to their immunodeficiency. In contrast, mice transplanted with cord blood CD34+ cells showed tissue swelling and a doubling of the diameter of their left paw. Fig. 16B Is a graphical assessment of the Fig.16A results shown.

[0043] Fig.17 Show the potential of HSCs to differentiate into T cell subtypes. After a 35-day differentiation period, the presence of CD4+, CD8+, and AB+ T cell populations in pro-T cells was evaluated by cell sorting. Fig.17The differentiation potential of bone marrow-derived CD34+ cells, embryoid body CD34+ cells, and HSCs ("gHSCs") (e.g., using Piezo1 activation) prepared according to the present invention was compared.

[0044] Fig.18 The degree of T cell-mediated cytotoxicity measured in co-cultures of T cells derived from HSCs and CD19+ lymphoma cells in the presence of an anti-CD3 / CD-19 bispecific antibody is shown. According to the present disclosure, T cells prepared from HSCs ("gHSCs") exhibit a high level of cytotoxicity against target cells.

[0045] Fig.19 It is shown that T cells derived from HSCs (pro-T cells) can be efficiently transduced. Pro-T cells were subjected to transduction with a lentivirus (LV) transgenic for an anti-CD-19 chimeric antigen receptor (CAR) (left panel), where the LV transduction efficiency was measured by cell sorting based on anti-CD19scFv staining (right panel). The results indicate that the transduction efficiency of T cells derived from HSCs reached approximately 85%.

[0046] Fig. 20 It is shown that LV-transduced T cells derived from HSCs (pro-T cells) can be efficiently matured into CD4+ / CD8+ T cells via CAR transduction.

[0047] Fig.21 The ability of T cells derived from HSCs transduced with anti-CD19 CAR (CAR pro-T cells) to exert their function via receptor-mediated cytotoxicity is shown. Luc+ NALM6 leukemia cells were co-cultured with CAR pro-T cells, and cell-mediated cytotoxicity was measured by luciferase assay.

[0048] Fig. 22 The ability of HSCs to develop into pro-T cells, as measured by their CD34-CD7+ markers, is shown.

[0049] Fig.23A and 23B It is shown that according to the present disclosure, the expression of T cell-specific transcription factors and thymic engraftment molecules is increased in pro-T cells derived from HSCs. Fig.23A TCF7 mRNA expression is shown, and Fig. 23B CCR7 mRNA expression is shown.

[0050] Fig.24A and 24B It is shown that pro-T cells derived from HSCs engraft and differentiate in the thymus. Fig.24A The engraftment and analysis procedures are demonstrated. Fig. 24BShows FACS analysis of the CD3 cell population gated on the CD45+ cell population, indicating that HSC-derived pro-T cells have excellent engraftment and differentiation potential in the thymus.

[0051] Fig.25 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 HSCs prepared according to the present disclosure. The T cells of the present disclosure exhibit comparable or superior activation, as measured by increased expression of CD107. The bottom panel shows Dynabeads activation, where the activated T cells express inflammatory cytokines. HSC-derived T cells express higher levels of inflammatory cytokines, as exemplified by the expression levels of TNF-α and interferon γ.

[0052] Fig.26 Shows that CCR5-knockout HSCs can differentiate comparably into pro-T cells compared to their wild-type (gHSC) counterparts, HSCs (retaining CCR5).

[0053] Fig. 27 Shows that CCR5-knockout HSCs can differentiate comparably into double-positive (CD4+CD8+) T cells when compared to their wild-type (gHSC) counterparts, HSCs (retaining CCR5).

[0054] The term "gHSC" is used herein to refer to iPSC-derived hematopoietic stem cells of the present disclosure.

[0055] The terms "wild-type" (WT), "unedited", "non-HLA-edited" are used interchangeably herein to refer to non-gene-edited cells of the present disclosure.

[0056] EB34+ cells refer to embryoid body-derived CD34+ cells. These include hematopoietic endothelial cells. Detailed Description

[0057] The present disclosure provides, in various aspects and embodiments, methods for generating T cell populations for use in cell therapy, including T lymphocytes (T cells) and progenitor T cells. In various embodiments, the present invention provides methods for the efficient in vitro development of progenitor T cells and T cell populations (including but not limited to precursor T cells, CD4+CD8+ "double-positive" T cells, CD4+ helper T cells, CD8+ cytotoxic T cells, and regulatory T cells) from human induced pluripotent stem cells (iPSCs). The cells generated according to the present disclosure in various embodiments are functional and / or more closely resemble their corresponding lineages isolated from peripheral blood or lymphoid organs. The present invention provides, in some aspects, isolated cells and cell compositions, including those generated by the methods disclosed herein, and methods for cell therapy.

[0058] According to aspects and embodiments of the present disclosure, the ability of human induced pluripotent stem cells (hiPSCs) to generate essentially unlimited pluripotent stem cell (PSC) populations is used to generate an unlimited supply of T cell populations or their precursors. The use of primary T cells as therapeutic lymphocytes is limited by their limited availability, cell numbers, restricted expansion potential, and histocompatibility issues. For example, Tregs are present in low numbers in the circulation and are challenging to isolate and expand in vitro. In addition, hiPSCs can be more readily genetically modified in vitro compared to primary cells, thus providing improved cell targeting specificity, cell numbers, and bypassing issues such as HLA matching. In addition, fully engineered hiPSC clones can serve as stable and safe sources (Nianias and Themeli, 2019). Moreover, since hiPSCs are of non-embryonic origin, unlike human embryonic stem cells (hESCs), they also do not have ethical concerns. Thus, according to the present disclosure, the use of hiPSCs has 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).

[0059] In one aspect, the present disclosure provides a method for preparing a population of T cells or progenitor T cells. The method includes enriching CD34+ cells from a population of differentiated pluripotent stem cells to prepare a CD34+-enriched population. Endothelial-to-hematopoietic transition (EHT) is induced in the CD34+-enriched population for at least two days but not more than twelve days to prepare a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem cell progenitors (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 fractions thereof) is differentiated into a population comprising a T cell population or a progenitor T cell population.

[0060] Traditionally, hematopoietic lineages are prepared by differentiating iPSCs into embryoid bodies until day 8 to harvest CD34+ cells. CD34 is commonly used as a marker for hematopoietic endothelial cells, hematopoietic stem cells, and hematopoietic progenitors. According to aspects and embodiments of the present disclosure, it has been found that inducing endothelial-to-hematopoietic transition (EHT) in a population of CD34+ cells (and which can be derived from iPSC-embryoid bodies) can be used to generate advanced T progenitor and T cell populations in vitro, including but not limited to regulatory T cells (Tregs).

[0061] 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 thereto)) is used to generate CD34+ cells, from which HSCs and / or HSPCs are obtained by inducing EHT. The HSC and / or HSPC population (e.g., CD34+ cells that have undergone EHT) is then cultured in T cell medium supplemented with, for example, recombinant human fibrin fragment and DLL-4 to generate Tpro cells, which are 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). Expression of Foxp3 in α / β T cells leads to the in vitro generation of Tregs.

[0062] For example, the earliest intrathymic progenitors express high levels of CD34 and CD7, do not express CD1a, and are triple-negative (TN) for the mature T cell markers CD4, CD8, and CD3. Specification of the T cell lineage is associated with the expression of CD1a by prothymocytes that express CD7. Thus, the immature stages of T cell development are generally divided into CD34 + CD1a - (the most immature) and CD34 + CD1a + cells. Early thymocytes progress from CD34 + CD7 + CD1a - to CD34 + CD7 + CD1a + and this transition is associated with T cell specification. CD34 + CD7 + CD1a + cells may be committed to the T-lineage. After this stage, thymocytes progress to the CD4 immature single-positive stage, at which time CD4 is expressed in the absence of CD8. Thereafter, a subset of cells differentiates into CD4 + CD8 + double-positive (DP) stage. Finally, after TCRα rearrangement, DP thymocytes expressing TCRαβ undergo positive and negative selection and give rise to CD4 + CD8 - and CD4 - CD8 + single-positive (SP) T cells.

[0063] In various embodiments, as described in detail herein, a population comprising HSCs and / or HSPCs differentiates into a population 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). The cells can optionally be harvested or recovered at certain steps, or in some embodiments, differentiating into the desired T cell population does not include a harvest / recovery step. That is, differentiation can occur continuously in culture.

[0064] In some embodiments, a population comprising HSCs and / or HSPCs differentiates 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 can be further differentiated without first being recovered 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. Prior to such differentiation, the progenitor T cells are optionally recovered from the culture.

[0065] In some embodiments, a population comprising HSCs and / or HSPCs differentiates into a population comprising double positive T cells (CD4+CD8+) and / or single positive T cells (CD4+CD8−; CD8+CD4−), which can optionally occur in continuous culture (i.e., without harvesting or recovering intermediate cell populations). In various embodiments, the double positive cells and / or single positive T cells differentiate into regulatory T cells, optionally including the step of recovering the double positive cells and / or single positive cells from the culture prior to differentiating into regulatory T cells.

[0066] In some embodiments, a population comprising HSCs and / or HSPCs differentiates into a population comprising regulatory T cells, which can optionally occur in continuous culture (i.e., without harvesting or recovering intermediate cell populations).

[0067] In some aspects and embodiments, the present disclosure provides a method for generating a CD7+ progenitor T cell population or a derivative of such population. For example, the method includes generating a population of HSCs and / or HSPCs, which may include human long-term hematopoietic stem cells (LT-HSCs) from iPSCs (e.g., hiPSCs). The population of HSCs and / or HSPCs is obtained by inducing the endothelial-to-hematopoietic cell transition of CD34+ cells (e.g., CD34+ cells derived from embryoid bodies). The population of HSCs and / or HSPCs (or cells isolated therefrom) is cultured with a partial or full set of Notch ligands, Sonic Hedgehog (SHH), recombinant human fibrin fragments (or other extracellular matrix components), and / or combinations thereof to generate a population comprising CD7+ progenitor T cells or a population of derivative cells (e.g., a T cell population).

[0068] The Notch signaling pathway regulates the formation, differentiation, and function of progenitor T cells, pre-T cells, and / or mature T lymphocytes. In vivo, T cell development occurs after lymphoid progenitors differentiate from hematopoietic stem cells in the bone marrow and migrate to the thymus. Specialized thymic epithelial cells induce T cell development along a controlled pathway. Notch signaling plays a key role during thymic T cell lineage commitment. When lymphoid progenitors enter the thymus, they encounter dense expression of Notch ligands on thymic epithelium, which drives thymopoiesis. The present disclosure provides populations of HSCs and / or HSPCs generated in vitro from iPSCs, and these populations of HSCs and / or HSPCs respond to Notch ligands, SHH, and / or extracellular matrix components by generating T progenitors and T cell lineages in vitro in large quantities.

[0069] In various embodiments, iPSCs are prepared by reprogramming somatic cells. The term "induced pluripotent stem cell" or "iPSC" refers to a cell derived from a somatic cell, such as a skin or blood cell that has 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 from umbilical cord blood), PBMCs, CD34+ cells, or other human primary tissues. In some embodiments, iPSCs are derived from CD34+ cells isolated from peripheral blood. In various embodiments, the iPSCs are autologous or allogeneic (e.g., HLA-matched at one or more loci) to the recipient (the subject in need of treatment as described herein). In various embodiments, the iPSCs can be gene-edited to facilitate HLA matching (such as deleting one or more HLA class I and / or class II alleles or their master regulators, including but not limited to β-2-microglobulin (B2M), CIITA, etc.), or gene-edited to cause other loss-of-function or expression of other functions. For example, the 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, the iPSCs retain the expression of at least one HLA class I and at least one HLA class II complex.

[0070] In various embodiments, a T cell population is derived from iPSCs that are gene-edited to be one of the following: (i) HLA-A - B + C + DP - DR + DQ + , (ii) HLA-A - B + C + DP + DR + DQ - , (iii) HLA-A - B + C + DP - DR + DQ - ; (iv) HLA-A - B - C + DP - DR + DQ + ; (v) HLA-A- B - C + DP + DR + DQ - , (vi) HLA-A - B - C + DP - DR + DQ - 。For the retained HLAs (such as HLA-B, HLA-C, and HLA-DR), the cells can be homozygous or retain only a single copy of the gene. For example, the modified cells are at least identified as (a) HLA-C+ and HLA-DR+, and optionally identified 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-.

