Modified stem cells matched to patient HLA type

Abstract

This disclosure relates to stem cells lacking endogenous expression of class I and / or class II human leukocyte antigen (HLA) genes and optionally comprising exogenous HLA-A, HLA-B, and / or HLA-C genes matched to those of a patient in need thereof. In some embodiments, these stem cells also comprise one or more gene edits, including stem cells that are protected from autoimmune attack or that express a chimeric antigen receptor, as well as stem cells for other treatments. Also disclosed are methods of preparing patient-matched stem cells.

Description

MODIFI ED STEM CELLS MATCH ED TO PATIENT HLA TYPECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 637,950, filed April 24, 2024, the disclosure of which is hereby incorporated by reference in its entirety.ELECTRONIC SEQUENCE LISTING

[0002] This application contains a Sequence Listing in computer readable form entitled “01123-0016-00PCT”, created April 16, 2025, having a size of 49,244 bytes, which is incorporated by reference herein.DESCRIPTIONFIELD

[0003] This disclosure relates to stem cells lacking endogenous expression of class I and / or class II human leukocyte antigen (HLA) genes and optionally comprising exogenous HLA-A, HLA-B, and / or HLA-C genes matched to those of a patient in need thereof. In some embodiments, these stem cells also comprise one or more gene edits, including stem cells that are protected from autoimmune attack or that express a chimeric antigen receptor, as well as stem cells for other treatments. Also disclosed are methods of preparing patient-matched stem cells.BACKGROUND

[0004] Therapy with stem cells has been used to treat a variety of different diseases, such as blood-related cancers including leukemia and lymphoma. Further, stem cells can be differentiated into cells for treatment, such as chimeric antigen receptor (CAR) T cells for cancer treatment or pancreatic beta cells for treatment of type I diabetes.

[0005] However, stem cells need to be matched to the human leukocyte antigen (HLA) profile of the patient to avoid attack by the patient’s immune system, and it remains difficult and expensive to prepare stem cells matched to a specific patient. For example, stem cells are commonly harvested from the patient for treatment and then modified in vitro before being administered back to the same patient (i.e., autologous stem cells), which is expensive and time-consuming. While means of preparing hypoallergenic stem cells lacking HLAprotein expression are known, these cells may be attacked by natural killer cells after transplantation. Further, stem cells lacking HLA proteins could avoid normal immunosurveillance mechanisms, and thus any mutation in these cells could lead to a tumor that the patient’s immune system is unable to detect and attack. The improved strategy described herein allows for normal antigen presentation on class I HLA molecules that are matched to a patient’s HLA profile using off-the-shelf stem cells to avoid the need for preparation of autologous stem cells.

[0006] The methods and cells described herein keep the class I HLA polymorphism intact and matched to the patients, while also allowing quick and cost-efficient preparation of patient-matched stem cells for treatment as compared to autologous stem cell generation. The present approach is superior to generating autologous stem cells by reprograming in term of cost and time, as “off-the-shelf’ stem cells can be used to prepare patient-matched cells for treatment of any patient. The present approach is also safer than generating hypoimmunogenic cells (such as those lacking HLA-A, B, and C) since the present cells would still be patient to immunosurveillance.

[0007] An exemplary disease that may be treated with the present patient-matched stem cells is Type I diabetes (T1D). T1D is caused by immune-mediated destruction of insulin-producing pancreatic beta cells. The loss of pancreatic beta cells leads to insulin deficiency that can only be treated by daily glucose monitoring and insulin injections, a treatment T1D patients depend on for survival for the rest of their lives. Currently there is no cure for T1D. To cure T1D, one has to replenish lost pancreatic beta cell mass in the patients. Several groups have developed effective differentiation protocols to generate insulinproducing beta-like cells from human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSC) (see Odorico et al., Transplantation. 2018; 102(8): 1223-9). These advances have raised the prospect of replacing lost beta cells in T1D patients with autologous stem cell-derived beta-like cells, a strategy with the potential to provide an unlimited supply of cells while circumventing issues of transplant rejection.

[0008] However, large hurdles to using stem cells to treat diabetes and other conditions persist. For example, without immune suppression, recurrent autoimmunity in T1D patients will rapidly destroy transplanted beta cells. Therefore, to successfully cure T1D, the autoimmunity needs to be eliminated, or transplanted beta cells have to be protected against autoimmune destruction.