[0071] In some embodiments, the T cell population is derived from iPSCs that have been gene-edited to be 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.

[0072] As used herein, the term "negative" (-) or "negative" with respect to a particular HLA class I or class II molecule indicates that both copies of the gene have been disrupted in the cell line or population, and thus the cell line or population does not exhibit significant functional expression of the gene. Such cells can be generated by deleting all or part of the gene or alternatively using other techniques such as siRNA. As used herein, the term "deletion" in the context of genetic modification (i.e., gene editing) of a target gene refers to the abolition of the functional expression of the corresponding gene product (i.e., the corresponding polypeptide). Such gene editing includes deletion of all or part of the gene or deletion of key cis-acting expression control sequences.

[0073] In some embodiments, the 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 gRNA comprises a modification at or near the 5' end (e.g., within 1 to 10, 1 to 5, or 1 to 2 nucleotides of the 5' end) and / or a modification at or near the 3' end (e.g., within 1 to 10, 1 to 5, or 1 to 2 nucleotides of the 3' end). In some embodiments, the modified gRNA exhibits increased resistance to nucleases. In some embodiments, the gRNA comprises two separate RNA molecules (i.e., "dual gRNA"). The dual gRNA comprises two separate RNA molecules: "crispr RNA" (or "crRNA") and "tracr RNA", and is well known to those skilled in the art.

[0074] Generally, various gene editing techniques are known and can be applied according to various embodiments of the present disclosure. Gene editing techniques include, but are not limited to, zinc fingers (ZF), transcription activator-like effectors (TALE), etc. Fusion proteins containing one or more of these DNA-binding domains and the cleavage domain of the Fokl endonuclease can be used to generate double-strand breaks in the desired region of DNA in cells (see, for example, U.S. Patent Application Publication No. US 2012 / 0064620, U.S. Patent Application Publication No. US 2011 / 0239315, U.S. Patent No. 8,470,973, U.S. Patent Application Publication No. US 2013 / 0217119, U.S. Patent No. 8,420,782, U.S. Patent Application Publication No. US 2011 / 0301073, U.S. Patent Application Publication No. US2011 / 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 hereby incorporated by reference). In some embodiments, gene editing is performed using CRISPR-associated Cas systems known in the art (e.g., CRISPR-Cas9). See, for example, US8,697,359, US 8,906,616, and US 8,999,641, each of which is hereby incorporated by reference in its entirety. In various embodiments, gene editing employs type II Cas endonucleases (such as Cas9) or type V Cas endonucleases (such as Cas12a). Type II and type V Cas endonucleases are RNA-guided. gRNA design for guiding 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, FEBS J. September 2016; 283(17):3232–3238. In other embodiments, non-canonical type II or type V Cas endonucleases that are homologous (although with low primary sequence homology) to Streptococcus 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. April 2021; 22(7):3327.In other embodiments, gene editing employs base editing or prime editing to incorporate mutations without causing double-strand breaks. See, for example, Antoniou P, et al., Base and Prime Editing Technologies for Blood Disorders, Front. Genome Edit., Jan. 28, 2021; Matsuokas IG, Prime Editing: Genome Editing for Rare Genetic Diseases Without Double-Strand Breaks or Donor DNA, Front. Genet., Jun. 9, 2020. A variety of other gene editing processes are known, including using dead Cas (dCas) systems (e.g., Cas fusion proteins) to direct DNA modification enzymes to desired targets, using dCas as a guide RNA-directed system. Brezgin S, Dead Cas Systems: Types, Principles, and Applications, Int J Mol Sci. Dec. 2019;20(23):6041.

[0075] Base editors that can install precise genomic alterations without generating double-stranded DNA breaks can also be used for gene editing in cells (e.g., iPSCs) (e.g., engineering gene therapy vectors). Base editors essentially comprise catalytically disabled nucleases, such as Cas9 nickase (nCas9), which cannot generate DSBs, and are fused to a nucleobase deaminase and, in some cases, to a DNA glycosylase inhibitor. Currently, there are 2 main types of base editors, cytidine base editors (CBEs) and adenine base editors (ABEs), which catalyze C>T and A>G conversions. 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 that comprise a reverse transcriptase conjugated (e.g., fused) to a Cas endonuclease and a polynucleotide conjugated (e.g., fused) to a guide RNA that serves as a DNA synthesis template, as described in WO 2020 / 191153.

[0076] Exemplary vectors useful for genome editing applications include, but are not limited to, 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, coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses (including herpesviruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus) and poxviruses (e.g., canarypox virus, vaccinia virus, or modified vaccinia virus)). Vectors containing a nucleic acid molecule of interest can be delivered to cells (e.g., iPS cells, endothelial cells, hematopoietic endothelial cells, HSCs (ST-HSCs or LT-HSCs)) by any method known in the art, including but not limited to transduction, transfection, infection, and electroporation. Any of these vectors can include transposable elements (such as piggyBac transposons or Sleeping Beauty transposons). Transposons insert specific DNA sequences into the genomes of vertebrates. Once excised from the transposon, genes can be integrated into the genome of mammalian cells by transposase-catalyzed cleavage at similar excision sites present in the nuclear genome.

[0077] To improve efficiency, in some embodiments, Cas and gRNA can be combined before being delivered into cells. The Cas-gRNA complex is referred to as a ribonucleoprotein (RNP). Many methods have been developed for directly delivering RNPs to cells. For example, RNPs can be delivered into cells in culture by lipid transfection or electroporation. Electroporation using a nucleofection protocol can be employed, and this procedure allows the rapid entry of RNPs into the nucleus, so genome cleavage can begin immediately. See, for example, Zhang S, Shen J, Li D, Cheng Y. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR / Cas9 genome editing. Theranostics. January 1, 2021;11(2):614-648, which is hereby incorporated by reference in its entirety. In some embodiments, Cas9 and gRNA are electroporated into donor iPSCs and / or HSCs as RNPs.

[0078] Typically, a protospacer adjacent motif (PAM) is required for Cas nuclease cleavage, and the protospacer adjacent motif is typically found 3 to 4 nucleotides downstream of the cleavage site. The PAM is a short DNA sequence (usually 2 to 6 base pairs in length) that is located after 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 the haplotype or polymorphism of the HLA locus does not include four Gs, four Cs, GC repeat sequences, or combinations thereof.

[0079] In some embodiments, a CRISPR / Cas9 system specific for a unique HLA haplotype can be developed using the gRNAs described herein by designing a single gRNA targeting each of the donor-specific HLA-A, HLA-DPB1, and HLA-DQB1 genes (for example). For gene knockout, the gRNA targets the Cas9 protein to a suitable site for editing. Next, the Cas9 protein can perform a double-strand break (DSB), where the DNA is repaired by the non-homologous end joining (NHEJ) mechanism, which generates indels that result in frameshift mutations and terminate the function of the resulting protein. However, off-target gene modification can occur and alter the function of other intact genes. For example, even in the presence of a certain degree of mismatch, the Cas9 endonuclease can generate DSBs at unwanted off-target positions. Such off-target activity can generate genomic instability events such as point mutations and genomic structural variations. In various embodiments, the sgRNA targeting HLA-A can target a region of chromosome 6 defined as 29942532-29942626. In various embodiments, the sgRNA targeting HLA-DQB1 can target a region of chromosome 6 defined as 32665067-32664798. In various embodiments, the sgRNA targeting HLA-DPB1 can target a region of chromosome 6 defined as 33080672-33080935.

[0080] gRNAs can be used to develop cloned iPSCs. On-target editing, (ii) off-target editing, and (iii) translocation editing of such iPSC lines can be evaluated, for example, using sequencing as described herein. Specifically, such assays can be performed by multiplex PCR that utilizes primers designed to target and enrich regions of interest followed by next-generation sequencing (e.g., amplicon sequencing, AMP-seq). The on-target and translocation groups can amplify the expected edited regions, allowing selection of iPSC clones with the expected edits that do not have chromosomal translocations caused by fusion of unintended DSB cleavage sites. The off-target group can enrich any potential off-target regions identified by sequencing and allows selection of iPSC clones with negligible off-target mutations. Collectively, these assays enable screening of iPSC clones to select clones with desired edits while excluding potential CRISPR / Cas9-related genomic integrity issues.

[0081] In some embodiments, to further ensure genomic stability and integrity of the reprogrammed and edited iPSCs, genetic and genomic assays can be performed to select clones that, for example, have not undergone translocation and mutation events and have not integrated episomal vectors. For example, whole-genome sequencing (WGS) is performed on CD34+ cells and the reprogrammed iPSC clones, where genomic differences resulting from editing are compared. These analyses provide an assessment of which iPSC clone genomes differ from the CD34+ starting material, enabling informed selection of iPSC clones that do not generate mutations during reprogramming.

[0082] In some embodiments, karyotyping using a system such as the KARYOSTAT assay is used to select iPSC clones that do not generate indels and 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. May 15, 2021; 37:107140, which is hereby incorporated by reference in its entirety. The KARYOSTAT assay allows visualization of chromosomal aberrations at a resolution similar to that of G-banded karyotyping. For chromosomal gains, the size of the structural aberrations that can be detected is >2 Mb, and for chromosomal losses, the size of the structural aberrations that can be detected is >1 Mb. The KARYOSTAT array is functionalized to achieve balanced whole-genome coverage by low-resolution DNA copy number analysis, where the assay covers all 36,000 RefSeq genes, including 14,000 OMIM targets. The assay is capable of detecting aneuploidy, submicroscopic aberrations, and mosaic events.

[0083] In some embodiments, array comparative genomic hybridization (aCGH) analysis is used to select iPSC clones that do not generate copy number aberrations (CNAs) during reprogramming, such as described in Wiesner et al., “Molecular Techniques,” edited by Klaus J. Busam, Pedram Gerami, Richard A. Scolyer, “Pathology of Melanocytic Tumors,” Elsevier, 2019, pages 364 - 373, ISBN 9780323374576; and Hussein SM, et al., “Copy number variation and selection during reprogramming to pluripotency,” Nature. March 3, 2011; 471(7336):58 - 62, which are hereby incorporated by reference in their entirety. aCGH is a technique for analyzing the entire genome for CNAs by comparing sample DNA and reference DNA.

[0084] In some embodiments, targeted hematopoietic malignancy NGS panel analysis is used to select iPSC clones that do not generate hematopoietic malignancy mutations during reprogramming. For example, the targeted hematopoietic malignancy NGS panel can focus on myeloid leukemia, lymphoma, and / or other genes associated with hematopoietic malignancies to generate a smaller and more manageable dataset than a more extensive approach. Targeted hematopoietic malignancy NGS panel analysis includes using highly multiplexed PCR to amplify regions associated with hematopoietic malignancies, followed by next - generation sequencing.

[0085] In some embodiments, droplet digital PCR (ddPCR) is used to select iPSC clones that do not integrate episomal vectors and that have been passaged sufficiently for episomal vector clearance. As described herein, iPSC reprogramming of CD34+ cells can be achieved by delivery of episomal vectors encoding reprogramming factors. However, although rare, episomal vectors can randomly integrate into the cellular genome, which can disrupt developmental processes, homeostasis, etc. Thus, the ddPCR method can be used to detect residual episomal vectors in iPSC cultures and is capable of selecting iPSC clones that do not integrate episomal vectors.

[0086] In some embodiments, after assessing that the selected clone has no genome aberrations associated with editing, the clone can be additionally tested for spontaneous mutations that may occur during expansion. For example, mutations affecting hematological malignancy genes, indels, translocations, numerical aberrations, such as those 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 hematological malignancy NGS panel AMP-Seq analysis, and / or droplet digital PCR (ddPCR).

[0087] 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, for example, having known or unknown TCR specificities. In some embodiments, the T cells carry TCRs specific for one or more self-antigens or other antigens of interest. Exemplary self-antigens are described herein. In still 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.

[0088] Somatic cells can be reprogrammed by expressing 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. Patent 10,676,165; U.S. Patent 9,580,689; and U.S. Patent 9,376,664, which are hereby incorporated by reference in their entireties. In various embodiments, well-known viral vector systems such as lentivirus, Sendai virus, or measles virus systems are used to express the reprogramming factors. Alternatively, the reprogramming factors can be expressed by introducing mRNA encoding the reprogramming factors into somatic cells. In addition, iPSCs can be generated by introducing non-integrating episomal plasmids that express the reprogramming factors, i.e., for generating transgene-free and virus-free iPSCs. Known episomal plasmids can be employed that have limited replication ability and thus are lost after several cell generations.