[0009] There are several strategies currently being explored to protect transplanted beta cells. First, transplanted beta cells could be protected by a physical barrier (encapsulation), which allows glucose and insulin to diffuse across the capsule wall freely, but the physical contact between transplanted beta cells and recipient immune cells is prevented (Vegas AJ et al., Nat Med. 2016;22(3):306-l l). The disadvantages of this strategy include poor survival of transplanted beta cells in the capsule (due to lack of direct blood supply) and delayed glucose sensing and insulin secretion. Secondly, several research groups generated hypoimmunogenic stem cells where both class I and II HLA molecules are abolished / deleted, and HLA-E, PD-L1, or CD47 transgenes are introduced in the cells to prevent attack from natural killer (NK) cells, allo-reactive T cells, or macrophages (Han X et al., Proc Natl Acad Sci USA. 2019; 116(21): 10441-6). These hypoimmunogenic stem cells can then be differentiated into pancreatic beta-like cells and transplanted into T1D patients. This strategy and approach might shield transplanted beta cells from autoimmune and allo- immune attack, but it also prevents the transplanted beta cells from being recognized by all immune surveillance. If these hypoimmunogenic cells undergo spontaneous tumorigenic mutations or pathogen infection, the immune system will have no way to provide defense.

[0010] In T1D, pancreatic beta cells are also at fault in initiation of the autoimmune attack because they might have certain intrinsic stress, which leads to neo-antigen production and presentation, causing autoimmune recognition and destruction. Previously, through a genome-wide CRISPR screen, 11 gene mutants were identified capable of protecting beta cells from autoimmune killing (Cai EP et al. Nat Metab. 2020;2(9):934-45). Mutation of RNLS, a human T1D GWAS gene, reduces intrinsic stress in both human and mouse beta cells, and allows beta cells to survival in diabetic NOD mice (4). In addition, an FDA approved drug, pargyline, was found to target RNLS and provide the same protection against autoimmunity as RNLS mutation (4). The mutation of at least two other genes discovered from the CRISPR screen, Hivep2 and Zbed3, is able to protect beta cells from autoimmunity similarly to RNLS mutation, and it is possible that combined mutation of multiple genes here will provide even stronger protection. Interestingly, all these mutations only eliminate the autoimmune recognition and killing, but do not affect general immune response, as class I MHC molecule expression and alloimmune response are still unchanged. Described herein are patient-matched stem cells modified with protective gene edits, wherein the differentiated beta-like cells may not trigger an autoimmune reaction by the patient, but the intact class IMHC antigen presentation on the differentiated beta-like cells still allows them to be protected from tumorigenesis and pathogen invasion.

[0011] Using similar methods, described herein are patient-matched stem cells that can be prepared expressing chimeric antigen receptors (CARs) and differentiated into T cells for the treatment of cancer.SUMMARY

[0012] In accordance with the description, described herein are human stem cells (such as iPSC cells) that lack endogenous class I and II HLA and can maintained / stored for later use. These “off the shelf’ stem cells can be later modified to express exogenous class I and II HLA matched to a given patient. In some embodiments, a recombinase-mediated cassette exchange (RMCE) element is inserted into the genome at the AAVS1 or Pansio-1 locus allows for expression of exogenous class I and II HLA. For a specific patient, his / her / their class I HLA alleles can be typed, and an expression construct containing multiple or all of their HLA genes can be assembled in vitro and shuttled into the RMCE locus for expression. DNA for various HLA alleles can be synthesized and banked for easy access, allowing for “off-the-shelf’ preparation of HLA-matched stem cells for administered to a patient.

[0013] In one representative example, for T1D patients, the RMCE-ready HLA7' stem cells can be further modified with the beta cell protective gene edits to prevent autoimmune attack. When transplanted, these patient-matched stem cells could lead to release of insulin from the transplanted cells while avoiding an autoimmune response from the patient’s immune system, while still allowing for immune surveillance of the transplanted cells. Similarly, differentiated T cells expressing CARs can be prepared from patient-matched stem cells described herein.

[0014] Each of the following embodiments are provided herein:

[0015] Embodiment 1. A method of preparing a patient-matched stem cell comprising (a) modifying a stem cell to lack expression of class I and class II human leukocyte antigen (HLA) genes; (b) storing said modified stem cell; (c) retrieving said modified stem cell after storage; (d) inserting HLA-A, HLA-B, and / or HLA-C genes matched to those of a patient in need thereof, thereby preparing a patient-matched stem cell.

[0016] Embodiment 2. A stem cell produced by the method of embodiment 1.

[0017] Embodiment 3. A patient-matched stem cell, wherein the stem cell (a) is not autologous; (b) lacks endogenous expression of class I and class II HLA genes; and (c) comprises exogenous HLA-A, HLA-B, and / or HLA-C genes matched to those of a patient in need thereof.

[0018] Embodiment 4. The method of embodiment 1 or stem cell of embodiment 2 or 3, wherein the exogenous HLA-A, HLA-B, and / or HLA-C genes comprise one allele or two alleles for each gene.