[0089] In some embodiments, human pluripotent stem cells (e.g., iPSCs) are gene - edited. Gene editing includes, but is not limited to, modification of, for example, HLA genes (e.g., deletion of one or more HLA class I and / or class II genes), deletion of β2 - microglobulin (β2M), deletion of CIITA, deletion or addition of T - cell receptor (TCR) genes, or addition of a chimeric antigen receptor (CAR) gene. Exemplary CARs can target any desired organ - or tissue - specific antigen, and in some embodiments, antigens specific to the donor organ. For example, the iPSCs can be T - cell receptor (TCR) - transduced iPSCs. Exemplary TCRs can be specific for an autoantigen of interest. Such embodiments enable large - scale production of regenerative Tregs with the desired antigen specificity. Alternatively, engineered iPSCs with one or more HLA knockouts and TCR knockouts can be placed in a bioreactor and differentiated in feeder - free and serum - free conditions under GMP - grade conditions to produce fully functional and tissue - compatible T cells.

[0090] In some embodiments, the iPSCs are prepared from CD3 + cells, or in some embodiments, from T lymphocytes (T - iPSCs). For example, T lymphocytes with the desired antigen specificity can be isolated (using, for example, cell sorting with HLA peptide ligands) and reprogrammed into T - iPSCs. Then, according to the present disclosure, these T - iPSCs are redifferentiated into a population containing the desired T - cell population or T - progenitor cell population. When T - iPSCs are generated from antigen - specific T cells, the T - iPSCs inherit the rearranged T - cell receptor (TCR) genes. In these embodiments, the T cells redifferentiated from T - iPSCs exhibit the same antigen specificity as the original T cells.

[0091] 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 from iPSC differentiation are three - dimensional aggregates of iPSCs and contain three (or two or one) embryonic germ layers based on the differentiation method. For example, the preparation of EBs is described in US2019 / 0177695, which is hereby incorporated by reference in its entirety. In some embodiments, EBs prepared by iPSC differentiation are expanded in a bioreactor, such as described by Abecasis B. et al., Expansion of 3D human induced pluripotent stem cells aggregates in bioreactors:Bioprocess intensification and scaling-up approaches .J.of Biotechnol.246(2017)81 - 93. The EBs can be used to generate any desired cell type. Other methods for the expansion or differentiation of EBs, including 3D suspension culture, are described in WO 2020 / 086889, which is hereby incorporated by reference in its entirety.

[0092] In some embodiments, methods according to each aspect can include generating CD34+-enriched cells from pluripotent stem cells (e.g., EBs) and inducing endothelial-to-hematopoietic cell differentiation. HSCs containing a relatively high frequency of LT-HSCs can be generated from a cell population using: various stimuli or factors, including mechanical, biochemical, metabolic, and / or topographical stimuli, and factors such as induction of the extracellular matrix, niche factors, cell-extrinsic factors, cell-intrinsic properties; and including pharmacological and / or genetic means.

[0093] In some embodiments, the method includes preparing endothelial cells with hematopoietic potential from pluripotent stem cells prior to EHT induction. In some embodiments, overexpression of GATA2 / ETV2, GATA2 / TAL1, or ER71 / GATA2 / SCL can result in the formation of endothelial cells with hematopoietic potential from PSC sources. In some embodiments, the method includes overexpressing the E26 transformation-specific variant 2 (ETV2) transcription factor in iPSCs. After CD34+ enrichment, HSCs are generated from endothelial cells using mechanical, biochemical, pharmacological, and / or genetic stimuli or modifications. ETV2 can be expressed by introducing a non-integrating episomal plasmid encoding for constitutive or inducible expression of ETV2, and for generating transgene-free hematopoietic ECs. In some embodiments, ETV2 is expressed from mRNA introduced into iPSCs. Any available method for introducing mRNA can be used, including electroporation or lipofection. Differentiation of cells expressing ETV2 can include the addition of VEGF-A. See Wang K, et al. Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with mRNA .Sci. Adv. Vol. 6 (2020). Cells generated in this manner can be used to stably generate CD34+ cells, followed by EHT induction according to embodiments of the present disclosure.

[0094] 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 to 14 of iPSC differentiation (such as day 8, day 9, day 10, day 11, day 12, day 13, or day 14). Differentiation of iPSCs can be carried out according to known techniques. In some embodiments, iPSC differentiation involves a combination of factors such as, but not limited to, bFGF, Y27632, BMP4, VEGF, SCF, EPO, TPO, IL-6, IL-11, and / or IGF-1. In some embodiments, feeder-free, serum-free, and / or GMP-compatible materials are used to differentiate hPSCs. In some embodiments, hPSCs are co-cultured with mouse bone marrow-derived feeder cells (such as the OP9 or MS5 cell lines) in a serum-containing medium. The culture can contain growth factors and cytokines to support the differentiation of embryoid bodies or monolayer systems. The OP9 co-culture system can be used to generate pluripotent HSPCs, which 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 11, article 481 (2020). Alternatively, a stepwise process using defined conditions with specific signals can be used. For example, the expression of HOXA9, ERG, RORA, SOX4, and MYB in human PSCs favors direct differentiation into CD34+ / CD45+ progenitor cells with multilineage potential. In addition, the 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. October 3, 2013; 13(4).

[0095] EHT can be induced by any known method. In some embodiments, induction of EHT generates a population of HSCs that includes LT-HSCs. In some embodiments, EHT generates a cell population that includes HSPCs. In some embodiments, EHT generates HSCs and / or HSPCs from endothelial cells or hematopoietic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological, and / or genetic means (e.g., by stimulation, inhibition, and / or genetic modification). In some embodiments, EHT generates a stem cell population that includes 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.

[0096] In some embodiments, a population of HSCs and / or HSPCs or a fraction thereof differentiates into T cells or progenitors or derivatives thereof without reliance on the use of an agonist of a mechanosensitive receptor or mechanosensitive channel (such as Yoda1). In some embodiments, the use of a mechanosensitive receptor or mechanosensitive channel agonist (e.g., Yoda1) is optional. Thus, in some embodiments, CD34+ cells are enriched from a population of differentiated pluripotent stem cells to prepare a CD34+-enriched population. The endothelial-to-hematopoietic cell transition of the CD34+-enriched cell population is induced for at least two days but not more than 12 days, with optional use of a mechanosensitive receptor or mechanosensitive channel agonist such as Yoda1, jedi1, jedi2, or ssRNA40. The HSCs and / or HSPCs differentiate into a progenitor T cell population or a T cell population (e.g., as described herein). In some embodiments, the endothelial-to-hematopoietic cell transition of the CD34+-enriched cell population is induced for at least two days and further continues 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. The total EHT differentiation proceeds for not more than 12 days.

[0097] In some embodiments, the method includes increasing the expression or activity of dnmt3b in PSCs, embryoid bodies, CD34+ cells, ECs, HECs, or HSCs, which can be performed by mechanical, genetic, biochemical, or pharmacological means. In some embodiments, the method includes increasing the activity or expression of DNA (cytosine-5-)-methyltransferase 3 beta (Dnmt3b) and / or GTPase IMAP family member 6 (Gimap6) in cells. See WO 2019 / 236943 and WO 2021 / 119061, which are hereby incorporated by reference in their entireties. In some embodiments, the induction of EHT includes increasing the expression or activity of dnmt3b.

[0098] In some embodiments, the cell is contacted with an effective amount of a mechanosensor or a mechanosensitive channel agonist that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensor is Piezo1. An exemplary Piezo1 agonist is Yoda1. In some embodiments, the mechanosensor 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 against the mechanosensitive ion channel Piezo1. Syeda R, Chemical activation of the mechanotransduction channel Piezol .eLife (2015). Yoda1 has the following structure:

[0099]

[0100] Derivatives of Yoda1 can be used in various embodiments. For example, in some embodiments, derivatives containing a 2,6-dichlorophenyl nucleus are employed. Exemplary agonists are disclosed in Evans EL, et al., Yoda1 analogue(Dooku1)which antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation , British J. of Pharmacology 175(1744 - 1759):2018. Other Piezo1 agonists include Jedi1, Jedi2, single-stranded (ss)RNA (e.g., ssRNA40) and its derivatives and analogs. See Wang Y., et al., A lever-like transduction pathway for long-distance chemical-and mechano-gating of the mechanosensitive Piezo1 channel . Nature Communications (2018) 9:1300; Sugisawa, et al., RNA Sensing by Gut Piezo1 Is Essential for Systemic Serotonin Synthesis ,Cell,Volume 182, Issue 3, 2020, pp. 609 - 624, which is hereby incorporated by reference in its entirety. These Piezo1 agonists are commercially available. In various embodiments, the effective amount of the 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, in the range of 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., ssRNA40) and its derivatives and analogs can be used for Piezo1 activation.

[0101] In various embodiments, pharmacological Piezo1 activation is applied to CD34+ cells (i.e., CD34+-enriched cells). In certain embodiments, pharmacological Piezo1 activation can be further applied to iPSCs, embryoid bodies, ECs, hematopoietic endothelial cells (HECs), HSCs, hematopoietic progenitor cells, and hematopoietic lineages. In certain embodiments, Piezo1 activation is applied at least to EBs generated from iPSCs, CD34+ cells isolated from EBs, and / or combinations thereof, which, according to various embodiments, allows for better generation of T progenitor cells and T cell lineages derived therefrom (e.g., Tregs and mature α / β T cells) compared to other methods used to induce EHT. Advantageously, progenitor T cells generated by Piezo1 activation during EHT have a greater engraftment potential than progenitor T cells prepared without Piezo1 activation during EHT.

[0102] In some embodiments, a pharmacological agent (such as, but not limited to, retinoic acid, retinoic acid receptor (RAR) agonist, dibutyl cyclic AMP, 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 can be further applied to iPSCs, embryoid bodies, ECs, hematopoietic endothelial cells (HECs), HSCs, hematopoietic progenitor cells, and hematopoietic lineages (e.g., for expanding cell populations). In certain embodiments, pharmacological agent activation, such as retinoic acid or retinoic acid receptor (RAR) agonist activation, is applied at least to EBs generated from iPSCs, CD34+ cells isolated from EBs, and / or combinations thereof.

[0103] Alternatively or additionally, the activity or expression of Dnmt3b can be directly increased in cells (e.g., in CD34+-enriched cells). For example, the mRNA expression of Dnmt3b can be increased by delivering transcripts encoding Dnmt3b to the cells, or by introducing a transgene encoding Dnmt3b, or by a transgene-free method (not limited to introducing non-integrating episomes into cells). In some embodiments, gene editing is employed to introduce genetic modifications into the Dnmt3b expression element in the cell, such as but not limited to increasing promoter strength, ribosome binding, RNA stability, and / or affecting RNA splicing.

[0104] In some embodiments, the method includes increasing the activity or expression of Gimap6 in the cell, alone or in combination with Dnmt3b and / or other genes that are upregulated or downregulated upon cyclic strain or piezoelectric activation. To increase the activity or expression of Gimap6, an mRNA transcript encoding Gimap6 can be introduced into the cell, or a transgene-free method can be employed, including but not limited to introducing episomes into the cell; or alternatively a transgene encoding Gimap6. In some embodiments, gene editing is used to introduce genetic modifications into the Gimap6 expression element in the cell (such as one or more modifications to increase promoter strength, ribosome binding, RNA stability, or affecting RNA splicing).

[0105] In embodiments of the present disclosure that employ mRNA delivery to cells, known chemical modifications can be used to avoid the innate immune response in the cells. For example, synthetic RNA containing only canonical nucleotides can bind to pattern recognition receptors and can trigger an effective immune response in the cell. Such a response can lead to translation blockage, secretion of inflammatory cytokines, and cell death. RNA containing certain non-canonical nucleotides can evade detection by the innate immune system and can be efficiently translated into protein. See US 9,181,319, which is hereby incorporated by reference, particularly regarding nucleotide modifications to avoid the innate immune response.