[0019] Embodiment 5. The method or stem cell of any one of embodiments 1-4, wherein the stem cell does not trigger attack by natural killer cells.

[0020] Embodiment 6. The method or stem cell of any one of embodiments 1-5, wherein the HLA-A, HLA-B, and / or HLA-C genes are comprised in a cassette, wherein the cassette optionally further comprises (a) a promoter, optionally wherein the promoter is an HLA-A promoter or elongation factor 1 -alpha 1 promoter; (b) a marker of insertion, optionally wherein the marker of insertion is green fluorescent protein; and / or (c) a positive selection marker, optionally wherein the selection marker is neomycin.

[0021] Embodiment 7. The method or stem cell of embodiment 6, wherein the cassette is inserted into the genome of the stem cell with a high-fidelity integrase, optionally wherein the high-fidelity integrase is Bxbl integrase, or without introducing a doublestranded DNA break.

[0022] Embodiment 8. The method or stem cell of embodiment 6 or embodiment 7, wherein inserting the cassette into the genome of the stem cell without introducing a doublestranded DNA break is performed via a recombinase-mediated cassette exchange (RMCE) element, transposon-mediated DNA insertion, or use of a CRISPR nickase and homology directed repair.

[0023] Embodiment 9. The method or stem cell of any one of embodiments 6-8, wherein the cassette is inserted into the genome at the adeno-associated virus integration site 1 (AAVS1) locus in intron 1 of the gene that encodes protein phosphatase 1 regulatory subunit 12C (PPP1R12C) or in the Pansio-1 locus.

[0024] Embodiment 10. The method or stem cell of embodiment 8 or 9, wherein the RMCE element is introduced into the cell using a CRISPR / Cas9 system.

[0025] Embodiment 11. The method or stem cell of any one of embodiments 6-10, wherein the cassette further comprises a site-specific recombination site at a first end of the cassette and a different site-specific recombination site at the second end of the cassette.

[0026] Embodiment 12. The method or stem cell of embodiment 11, wherein a sitespecific recombination site comprises loxP, mutant loxP, flippase recognition target (FRT), or all.

[0027] Embodiment 13. The method or the stem cell of any one of embodiments 6- 12, wherein the cassette comprising HLA-A, HLA-B, and / or HLA-C genes is prepared using Gibson assembly, In-Fusion cloning, Golden Gate assemably or restriction cloning.

[0028] Embodiment 14. The method or stem cell of any one of embodiments 1-13, wherein the stem cell is an adult stem cell, pluripotent stem cell, embryonic stem cell, hematopoietic stem cell, bone marrow stromal stem cell, or mesenchymal stem cell.

[0029] Embodiment 15. The method or stem cell of embodiment 14, wherein the stem cell is a human embryonic stem cell or induced pluripotent stem cell.

[0030] Embodiment 16. The method or stem cell of any one of embodiments 1-15, wherein the beta 2-microglobulin gene is not modified in the stem cell.

[0031] Embodiment 17. The method or stem cell of any one of embodiments 1-16, wherein the major histocompatibility complex (MHC) Class II transactivator (CIITA) gene in the stem cell has been modified to block expression of class II HLA genes.

[0032] Embodiment 18. The method or stem cell of embodiment 17, wherein the class I HLA genes and / or CIITA gene have been modified by gene editing.

[0033] Embodiment 19. The method or stem cell of embodiment 18, wherein (a) two flanking gRNAs are used to delete both copies of HLA-B and HLA-C simultaneously; (b) two flanking gRNAs are used to delete both copies of HLA-A; and / or (c) one gRNA is used to introduce null mutations in both copies of CIITA.

[0034] Embodiment 20. The method or stem cell of embodiment 19, wherein the one or more of the flanking gRNAs comprise SEQ ID NOs: 1-4 and / or the sequence of the one gRNA is used to introduce null mutations in both copies of CIITA is SEQ ID: 27.

[0035] Embodiment 21. The method or stem cell of embodiment 20, wherein the flanking gRNAs each comprise one of SEQ ID NOs: 1-4.

[0036] Embodiment 22. The method or stem cell of any one of embodiments 19-21, wherein the null mutations are frameshift or indel mutations.

[0037] Embodiment 23. The method or stem cell of any one of embodiments 1-22, further comprising confirming deletion of class I and II HLA and / or CIITA by PCR and / or FACS analysis.

[0038] Embodiment 24. The method or stem cell of any one of embodiments 1-23, wherein the stem cell further comprises at least one gene edit to inhibit expression of one or more of (a) menin (SEQ ID No: 5); (b) transcription factor HIVEP2 (SEQ ID No: 6); (c) renalase (SEQ ID No: 7); (d) lengsin (SEQ ID No: 8); (e) eIF-2-alpha kinase activator GCN1 (SEQ ID No: 9); (f) perilipin-4 (SEQ ID No: 10); (g) mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 11); (h) protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 12); (i) zinc finger BED domain-containing protein 3 (SEQ ID No: 13); and metabotropic glutamate receptor 2 (SEQ ID No: 14).