[0106] In some embodiments, the expression of Dnmt3b and / or Gimap6 is increased by introducing a transgene into the cell, which can direct the desired overexpression level (with other options of different promoter strengths or expression control elements). Various viral vectors or transfection reagents known in the art (including lipid nanoparticles) can be used to introduce the transgene. In some embodiments, the expression of Dnmt3b and / or Gimap6 is increased by a transgene-free method (e.g., episomal delivery). In some embodiments, gene editing techniques are used to increase the expression or activity of Dnmt3b and / or Gimap6 or other genes disclosed herein, e.g., to introduce one or more modifications to increase promoter strength, ribosome binding, or RNA stability.

[0107] 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, a cell population is introduced into a bioreactor that provides cyclic strain biomechanical stretch, as described in WO 2017 / 096215, which is hereby incorporated by reference in its entirety. The cyclic strain biomechanical stretch can increase the activity or expression of Dnmt3b and / or Gimap6. In these embodiments, mechanical means apply a stretching force to the cells or to the cell culture surface on which the cells (e.g., ECs or HECs) are cultured. For example, a computer-controlled vacuum pump system or other components (e.g., FlexCell TM Tension System, Cytostretcher System) can be used to apply in vitro cyclic 2D, 3D, or 4D stretch to the cells under defined and controlled cyclic strain conditions. For example, the cyclic stretch applied can be a cyclic strain of about 1% to about 20% (e.g., a cyclic strain of about 6%), for several hours or days (e.g., about 7 days). In various embodiments, the cyclic strain is applied for at least about one hour, at least about two hours, at least about six hours, at least about eight 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.

[0108] Alternatively or additionally, EHT is stimulated through Trpv4 activation. Trpv4 activation can be carried out by contacting the cells (e.g., CD34+ enriched cells, ECs, or HECs) with one or more Trpv4 agonists, which are optionally selected from GSK1016790A, 4α-PDD, or analogs and / or derivatives thereof.

[0109] When a cell population is described herein as having a certain phenotype, it is understood that the phenotype represents a significant 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. Additionally, at each step, 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, fluorescence-activated cell sorting or magnetic beads that bind cells to certain cell surface antigens (including those described herein) can be used based on cell surface antigens to sort cells. A negative selection column can be used to remove cells that express an undesired cell surface marker. In some embodiments, cells are enriched for CD34+ cells (before and / or after undergoing EHT). In some embodiments, the cell population is cultured under conditions that promote the expansion of CD34+ cells, thereby generating an expanded stem cell population. In various embodiments, T progenitor cells in the enriched cells (using cell surface markers described herein, such as CD7+) are enriched, and these cells can optionally be further differentiated in culture. Additionally, for example, after progenitor T cell or T cell differentiation, cells can be enriched for markers such as CD3, CD4, and / or CD8, etc. For example, Tregs can be further enriched in the resulting population by CD25+ enrichment.

[0110] In various embodiments, CD34+ cells (e.g., floating cells and / or adherent cells) are harvested from a culture that undergoes an endothelial-to-hematopoietic cell transition between day 10 and day 20 of iPSC differentiation (such as between day 10 and day 17 of iPSC differentiation, or day 12 to 15).

[0111] In various embodiments, the HSC and / or HSPC population (e.g., or CD34+-enriched cells isolated therefrom) is further expanded. For example, cells can be expanded according to the methods disclosed in US 8,168,428; US 9,028,811; US 10,272,110; and US 10,278,990, which patents are hereby incorporated by reference in their entireties. In some embodiments, the in vitro expansion of HSC or CD34+-enriched cells uses prostaglandin E2 (PGE2) or a PGE2 derivative. In some embodiments of the present disclosure, the HSC contains at least about 0.01% LT-HSC, or at least about 0.05% LT-HSC, or at least about 0.1% LT-HSC, or at least about 0.5% LT-HSC, or at least about 1% LT-HSC.

[0112] Hematopoietic stem cells (HSCs) that 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 stem cell population comprising HSCs is enriched, e.g., as described in US 9,834,754, which is hereby incorporated by reference in its entirety. For example, the method can include sorting a cell population based on the expression of one or more of CD34, CD90, CD38, and CD43. Components that are one or more of + CD34 + CD90 - CD38 - CD43 - can be selected for further differentiation. In some embodiments, the stem cell population for differentiation into hematopoietic lineages is at least about 80% CD34 + or at least about 90% CD34 + or at least about 95% CD34

[0113] In some embodiments, an HSC / HSPC population, or a CD34+-enriched cell or fraction thereof, or a derived population is expanded as described in US2020 / 0308540, which is hereby incorporated by reference in its entirety. For example, cells are expanded by exposing the cells to an aryl hydrocarbon receptor antagonist including, e.g., 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 Sep 10, 2010;329(5997):1345–1348.

[0114] In some embodiments, compounds that promote CD34 + cell expansion include pyrimidoindole derivatives including, e.g., UM171 or UM729 (see US2020 / 0308540, which is hereby incorporated by reference).

[0115] In some embodiments, the stem cell population or CD34+-enriched cells are further enriched for cells expressing periostin and / or platelet-derived growth factor receptor alpha (pdgfra), or are modified to express periostin and / or pdgfra, as described in WO 2020 / 205969, which is hereby incorporated by reference in its entirety. Such expression can be carried out by delivering the coding transcript to the cells, or by introducing a coding transgene, or by a transgene-free method (not limited to introducing non-integrating episomes into the cells). In some embodiments, gene editing is employed to introduce genetic modifications into the expression elements in the cells, such as to modify promoter activity or strength, ribosome binding, RNA stability, or to affect RNA splicing.

[0116] In other embodiments, the stem cell population or CD34+-enriched cells are cultured with an inhibitor of the histone methyltransferase EZH1. Alternatively, EZH1 is partially or completely absent or inactivated or transiently silenced (e.g., by siRNA) in the stem cell population. Inhibition of EZH1 can direct bone marrow progenitors (e.g., CD34+CD45+) to the lymphoid lineage. See WO 2018 / 048828, which is hereby incorporated by reference in its entirety. In other embodiments, EZH1 is overexpressed in the stem cell population.

[0117] In various embodiments, a population comprising HSCs and / or HSPCs or fractions thereof differentiates into a population comprising T progenitors or T lymphocytes.

[0118] In some embodiments, a cell population is cultured in vitro with Notch ligand(s) (partially or fully), SHH, extracellular matrix components, and / or combinations thereof to differentiate HSCs into a T cell population (or a precursor thereof). Additionally, according to known methods, xenogeneic OP9-DL1 cells are commonly used to differentiate into T cells. The OP9-DL1 co-culture 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 for clinical application of the cells. There is a need for feeder-free cell systems capable of generating T lymphocytes from hiPSCs for clinical use, and in some embodiments the present invention achieves this goal.

[0119] As used herein, the term "Notch ligand" refers to a ligand capable of binding to a Notch receptor polypeptide present in the T cell membrane of hematopoietic stem or progenitor cells. Notch receptors include Notch-1, Notch-2, Notch-3, and Notch-4. Notch ligands typically have a DSL domain (D - delta, S - Serrate, and L - Lag2), which includes 20 to 22 amino acids at the amino terminus and 3 to 8 EGF repeats on the extracellular surface. In various embodiments, the Notch ligand includes delta - like 1 (DLL1), delta - like 4 (DLL4), SFIP3, delta Max (disclosed in PCT / US2020 / 041765 and PCT / US2020 / 030977, which are hereby incorporated by reference in their entireties) or at least one of its functional portions. The key signal transmitted by thymic stromal cells to incoming lymphocyte progenitors in vivo is mediated by DL4, which is expressed by cortical thymic epithelial cells.

[0120] The earliest intrathymic progenitors express high levels of CD34 and CD7, do not express CD1a, and are triple - negative (TN) for the mature T - cell markers CD4, CD8, and CD3. The commitment to the T - cell lineage is associated with the expression of CD1a by pro - thymocytes expressing CD7. Thus, the immature stages of T - cell development are generally divided into CD34 + CD1a - (the most immature) and CD34 + CD1a + cells. Early thymocytes transition from CD34 + CD7 + CD1a - to CD34 + CD7 + CD1a + is associated with T - cell commitment. CD34 + CD7 + CD1a + cells may be restricted to the T - lineage. After this stage, thymocytes progress to the CD4 immature single - positive stage, where CD4 is expressed in the absence of CD8. Thereafter, a portion of the cells differentiate into CD4 + CD8 + double - positive (DP) stage. Finally, after TCRα rearrangement, DP thymocytes expressing TCRαβ undergo positive and negative selection and give rise to CD4 + CD8 - and CD4 - CD8 + single - positive (SP) T cells.

[0121] In some embodiments, progenitor T cells are isolated by enriching for CD7 expression. In some embodiments, progenitor T cells are expanded as described in US 2020 / 0308540, which is hereby incorporated by reference in its entirety. For example, cells can be expanded by exposing the cells 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 compound that promotes expansion includes pyrimidoindole derivatives including, for example, UM171 or UM729 (see US2020 / 0308540, which is hereby incorporated by reference).

[0122] In some embodiments, differentiating into progenitor T cells can further include the presence of stem cell factor (SCF), Flt3L, and interleukin (IL)-7. In various embodiments, the resulting CD7+ progenitor T cells express CD1a. The CD7+ progenitor T cells do not express CD34 or express a reduced level 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 can comprise CD7 + CD1a + cells. In some embodiments, the phenotype of the progenitor T cells comprises CD7 + CD5 + cells. In some embodiments, the progenitor T cells comprise CD7 + CD1a + CD5 + cells, and optionally CD34 + .

[0123] In some embodiments, the progenitor T cells exhibit a reduced level of CD34 expression, a very low level of CD34 expression (compared to the HSC population), or no CD34 expression. In some embodiments, the CD34 expression in this population is reduced by at least about 50% or at least about 75% relative to the HSC population.

[0124] In some embodiments, the Notch ligand is an anti-Notch (agonist) antibody that can bind and participate in Notch signaling. In some embodiments, the antibody is a monoclonal antibody (including human or humanized antibodies), a single-chain antibody (scFv), a nanobody, or other antibody fragment or antigen-binding molecule capable of activating the Notch signaling pathway.

[0125] In some embodiments, the Notch ligand is a delta family Notch ligand. In some embodiments, the delta family ligand is delta-1 (Genbank accession number AF003522, Homo sapiens), delta-like 1 (DLL1, Genbank accession numbers NM_005618 and NP_005609, Homo sapiens; Genbank accession numbers X80903, 148324, Mus musculus), delta-4 (Genbank accession numbers AF273454, BAB18580, Mus musculus; Genbank accession numbers AF279305, AAF81912, Homo sapiens) and / or delta-like 4 (DLL4; Genbank accession numbers Q9NR61, AAF76427, AF253468, NM_019074, Homo sapiens; Genbank accession number NM 019454, Mus musculus). The Notch ligand is commercially available or can be produced by, for example, recombinant DNA technology.

[0126] 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 the human DLL1 or DLL4 Notch ligand. Functional derivatives of the Notch ligand (including fragments or portions thereof) will be capable of binding to and activating the Notch receptor. Binding to the Notch receptor can be determined by a variety of methods known in the art, including in vitro binding assays and receptor activation / cellular signaling assays.

[0127] In some embodiments, the Notch ligand is DLL4 having one or more affinity-enhancing mutations with respect to hDLL4, such as one or more (or all) of the following: 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).

[0128] In various embodiments, the Notch ligand is soluble and optionally immobilized on microparticles or nanoparticles, which are optionally paramagnetic to allow for magnetic enrichment or concentration processes. In other embodiments, the Notch ligand is immobilized on a 2D or 3D culture surface, optionally together with other adhesion molecules such as VCAM-1. See US 2020 / 0399599, which is hereby incorporated by reference in its entirety. In other embodiments, the beads or particles are polymers (e.g., polystyrene or PLGA), gold, dextran iron, or are composed of biomaterials such as particles formed from lipids and / or proteins. In various embodiments, the particles have a diameter or maximum dimension ranging from about 0.01 μm (10 nm) to about 500 μm (e.g., about 1 μm to about 7 μm). In other embodiments, polymer scaffolds with conjugated ligands can be employed, as described in WO 2020 / 131582, which is hereby incorporated by reference in its entirety. For example, the scaffold can be composed of polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivatives, gelatin, collagen, agarose, hyaluronic acid, poly(lysine), polyhydroxybutyrate, poly-ε-caprolactone, polyphosphazene, 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 includes pores with a diameter between about 1 pm and 100 pm.

[0129] In some embodiments, the C-terminus of the Notch ligand is conjugated to a selected support. In some embodiments, this can include adding a sequence at the C-terminus of the Notch ligand that can be enzymatically conjugated to the support, for example, via a biotin molecule. In another embodiment, a Notch ligand-Fc fusion is prepared such that the Fc fragment can be immobilized by binding to protein A or protein G, which is conjugated to the support. Of course, any known protein conjugation method can be employed.

[0130] Thus, in various embodiments, the Notch ligand is immobilized, functionalized, and / or embedded in a 2D or 3D culture system. The Notch ligand can be incorporated together with components of the extracellular matrix, such as one or more selected from fibronectin, recombinant human fibrin fragment, 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 the culture conditions, providing a topographical pattern and / or texture (e.g., roughness) that aids in cell differentiation and / or expansion.

[0131] In some embodiments, HSCs are cultured in a medium containing TNF-α and / or an aryl hydrocarbon / dioxin receptor antagonist (SR1) and differentiated into progenitor T cells in the presence of a Notch ligand. See US 2020 / 0390817, US2021 / 0169934, and US 2021 / 0169935, which are hereby incorporated by reference in their entireties. In some embodiments, the HSCs are cultured in a 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 fragment. In some embodiments, the cells are cultured with a recombinant human fibrin fragment, which is recombinant human fibronectin and contains three functional domains: the human fibronectin cell-binding domain (C domain), the heparin-binding domain (H domain), and the CS-1 sequence domain. In some embodiments, the cells are cultured in the presence of immobilized delta-like-4 ligand and the recombinant human fibrin fragment. In some embodiments, the cells are cultured in the presence of immobilized delta-like-4 ligand, TNF-α, and the recombinant human fibrin fragment. In some embodiments, the cells are cultured in the presence of immobilized delta-like-1 ligand and the recombinant human fibrin fragment. In some embodiments, the cells are cultured in the presence of SFIP3 and the recombinant human fibrin fragment. In some embodiments, the cells are cultured in the presence of immobilized delta-like-4 ligand and SHH molecule and / or its functional derivatives. Exemplary fibronectin fragments contain one or more RGDS, CS-1, and heparin-binding motifs. The fibronectin fragment can be free in solution or immobilized on the culture surface or on particles. In some embodiments, the cells are cultured for 5 to 7 days to prepare CD7+ progenitor T cells. In some embodiments, the 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 to 21 days to produce mature T cells (e.g., CD3+). In some embodiments, the cells are cultured for 21 days or longer to produce Tregs.

[0132] In various embodiments, the method produces a Treg population by culturing a cell population comprising HSCs and / or HSPCs with a Notch ligand (including any of the foregoing embodiments) in the presence or absence of extracellular matrix components, and optionally adding TNF-α to the culture at certain stages of differentiation. Thus, the cells produced in some embodiments are progenitor or precursor cells committed to the T cell lineage ("progenitor T cells"). In some embodiments, the cells are CD7+ progenitor 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) cells or CD4+CD8- single positive cells. In some embodiments, the cells are CD4+CD8- single positive (SP) cells and in some embodiments can be TCRhi. In some embodiments, the cells are TCRαβ+. In various embodiments, the cells are CD3+.

[0133] In various embodiments, the progenitor T cells are further cultured under suitable conditions to produce cells of the desired T cell population (e.g., Tregs), including with 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 sufficient time to form cells of the desired T cell population. In some embodiments, the HSCs / HSPCs or progenitor T cells are cultured together in suspension with a soluble Notch ligand or a Notch ligand conjugated to a particle or other support or a Notch ligand-expressing cell. In some embodiments, the progenitor T cell or HSC / HSPC population is cultured in a bioreactor in suspension or adherent format, optionally a closed or enclosed automated bioreactor, with soluble or conjugated Notch ligand in the suspension. One or more cytokines, extracellular matrix components, and thymic niche factors that promote commitment and differentiation to the desired T cell population can 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 produce progenitor T cells. In some embodiments, the stem cell population or its derivatives are cultured for at least about 21 days or at least about 28 days to produce 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 the Treg population.

[0134] In various embodiments, HSC / HSPC populations are cultured in artificial thymic organoids (ATO). See Hagen, M. et al. (2019). The ATO will contain a culture of HSCs (or HSC aggregates) with a stromal cell line expressing Notch ligands under serum-free conditions. Artificial thymic organoids are 3D systems that induce the differentiation of hematopoietic precursors into naive CD3+CD8+ and CD3+CD4+ T cells. In some embodiments, the artificial thymic organoid contains DLL4 and BMP2, or functional fragments thereof.

[0135] In various embodiments, the method includes generating Tregs. In certain embodiments, the Tregs express CD3 and the T cell receptor. In some embodiments, the Tregs contain CD4 + cells, which are optionally expanded in culture. In some embodiments, iPSCs, CD34+ cells, or derivatives thereof are modified to express a chimeric antigen receptor (CAR).

[0136] Regulatory T cells (Tregs) have extensive clinical application potential. For example, they can be used to control the harmful immune responses seen in patients with autoimmune diseases such as childhood (type I) diabetes, rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease; and to inhibit the rejection of transplanted organs in patients receiving heart, liver, or kidney transplants. However, their clinical application is limited due to the low frequency of Tregs in peripheral blood (about 1% to 2% of humans). Therefore, the successful in vitro generation of Tregs from iPSCs would meet a huge demand.

[0137] T lymphocytes and progenitor T cells can differentiate into Tregs by expressing FOXP3. The Tregs can optionally be further isolated or enriched by positive and / or negative selection. In various embodiments, the present disclosure provides a cell population 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, and it can be antigen- or tissue-specific. Tregs can be defined as CD3+CD4+CD25+ and FoxP3+ cells. In some embodiments, additional cell surface markers include CTLA-4, CD39, CD73, GITR, and / or LAG-3. In an exemplary embodiment, 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.

[0138] In some embodiments, pluripotent stem cells are cultured under conditions that permit the formation of embryoid bodies. The embryoid bodies are dissociated, and CD34+ cells are isolated and used to induce EHT, which is then differentiated into T cells or progenitor T cells by culturing the cells that have undergone EHT with at least one Notch ligand (as further described herein). A vector comprising a nucleic acid sequence encoding Foxp3 is introduced during or after T cell differentiation, thereby driving the formation of Tregs. In some embodiments, the T cell population 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 an islet-specific T cell receptor (TCR) to target the engineered Tregs to sites of pancreatic-related diseases. Optionally, the engineered Tregs include an inserted IL-2 signaling complex, which provides the engineered Tregs with a proliferation and functional advantage.

[0139] Tregs can be defined as CD4 + CD25 +Tregs control immune responses to self and foreign antigens and help prevent autoimmune diseases. In some embodiments, the differentiation of cells into Treg cells involves modifying Treg precursors (e.g., CD4+ αβ T cells or their precursors) 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 generation of regulatory T cells. In some embodiments, gene editing is performed on iPSCs, CD34+ cells (e.g., isolated before or after EHT), progenitor T cells, CD25+ T cells, CD4+ CD8+ cells, or CD4+ cells (e.g., αβ T cells) to provide expression of FOXP3, which can be constitutive and stable expression. In some embodiments, regulatory sequences containing strong enhancers and / or promoters are inserted to operably control the 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 to increase transcription of the FOXP3 gene. In some embodiments, a transcriptional activation domain is inserted, which contains specific DNA sequences that can be bound by transcription factors, whereby the transcription factors can thus control the transcription rate. Specific transcription factors 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 the coding sequence and constitutive expression control sequences) is inserted to provide constitutive expression of FOXP3. Various ways 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, which is hereby incorporated by reference in its entirety. In some embodiments, the CRISPR / Cas9 is used to insert a FOXP3 donor gene with constitutive expression control sequences.

[0140] In some embodiments, in the presence of a growth factor such as IL-2, Treg cells are expanded in culture. Additionally, the expansion protocol can include the use of anti-CD3 and agonist anti-CD28 antibodies, which can be conjugated to a matrix surface (including beads) or provided in soluble form. In some embodiments, expansion of Tregs is not required (or is minimal), thus avoiding loss of desired function through further culturing. Additionally, the proliferative capacity of Tregs may be limited. In some embodiments, Tregs are expanded in culture for 7 days or less, or about 4 days or less, or 2 days or less. In some embodiments, Tregs are engineered to have a proliferative advantage, for example, by expressing a signaling complex as described, for example, in US 2021 / 0253652, which is hereby incorporated by reference in its entirety. In some embodiments, the signaling complex involves interleukin-2 receptor components and optionally involves 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 hereby incorporated by reference in its entirety. For example, each chimeric protein component of the complex can have one half of a rapamycin-binding complex as an extracellular domain, which is fused to one half of an intracellular signaling complex (e.g., an IL-2 signaling complex). Delivery of a nucleic acid encoding the signaling complex to a host cell permits intracellular signaling within the cell, which can be controlled by the presence of rapamycin or a rapamycin-related compound.

[0141] In still other embodiments, the invention generates T cells (e.g., CTLs, helper T cells, or Tregs) that express a chimeric antigen receptor. The cells can be effectively transduced by a vector such as, but not limited to, a retroviral or non-integrating viral vector (e.g., adenovirus, adeno-associated virus, integrase-deficient lentivirus, poxvirus) or a non-viral vector (e.g., plasmid vector, artificial chromosome) or an episomal or episomal hybrid vector that carries a CAR targeting a tumor antigen (e.g., CD19, CD38, CD33, CD47, CD20, etc.). The CAR is intended to enhance the ability of the cell to recognize, bind, and kill target cells (e.g., cancer or tumor cells). In some embodiments, the CAR enhances the ability of the cell to recognize tumor cells. In some embodiments, the CAR enhances the anti-tumor activity of the cell. In some embodiments, but not limited to, the CAR is a G protein-coupled receptor 87 (GPR87) CAR and a solute carrier family 7 member 11 (SLC7A11 (xCT)) CAR, a TNF receptor superfamily member 17 (BCMA) CAR, a CD30 CAR, a CD19 CAR, a CD22-CAR, a CD33 CAR, a CD133-CAR, a mesothelin-CAR, a CD70 CAR, a CD73 CAR, for example targeting the following tumors or tumor antigens:

[0142] (i) Human epidermal growth factor receptor 2 (HER2) - ovarian cancer, breast cancer, glioblastoma, colon cancer, osteosarcoma, and medulloblastoma;

[0143] (ii) Epidermal growth factor receptor (EGFR)-positive malignancies, such as non-small cell lung cancer, epithelial cancer, cholangiocarcinoma, and glioma;

[0144] (iii) Mesothelin - mesothelioma, ovarian cancer, and pancreatic adenocarcinoma;

[0145] (iv) Prostate-specific membrane antigen (PSMA) - prostate cancer;

[0146] (v) Carcinoembryonic antigen (CEA) - pancreatic adenocarcinoma, breast cancer, and colorectal cancer;

[0147] (vi) Glypican-3 - hepatocellular carcinoma;

[0148] (vii) Epidermal growth factor receptor variant III (EGFRvIII) - glioblastoma;

[0149] (viii) Disialoganglioside 2 (GD2) - neuroblastoma and melanoma;

[0150] (ix) Carbonic anhydrase IX (CAIX) - renal cell carcinoma;

[0151] (x) Interleukin-13Ra2 - glioma;

[0152] (xi) Fibroblast activation protein (FAP) - malignant pleural mesothelioma;

[0153] (xii) L1 cell adhesion molecule (L1-CAM) - neuroblastoma, melanoma, and ovarian cancer;

[0154] (xiii) Cancer antigen 125 (CA 125) - epithelial ovarian cancer;

[0155] (xiv) Cluster of differentiation 133 (CD 133) - glioblastoma, cholangiocarcinoma, and adenocarcinoma;

[0156] (xv) Cancer / testis antigen 1B (CTAG1B) - melanoma and ovarian cancer;

[0157] (xvi) Mucin 1 - seminal vesicle cancer;

[0158] (xvii) Folate receptor-a (FR-a) - ovarian cancer;

[0159] (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 to 4.

[0160] (xix) EGFRvIII - glioblastoma.

[0161] (xx) Claudin 18.2 - solid tumors, advanced gastric adenocarcinoma, pancreatic adenocarcinoma.

[0162] (xxi) Mesothelin - mesothelioma, metastatic pancreatic cancer, ovarian cancer, cervical cancer, lung cancer.

[0163] See, for example, Zhou Z et al., Chimeric antigen receptor T cells applied to solid tumors. Front Immunol. October 31, 2022; 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.

[0164] In some embodiments, the CAR comprises an intracellular domain from the Fcε receptor γ (FcεRIγ). However, in further contemplated embodiments, the CAR may also comprise the intracellular domain of the T cell receptor (TCR) CD3ζ (CD3ζ), alone or in combination with additional components from second - or third - generation CAR constructs (such as CD28, CD134, CD137, and / or ICOS).

[0165] In some embodiments, the present invention generates T cells that exhibit a similar degree of T cell activation and subsequent T cell - mediated cytotoxicity as non - immunocompatible T cells (e.g., a pan - T cell line and other T cells used as experimental controls for assessing T cell - mediated cytotoxicity). Compared to CD34+ - derived T cells, the T cells generated herein exhibit significantly superior performance in T cell - mediated cytotoxicity.

[0166] On the other hand, the present disclosure provides a cell composition comprising HLA - A neg , HLA - DPB1 neg and HLA - DQB1 negA T cell population (or its precursor, such as a progenitor T cell population). Despite such gene deletions and / or gene editing, the T cell composition still retains intact antigen presentation function and the ability to differentiate from precursors (as described herein) and into a more mature phenotype. The cell compositions of this aspect provide an advantage in terms of HLA matching in the recipient to avoid, for example, GVHD. In various embodiments, the T cell population is homozygous for both HLA-B and HLA-C. In some embodiments, the T cell population is homozygous for HLA-DRB1.

[0167] In some embodiments of this aspect, the T cell population is a T progenitor cell population. In various embodiments, the T progenitor cell population implants into the thymus, spleen, or secondary lymphoid organs after 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 can express a chimeric antigen receptor (CAR). The cell populations according to this aspect can be prepared according to other aspects of the present disclosure.

[0168] The present invention provides a cell population or a pharmaceutically acceptable composition thereof, which is as described herein or produced by the methods described herein. In some embodiments, the cell population is a progenitor T cell population that is capable of implanting into the thymus, spleen, or secondary lymphoid organs after administration to a subject in need. In other embodiments, the cell population is an α / β T cell population, a CAR-T cell population, a CTL population (which can express CAR), a helper T population, or a Treg population (each as described herein). In various embodiments, a composition for cell therapy is prepared, which comprises a cell population and a pharmaceutically acceptable excipient. The pharmaceutical composition can comprise at least about 10 2 cells, or at least about 10 3 、or at least about 10 4 、or at least about 10 5 、or at least about 10 6 、or at least about 10 7 、or at least about 10 8 cells, or at least about 10 9 cells、or at least about 10 10 cells、or at least about 10 11 cells、or at least about 10 12 cells、or at least about 10 13 cells、or at least about 10 14 cells. For example, in some embodiments, a pharmaceutical composition is administered, which comprises about 100,000 to about 400,000 cells per kilogram (e.g., about 200,000 cells / kg) of T progenitor cells. In other embodiments, at about 10 5from about 0 to about 5×10 5 cells (e.g., about 2.5×10 5 cells / kg), or about 10 6 to about 5×10 6 cells (e.g., about 2.5×10 6 cells / kg), or about 5×10 6 to about 10 7 cells (e.g., about 5×10 6 cells / kg), or about 10 7 to about 10 8 cells (e.g., about 5×10 7 cells / kg), or about 10 8 to about 10 9 cells (e.g., about 5×10 8 cells / kg), or about 10 9 to about 10 10 cells, or about 10 10 to about 10 11 or about 10 11 to about 10 12 cells, or about 10 12 to about 10 13 cells, or about 10 13 to about 10 14 cells are administered per kg of recipient body weight.

[0169] The cell compositions of the present disclosure may further comprise a pharmaceutically acceptable carrier or excipient suitable for intravenous infusion or other routes of administration, and the compositions may comprise a suitable cryoprotectant. Exemplary carriers are 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 one fluid ounce to one pint.

[0170] In some embodiments, the cell population is a Treg population useful for adoptive cell therapy, e.g., for a human subject suffering from a disorder selected from autoimmune or inflammatory disorders or diseases or graft-versus-host disease (GVHD). Additionally, various genetic disorders can affect the immune system, presenting as autoimmune or pro-inflammatory states. In some embodiments, the Treg population is CAR-T cells. In various embodiments, regulatory T cells can express tissue- or cell-specific CARs. That is, the cells can express a CAR that is specific for a target organ or tissue, 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 islets of Langerhans, e.g., for the treatment of type 1 diabetes or prediabetes.

[0171] In some embodiments, the cell population is derived from autologous cells, universal compatible donor cells, or HLA-modified or HLA-null cells (e.g., as described herein). That is, the cell population is generated from iPSCs prepared from recipient subject cells or from iPSCs prepared from donor cells (e.g., universal donor cells, HLA-matched cells, HLA-modified cells, or HLA-null cells).

[0172] In other aspects, the present invention provides a method of cell therapy, comprising administering to a human subject in need thereof the cell population described herein or a pharmaceutically acceptable composition thereof. In various embodiments, the methods described herein are used to treat blood (malignant and non-malignant), bone marrow, and immune diseases. In various embodiments, the human subject suffers from a disorder comprising one or more of the following: lymphopenia, cancer, infectious diseases (e.g., viral diseases such as HPV or HIV), immunodeficiency, autoimmune diseases, skeletal dysplasia, hemoglobinopathies, anemia, bone marrow failure syndromes, and genetic disorders (e.g., genetic disorders affecting the immune system).

[0173] In some embodiments, the subject suffers from cancer, such as a hematological malignancy or a solid tumor. In such embodiments, T progenitor cells or T cells with anti-tumor specificity (such as CTLs that recognize tumor antigens) are administered to the subject.

[0174] In some embodiments, the subject has a disorder selected from the following: acute myeloid leukemia; acute lymphoblastic leukemia; chronic myeloid leukemia; chronic lymphocytic leukemia; myeloproliferative disorders; myelodysplastic syndromes; multiple myeloma; non-Hodgkin lymphoma; Hodgkin 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 lymphohistiocytosis; congenital metabolic defects; severe congenital neutropenia; Shwachman-Diamond syndrome; Diamond-Blackfan anemia; and leukocyte adhesion deficiency. The T cells generated using the methods described herein are administered to the subject, for example, by intravenous infusion. In some embodiments, the method can be performed after a myeloablative, non-myeloablative, or immunotoxin-based (e.g., anti-c-Kit, anti-CD45, etc.) conditioning regimen.

[0175] In other embodiments, the invention provides a method for cell therapy, comprising administering to a human subject in need thereof a population of Treg cells or a pharmaceutically acceptable composition thereof as described herein. In various embodiments, the subject has an autoimmune disease, an alloimmune disease, or an inflammatory disease. In some embodiments, the subject is a tissue or organ transplant recipient, 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 developing 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 HSC, cornea, skin, heart valve, nerve, and / or vein. Kidneys, livers, and hearts are the most common transplanted organs. Corneal and musculoskeletal transplants are the most common transplanted tissues.

[0176] In some embodiments, the subject has an autoimmune disorder, and in some embodiments, the autoimmune disorder 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.

[0177] In some embodiments, the subject has an immune disorder, such as celiac disease, hyperimmunoglobulin E syndrome, and IPEX syndrome.

[0178] As used herein, the term "about" means ±10% of the relevant value.

[0179] Certain aspects and embodiments of the present disclosure are further described with reference to the following examples.

[0180] Example

[0181] Example 1 – ETV2 overexpression increases the yield of hematopoietic endothelial cells and enhances the formation of CD34+ cells during iPSC differentiation, but does not affect pluripotency.

[0182] 方法

[0183] iPSCs were developed from hCD34+ cells by episomal reprogramming known in the art and were substantially as described by 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 body and hematopoietic endothelial differentiation were substantially as described in the following references: R. Sugimura, et al., Haematopoietic 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).

[0184] Briefly, hiPSCs were dissociated and resuspended in a medium supplemented with L-glutamine, penicillin / streptomycin, ascorbic acid, human holo-transferrin, monothioglycerol, BMP4, and Y-27632. Next, the cells were seeded in 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 a medium containing SB431542, CHIR99021, bFGF, and BMP4. On day 4, the cell medium was replaced with a medium supplemented with VEGF and bFGF. On day 6, the cell medium was replaced with a medium supplemented with bFGF, VEGF, interleukin (IL)-6, IGF-1, IL-11, SCF, and EPO. The cells were maintained in an incubator with 5% CO2, 5% O2, and 95% humidity. To harvest CD34+ cells, the EBs were dissociated on day 8, the cells were filtered through a 70-μm filter, and the CD34+ cells were isolated by CD34 magnetic bead staining.

[0185] 结果

[0186] An adenoviral vector containing the ETV2 and GFP sequences under the control of the EF1A promoter was used to transduce induced pluripotent stem cells (iPSCs). After transduction, approximately 45% of the iPSC cultures were observed to be GFP-positive, confirming ETV2 overexpression (ETV2-OE). Further observations showed that ETV2-OE in iPSC cells retained the pluripotent characteristics of iPSCs, as demonstrated by the expression of the stem cell marker TRA-1-60 ( 图1 ). 图1 FACS plots showing the transduction efficiency of iPSCs overexpressing the ETV2 and GFP sequences with an adenoviral vector.

[0187] Next, ETV2-OE-iPSCs (as well as control iPSCs transduced with a vector carrying the GFP sequence but not ETV2) were differentiated into embryoid bodies and subsequently into hematopoietic endothelial cells (Strugeon et al., 2014). The results showed that overexpression of ETV2 promoted the formation of hematopoietic endothelial cells, as demonstrated by the expression of CD235a - within the CD34 + and CD31 + markers ( 图2 ). Specifically, 图2 representative flow cytometric analysis of hematopoietic endothelial cells (defined here as CD235a-CD34+CD31+) is shown, and relative quantification indicates that ETV2-OE enhances the formation of hematopoietic endothelial cells compared to the control.

[0188] In addition, the results showed that ETV2-OE enhanced the formation of CD34 + cells ([ 图3 图3 ). 图3 Representative flow cytometry analysis showing CD34+ cells, and relative quantification indicated that ETV2-OE enhanced the formation of CD34+ cells.

[0189] Overall, these data indicate that overexpression of ETV2 in iPSCs does not affect their pluripotency and promotes their ability to undergo hematopoietic endothelial and hematopoietic differentiation.

[0190] Example 2 - iPSC-derived HSCs generated using Piezo1 activation undergo T cell differentiation similar to bone marrow-derived HSCs.

[0191] 方法

[0192] To analyze EHT, EB-derived CD34+ cells were suspended in a 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 wells for approximately 4 to 18 hours (by visual inspection), Yoda1 was added to the cultures for some experiments. After 4 to 7 days, the cells were collected for analysis.

[0193] iPSCs were differentiated into embryoid bodies for 8 days. On day 8, CD34+ cells were harvested from iPSC-derived embryoid bodies and cultured for an additional 5 to 7 days to induce the endothelial-to-hematopoietic cell (EHT) transition (with or without Yoda1). Then, CD34+ cells were harvested from the EHT cultures between days 5 and 7 for further hematopoietic lineage differentiation and analysis.

[0194] CD34+ cells harvested from the EHT cultures between days 5 and 7 (or a total of days 13 to 21 from iPSC differentiation) were seeded into 48-well plates pre-coated with rhDL4 and recombinant human fibrin fragments. T lineage differentiation was induced in a medium containing aMEM, FBS, ITS-G, 2BME, ascorbic acid-2-phosphate, Glutamax, rhSCF, rhTPO, rhIL7, FLT3L, rhSDF-1a, and SB203580.

[0195] On days 2 to 6, 80% of the medium was replaced every other day. On D7, the cells were transferred to a new coated plate and the presence of pro-T cells (CD34+CD7+CD5+ / -) was analyzed.

[0196] From day 8 to day 13, replace 80% of the culture medium every other day. On D14, transfer 100,000 cells / well to a new coated plate and analyze the cells for the presence of pre-T cells (CD34-CD7+CD5+ / -).

[0197] From day 15 to day 20, replace 80% of the culture medium every other day. Harvest the cells on day 21 and analyze the expression of CD3, CD4, CD8, CD5, CD7, and TCRab (as a surrogate for T cells) in the cells via FACS, and / or activate with CD3 / CD28 beads to evaluate their functional characteristics.

[0198] After 21 days of differentiation, collect the cells and re-seed them at approximately 80,000 cells per well into a new 96-well culture plate in RPMI 1640 (without L-glutamine; without phenol red) plus FBS, L-glutamine, IL-2, and then activate with 1:1 CD3 / CD28 beads. After activating with CD3 / CD28 beads for 72 hours, analyze the expression of CD3, CD69, and CD25 in the cells by FACS and analyze the IFN-γ expression using RT-qPCR. Analyze the supernatant by ELISA.

[0199] 结果

[0200] 图4A and 图4B showed that iPSC-derived HSCs from EHT of CD34+ cells from differentiated iPSCs (e.g., including Piezo1 activation in this example) undergo pre-T cell differentiation similar to bone marrow (BM)-HSCs. In addition, Figure 5A and Figure Figure 5B showed that iPSC-derived HSCs generated from EHT of CD34+ cells from differentiated iPSCs (involving Piezo1 activation in this example) undergo T cell differentiation and can be activated by CD3 / CD28 beads similar to BM-HSCs. Figure 6 showed that iPSC-derived HSCs (generated with Piezo1 activation in this example) can differentiate into functional T cells, as confirmed by the expression of INFγ after stimulation with CD3 / CD28 beads. Collectively, these results demonstrate that iPSC-derived HSCs (i.e., from EHT of CD34+ cells from differentiated iPSCs) enhance the ability of HSCs to further differentiate into progenitor T cells and functional T cells in vitro. Figure 6 The experiments shown involved the activation of Piezo1 during HSC formation.

[0201] Figure 7Shown is that HSCs generated according to the present disclosure (labeled D8+7iPSC-CD34+) successfully differentiated into CD4+CD8+ (“double positive”) T cells and TCRα / β T cells. The methods of the present disclosure are significantly superior to bone marrow CD34+ cells in T cell maturation. Figure 7 Results are shown during HSC formation with (“+Y”) and without (“-Y”) Yoda1.

[0202] Figure 8 Shown is that HSCs generated according to the present disclosure (D8+7iPSC-CD34+ cells (+ or -Yoda1)) successfully rearranged the TCR and were superior to bone marrow CD34+ cells. Shown are iPSC and EB negative controls, peripheral blood T cells as positive controls, T cells generated from BM CD34+ cells, and T cells generated according to the present disclosure with or without Yoda1.

[0203] Example 3 – Assessment of off-target editing in HLA-disrupted HSCs

[0204] HLA typing of triple-knockout (HLA-edited) HSC clones was performed to examine unwanted editing and to ensure that no major editing events, such as deletions, occurred in other regions of chromosome 6. Sequencing methods and analyses were performed to evaluate the extent of gRNA off-target activity, and gRNAs representing a low risk of affecting non-target HLA genes were selected.

[0205] Sequencing was performed using in situ break labeling in fixed and permeabilized cells by ligating full-length P5 sequencing adapters to ends-prepared DSBs. Genomic DNA was extracted, fragmented, ends-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). Subsequently, DNA sequencing of the DNA library enriched for DNA-labeled fragments was performed, removing all foreign, non-functional DNA. Since library preparation did not contain PCR, each sequencing read obtained corresponded to a single labeled DSB end from a cell. This produced DNA break reads enabling direct detection and quantification of genomic DSBs by sequencing without the need for error correction and enabling the generation of a clear list of off-target mutations.

[0206] Table 1 below summarizes the editing strategy results for two representative HLA-edited clones relative to wild-type cells (gHSCs).

[0207] Table 1: Clonal HSC HLA knockout.

[0208]

[0209] Table 2 provides non-limiting examples of gRNAs used in the experiments, which can be used to knock out the expression of the designated HLA genes.

[0210] Table 2: Exemplary gRNA sequences

[0211]

[0212]

[0213] The results showed that the editing strategy successfully and selectively targeted the HLA-A, DPB1, and DQB1 genes without affecting other HLA genes or introducing major deletions elsewhere.

[0214] These results were confirmed by phenotypic analysis of HLA-edited clones by FACS and immunofluorescence. As Figure 9A and 9B shown, the overall expression of HLA class I molecules in HLA-edited cells was tested positive compared to the overall expression of HLA class I molecules in wild-type cells (gHSCs). The specific expression of HLA-A by immunofluorescence confirmed that HLA-A was not expressed in HLA-edited cells, confirming the finding that the gene editing strategy only successfully deleted the HLA-A gene. Specifically, Figure 9A showed that HLA-edited cells were positive for class I-like HLA to the same extent as wild-type cells (i.e., gHSCs). This result indicates that although HLA-A is deleted, other class I molecules such as HLA-B and C are expressed and are not affected by the gene editing strategy.

[0215] To confirm the deletion of the HLA-A gene, the specific expression of HLA-A was analyzed by immunofluorescence. As Figure 9B can be seen, HLA-A was not expressed in HLA-edited clones, indicating that the gene editing strategy was efficient only in specifically deleting the HLA-A gene. Such retention of all class I expression and HLA-A deletion would facilitate patient matching while avoiding NK cell-mediated rejection.

[0216] Example 4 - Evaluation of the pluripotency and immunocompatibility of HLA-edited HSCs

[0217] Evaluate the ability of HLA-edited cells to retain pluripotency. As Figure 10 shown, immunofluorescence evaluation of HLA-edited iPSC clones indicated that they maintained trilineage differentiation, where ectodermal differentiation was indicated by nestin-488 and PAX6-594 staining, mesodermal differentiation was indicated by GATA-488 staining, and endodermal differentiation was indicated by CXCR4-488 and FOX2A-594 staining.

[0218] HLA class I molecules are expressed on the surface of all nucleated cells, and if the HLA class I molecules are mismatched between the donor and the recipient, the cells can be recognized and killed by CD8+ T cells. In addition, HLA mismatch may lead to cytokine release syndrome (CRS) and graft-versus-host disease (GVHD). In contrast, complete deletion of HLA-I molecules by B2M KO will make the cells targets of NK cell-mediated cytotoxicity. Retaining all class I expression and deletion of HLA-A can facilitate patient matching while preventing NK cell-mediated rejection. Therefore, the immunocompatibility of HLA-edited HSCs was tested by co-culture with peripheral blood mononuclear cells (PBMCs) to evaluate whether immune cells would reject the grafts of HLA-edited and wild-type HSCs (gHSCs).

[0219] Wild-type (gHSCs) and HLA-edited HSCs were co-cultured with PBMCs that were matched for HLA-B and HLA-C markers but mismatched for HLA-A. B2M KO HSCs lacking HLA class I molecule expression and CIITA KO HSCs lacking HLA class II molecule expression were used as controls to compare the degrees of cytotoxicity mediated by HLA-null and HLA-mismatched PBMCs, respectively. Figure 11 Results of PBMC-mediated cytotoxicity assays in co-cultures measured by Annexin V staining are shown. The results show that deletion of HLA-A in HLA-edited HSCs protects the cells from PBMC-mediated cytotoxicity, while WT, B2M KO, and CIITA KO are vulnerable to PBMC-mediated cytotoxicity. HSCs co-cultured with sorted CD8+ T cells from the same PBMC donor protect HLA-edited and B2M KO HSCs from CD8+ T cell cytotoxicity. In contrast, HSCs co-cultured with sorted NK cells protect only WT and HLA-edited cells from NK cell-mediated cytotoxicity.

[0220] In summary, the immunocompatibility results show that CD8+ T cells present in the PBMC samples are responsible for killing cells mismatched for HLA molecules (WT) and CIITA KO, while NK cells present in the PBMCs are responsible for killing HLA-null cells (B2M KO). However, HLA-edited HSCs are protected from CD8+ T cell-mediated cytotoxicity (because the mismatched HLA-A has been knocked out) and from NK cell-mediated cytotoxicity (because the expression of HLA class I molecules is largely retained).

[0221] Example 5 - Evaluation of the in vivo engraftment potential of HLA-edited HSCs

[0222] To evaluate the transplantation potential of HLA-edited HSCs, the in vivo engraftment ability of the cells was assessed by competitive transplantation against WT HSCs. Equal proportions of mCherry HLA-edited HSCs and wild-type HSCs (gHSCs) were mixed and transplanted into mice, and bone marrow (BM) and peripheral blood samples were recovered and evaluated by FACS from the mice to compare the relative amounts of each cell type present in the samples. As Figure 12 shown, both HLA-edited HSCs and WT HSCs contributed to roughly equal engraftment in BM and peripheral blood samples. These results confirm that HLA-edited HSCs (prepared according to the present disclosure) are comparable to WT HSCs in terms of their transplantation and reconstitution potential. Thus, the properties of WT HSCs are expected to be consistent with those of the HLA-edited HSCs of the present disclosure for generating T cell lineages.

[0223] Example 6 - Differentiation of HLA-Edited HSCs into CD4+ / CD8+ T Cells

[0224] Antigen-presenting cells (APCs) present antigens to helper CD4+ T cells via HLA-II molecules. Activation of helper CD4+ T cells promotes the generation of antigen-specific CD8+ T cells, which further develop into antigen-specific CTLs. Similarly, HLA class I molecules are expressed on the surface of all nucleated cells and display peptide fragments of intracellular proteins to CD8+ CTLs. After recognizing the HLA-I-peptide complex expressed on the cell surface, CTLs induce cytotoxic killing of target (infected) cells. Thus, studies were conducted to determine whether the deletion of HLA-A affects class I peptide presentation by edited HSCs. As Figure 13A and 13B shown, immunopeptidome analysis revealed that the deletion of HLA-A does not affect overall class I peptide presentation. When compared to wild-type HSCs (gHSCs), HLA-A-edited cells showed comparable peptide and protein presentation. In addition, as Figure 14A and 14B shown, the deletion of HLA-DQB1 and HLA-DPB1 does not affect overall class II peptide presentation by macrophages differentiated from HSCs. Collectively, these data indicate that these cells (and the lineages derived from them) retain their ability to present a broad spectrum of class I and class II peptides despite the deletion of HLA-A, HLA-DQ, and HLA-DP molecules.

[0225] Example 7 - In Vivo Testing of Antigen-Mediated Immune Responses.

[0226] Figure 15It is a schematic diagram of delayed-type hypersensitivity, showing the sensitization and elicitation phases of antigen presentation. Briefly, after 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 the peptide-MHC on the antigen-presenting cell (APC). When challenged by antigen, CD4+ helper T cells are activated, and cytokines recruit macrophages and other immune cells, which induces tissue swelling.

[0227] The delayed-type hypersensitivity assay was performed on transplanted mice. Specifically, the mice were sensitized by subcutaneous injection of sheep red blood cells as the antigen. If the mice have a functional immune system, the APCs process the antigen and present the peptide antigen to CD4+ T cells. Next, the challenge was performed by subcutaneous injection of the same antigen into the left paw of the mice. At this time, the T cells are activated and secrete cytokines, which recruit macrophages and other immune cells at the antigen injection site, thus causing tissue swelling. In this assay, a functional immune system results in swelling of the left paw, as measured with a micrometer caliper.

[0228] As Figure 16A and 16B can be seen, the control (non-transplanted) mice did not show swelling of the left paw due to their immunodeficiency. In contrast, the mice transplanted with cord blood CD34+ cells showed tissue swelling, and the diameter of their left paw doubled. Similar immune system responses were found in mice transplanted with both WT (unedited HSCs) and HLA-edited HSCs (HLA-edited).

[0229] Example 8 - Evaluation of various T cells and pro-T cells derived from HSCs

[0230] Next, the ability of various T cells and pro-T cells derived from HSCs to differentiate into mature T cells was tested. After a 35-day differentiation period, the presence of CD4+, CD8+, and αβ+ T cell populations in T cell precursors and their derivatives was evaluated by cell sorting. As Figure 17 shown, pro-T cells differentiated more efficiently into CD4+, CD8+, and αβ+ T cells than CD34+ cells derived from bone marrow (BM) and CD34+ cells derived from embryoid bodies (EB).

[0231] Next, to test the functional characteristics, each T cell population was co-cultured with a CD19+ lymphoma cell line and an anti-CD3 / CD-19 bispecific antibody. In this experimental model, the bispecific antibody acts simultaneously on the CD3 receptor on the T cells and the CD19 cell surface receptor on the lymphoma cells, thus triggering T cell activation. The degree of activation was evaluated by measuring the subsequent T cell-mediated cytotoxicity compared to a pan-T cell positive control. As Figure 18As shown, compared with both BM CD34+ T cells and EB CD34+ T cells, T cells showed statistically significantly superior characteristics in terms of cytotoxicity.

[0232] Since the entire differentiation process of T cells takes up to 35 days, transduction experiments were conducted to test whether the time required for differentiating HSCs could be shortened. The primary T cells were cultured in activation medium (for approximately 7 days) to improve the transduction efficiency of the cells. Next, the cells were transduced with lentiviral (LV) particles encoding the anti-CD19 CAR transgene. The cells were cultured for another 4 to 5 days (for a total of 12 days), and their maturation and killing capabilities were evaluated. As Figure 19 shown, primary T cells derived from HSCs could be efficiently transduced, with over 80% of the cells expressing CD19 CAR, as confirmed by cell sorting.

[0233] Next, the ability of primary T cells to be effectively matured into CD4+ / CD8+ T cells via CAR transduction was evaluated. The primary T cells were transduced with anti-CD19 CAR together with CD34+ cells derived from bone marrow (BM) and CD34+ cells derived from embryoid bodies (EB) (as well as pan T cells as a positive control). T cell subsets were screened by cell sorting to determine the presence of CD4 or CD8 cell surface marker expression. As Figure 20 shown, the results indicated that CAR transduction promoted T cell maturation, and an increase in T cell maturity was observed in primary T cells compared with CD34+ cells derived from bone marrow (BM) and CD34+ cells derived from embryoid bodies (EB).

[0234] The ability of LV-transduced primary T cells to function via anti-CD19 receptor-mediated cytotoxicity was evaluated. T cell subsets were co-cultured with a CD19+ leukemia cell line (NALM6) expressing a luciferase reporter gene (Luc+) to measure the degree of T cell-mediated cell lysis, with untransduced cells and pan T cells serving as negative and positive controls, respectively. As Figure 21 shown, CAR T cells functioned efficiently via T cell-mediated lysis, indicating that the degree of cytotoxicity was comparable to that of CAR-primary T cells derived from BM CD34+ cells. In contrast, CAR T cells derived from EB CD34+ cells did not show the ability to kill target cells.

[0235] Example 9 – Evaluation of the characteristics of HSCs developing into primary T cells.

[0236] The ability of HSCs to develop into primary T cells was evaluated by measuring the CD34-CD7+ markers on primary T cells. As Figure 22As shown, FACS analysis showed that HSCs generated according to the present disclosure successfully differentiated into CD34-CD7+ pro-T cells compared to bone marrow-derived CD34+ cells or EB-derived CD34+ cells.

[0237] Next, the expression of T cell-specific transcription factors and thymic engraftment molecules was measured. Figure 23A It was shown that the expression of TCF7 increased in the HSC-derived pro-T cells of the present disclosure, and Figure 23B it was shown that the expression of CCR7 increased. Figure 24A It was shown that the HSC-derived pro-T cells engrafted and differentiated in the thymus. Figure 24B Shown is FACS analysis of the CD3 cell population gated on the CD45+ cell population, which showed that the HSC-derived pro-T cells had excellent engraftment and differentiation potential in the thymus. The pro-T cells of this example were prepared from HSCs using Piezo1 activation as described above.

[0238] As Figure 25 shown, the in vitro activation of HSC-derived T cells was also measured. Figure 25 The top panel shows FACS analysis of activated T cells from different sources, including HSCs of the present disclosure (e.g., prepared using Piezo1 activation). T cells prepared from the HSCs of the present disclosure exhibited comparable or better activation, as measured by increased CD107 expression. The lower panel shows Dynabeads activation, in which the activated T cells expressed inflammatory cytokines. According to the present disclosure, HSC-derived T cells (e.g., prepared using Piezo1 activation) expressed higher levels of inflammatory cytokines, as exemplified by the expression levels of TNF-α and interferon γ.

[0239] Example 10 – Evaluate the properties of CCR5 knockout HSCs developing into pro-T cells.

[0240] To determine whether CCR5 knockout (CCR5-KO) HSCs differentiated into pro-T cells as well as their wild-type counterparts from which they were derived (i.e., the HSCs of the present disclosure), a study was conducted in which the CD34, CD7, and CD5 expression of HSCs and CCR5-KO was measured. As Figure 26 can be seen, comparable to bone marrow-derived CD34+ cells, the HSCs successfully differentiated into CD34+CD7+CD5+ pro-T cells. Similarly, the CCR5-KO also successfully differentiated into CD34+CD7+CD5+ pro-T cells like its wild-type counterpart.

[0241] Next, the properties of CCR5 knockout HSCs differentiating into double-positive (CD4+CD8+) T cells were evaluated, and whether they were comparable to the HSCs from which they were derived. As Figure 27As can be seen, when compared to the wild-type counterparts from which the HSCs are derived (i.e., the HSCs of the present disclosure), the CCR5-knockout HSCs are significantly differentiated into double-positive (CD4+CD8+) T cells.

[0242] References

[0243] 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).

[0244] 2. Brauer, P.M., Singh, J., Xhiku, S. & - Pflücker, J.C. T Cell Genesis: In Vitro Veritas Est? Trends Immunol 37, 889–901 (2016).

[0245] 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).

[0246] 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).

[0247] 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).

[0248] 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).

[0249] 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).

[0250] 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).

[0251] 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).

[0252] 10. Guo, R. et al. Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors. Cell Research 30, 21–33 (2020).

[0253] 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).

[0254] 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. A method for preparing a T cell population or its progenitor cells, the method comprising: Enriching CD34+ cells from a population of differentiated pluripotent stem cells to prepare a CD34+-enriched population; Inducing the CD34+-enriched cell population to undergo an endothelial-to-hematopoietic cell transition for at least two days but not more than 12 days to prepare a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem cell progenitors (HSPCs); and Differentiating the population comprising HSCs and / or HSPCs into a progenitor T cell population or a T cell population.

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

3. The method according to claim 1 or 2, wherein the population comprising HSCs and / or HSPCs differentiates into a population comprising one or more of T progenitors, precursor T cells, double-positive T cells, single-positive T cells, and regulatory T cells.

4. The method according to claim 3, wherein the population comprising HSCs and / or HSPCs differentiates into a population comprising T progenitors.

5. The method according to claim 4, wherein T progenitors are recovered from the culture.

6. The method according to claim 4 or 5, wherein the T progenitors further differentiate 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 according to claim 6, wherein the T progenitors further differentiate into a population comprising double-positive T cells.

8. The method according to claim 6, wherein the T progenitors further differentiate into a population comprising regulatory T cells.

9. The method according to claim 3, wherein the population comprising HSCs and / or HSPCs differentiates into a population comprising double-positive T cells and / or single-positive T cells.

10. The method according to claim 9, wherein the double-positive T cells and / or single-positive T cells differentiate into regulatory T cells, optionally including the step of recovering the double-positive cells and / or single-positive cells from the culture before differentiating into regulatory T cells.

11. The method according to claim 3, wherein the population comprising HSCs and / or HSPCs differentiates into a population comprising regulatory T cells.

12. The method according to 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 human primary tissue.

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

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

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

16. The method according to claim 12 or 13, wherein the iPSC is gene - edited to delete one or more class I HLA genes, delete one or more class II genes, and / or delete one or more genes that govern the ability of HLA or MHC expression or presentation.

17. The method according to claim 16, wherein the iPSC contains a deletion of HLA - A.

18. The method according to claim 16 or 17, wherein the iPSC contains a deletion of HLA - DPB1 and / or HLA - DQB1.

19. The method according to claim 16, wherein the one or more genes that govern the ability of HLA or MHC expression or presentation are β2 - microglobulin and / or CIITA.

20. The method according to any one of claims 14 to 18, wherein the iPSC contains a deletion of HLA - A, is homozygous for both HLA - B and HLA - C, contains deletions of HLA - DPB1 and HLA - DQB1, and is homozygous for HLA - DRB1.

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

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

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

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

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

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

27. The method according to claim 26, wherein the induction of EHT includes applying cyclic stretching to the CD34+ - enriched cells.

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

29. The method according to claim 26, wherein the induction of endothelial - to - hematopoietic cell transition includes Piezo1 activation.

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

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

32. The method according to claim 31, wherein the Trpv4 activation is carried out by contacting the CD34+ enriched cells with one or more Trpv4 agonists, which are optionally selected from GSK1016790A, 4α-PDD or analogs or derivatives thereof.

33. The method according to any one of claims 1 to 32, wherein the HSC and / or HSPC population or a fraction thereof is cultured with a part or all of the Notch ligands to generate a population comprising a CD7+ progenitor T cell or a derivative cell population.

34. The method according to claim 33, wherein the CD7+ progenitor T cell does not express CD34 or expresses a reduced level of CD34 compared to the HSC population.

35. The method according to claim 33 or 34, wherein the CD7+ progenitor T cell expresses CD5.

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

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

38. The method according to 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. The method according to any one of claims 33 to 38, wherein the Notch ligand is incorporated with an extracellular matrix component, which is optionally selected from fibronectin, recombinant human fibrin fragment and laminin, derivatives or analogs thereof and / or combinations thereof.

40. The method according to claim 39, wherein the Notch ligand and / or the extracellular matrix component is embedded in an inert material providing 3D culture conditions, which is optionally selected from cellulose, alginate and combinations thereof.

41. The method according to claims 39 to 40, wherein the Notch ligand, the extracellular matrix component or a combination thereof is contacted with culture conditions providing a topographical pattern and / or roughness for the cells.

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

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

44. The method according to claim 43, wherein the T cell expresses at least one of CD3 and a T cell receptor.

45. The method according to any one of claims 33 to 44, which comprises generating regulatory T cells from said progenitor T cells or αβCD3+ and / or CD4+ T cells produced therefrom.

46. The method according to claim 45, wherein said regulatory T cells express CD8 and / or CD4.

47. The method according to claims 45 and 46, wherein differentiating into Tregs comprises modifying Treg precursors to express FOXP3.

48. The method according to claim 47, wherein differentiating into Tregs comprises modifying Treg precursors to constitutively express FOXP3.

49. The method according to claim 47 or 48, wherein the cells modified to express FOXP3 are iPSCs, CD34+ cells before EHT, CD34+ cells after EHT, progenitor T cells, CD4+ / CD8+ T cells, CD8+ αβ T cells or CD4+ αβ T cells.

50. The method according to any one of claims 1 to 49, wherein said T cell population expresses a chimeric antigen receptor (CAR) or optionally a T cell receptor with cell or tissue specificity.

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

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

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

54. The cell composition according to any one of claims 51 to 53, wherein said T cell population is a T progenitor cell population.

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

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

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

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

59. The cell composition according to any one of claims 51 to 58, which comprises at least about 10 4 cells.

60. The cell composition according to any one of claims 51 to 58, which comprises 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 cells.

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

62. The method according to claim 61, wherein said 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. The method according to claim 61, wherein said human subject has cancer, which is optionally a hematological malignancy or a solid tumor, and said T cell population comprises progenitor T cells, CD8+ cytotoxic T cells or CD4+ helper T cells.

64. The method according to any one of claims 61 to 63, wherein the T cell population or T cell progenitor cells comprise a CAR targeting a tumor-associated antigen.

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

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

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

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

69. The method according to claim 68, wherein the autoimmune disorder 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. The method according to claim 61, wherein the human subject has an immune disorder selected from celiac disease, hyperimmunoglobulin E syndrome and IPEX syndrome and the T cell population comprises regulatory T cells.

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

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

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