[0039] Embodiment 25. The method or stem cell of any one of embodiments 17-24, wherein the gene editing is performed using a CRISPR / Cas9 system, zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, or group one intron encoded endonucleases (GIIEEs).

[0040] Embodiment 26. The method or stem cell of embodiment 25, wherein the gene editing is CRISPR gene editing.

[0041] Embodiment 27. The method or stem cell of any one of embodiments 17-26, wherein the gene editing is within or outside of the coding region of the gene, and wherein no gene product is expressed, or wherein a non-functional gene product is produced.

[0042] Embodiment 28. The method or stem cell of any one of embodiments 1-27, wherein the patient is a mammal.

[0043] Embodiment 29. The method or stem cell of embodiment 28, wherein the mammal is a human.

[0044] Embodiment 30. The method or stem cell of any one of embodiments 1-29, wherein the patient has cancer, heart disease, a neurodegenerative disease, type I or type II diabetes, or a blood diseases.

[0045] Embodiment 31. The method or stem cell of embodiment 30, wherein the patient has type I diabetes.

[0046] Embodiment 32. The method or stem cell of embodiment 31, wherein the patient-matched stem cell is differentiated so that it is capable of releasing insulin.

[0047] Embodiment 33. The method or stem cell of embodiment 32, wherein the differentiated cell is a beta cell.

[0048] Embodiment 34. The method or stem cell of any one of embodiments 1-29, wherein the stem cell further comprises at least one gene edit to introduce a chimeric antigen receptor.

[0049] Embodiment 35. The method or stem cell of embodiment 34, wherein the patient has cancer.

[0050] Embodiment 36. The method or stem cell of embodiment 34 or 35, wherein the stem cell is differentiated into a T cell or natural killer cell.

[0051] Embodiment 37. The method or stem cell of any one of embodiments 1-29, wherein the stem cell further comprises at least one gene edit to introduce one or more MHC class II proteins.

[0052] Embodiment 38. The method or stem cell of embodiment 37, wherein the stem cell is differentiated into a dendritic cell, mononuclear phagocyte, endothelial cell, thymic epithelial cell, or B cell.

[0053] Embodiment 39. The method or stem cell of any one of embodiments 1-38, wherein the stem cell further comprises at least one gene edit to introduce a suicide gene and / or a tumor suppressor gene.

[0054] Embodiment 40. The method or stem cell of embodiment 39, wherein the suicide gene encodes inducible caspase-9, cytosine deaminase, or herpes simplex virus thymidine kinase and / or the tumor suppressor gene encodes p53.

[0055] Embodiment 41. A method of treating a patient with type I or type II diabetes comprising administering stem cells of any one of embodiments 1-31 or cells capable of releasing insulin of embodiment 32 or 33, to the patient.

[0056] Embodiment 42. The method of embodiment 41, wherein the treating lowers blood glucose, increases insulin secretion in response to glucose, and / or ameliorates other symptoms of diabetes.

[0057] Embodiment 43. A method of treating or preventing diabetes in a patient, comprising (a) determining the HLA-A, HLA-B, and / or HLA-C genes endogenously expressed by the patient; (b) preparing a shuttle vector comprising a cassette comprising exogenous HLA-A, HLA-B, and / or HLA-C genes that match those endogenously expressed by the patient; (c) patient contacting the shuttle vector with modified stem cells of any one ofembodiments 1-31 in culture to allow insertion of the cassette comprising HLA-A, HLA-B, and / or HLA-C genes into the genome of the modified stem cell, thereby preparing patient- matched stem cells; selecting for patient-matched stem cells with insertion of the cassette; transplanting the selected patient-matched stem cells into the patient; and allowing the transplanted stem cells to release insulin in the patient in response to glucose.

[0058] Embodiment 44. The method of embodiment 43, wherein the stem cells are differentiated into cells capable of releasing insulin in culture after step (d) and before step (e).

[0059] Embodiment 45. The method of embodiment 44, wherein the cells capable of releasing insulin are beta cells.

[0060] Embodiment 46. The method of any one of embodiments 43-45, for use with an additional treatment.

[0061] Embodiment 47. The method of any one of embodiments 46, wherein the additional treatment is insulin, a glucagon-like peptide analog or agonist, dipeptidyl peptidase-4 inhibitor, amylin analog, biguanide, thiazolidinedione, sulfonylurea, meglitinide, alpha-glucosidase inhibitor, or sodium / glucose transporter 2 inhibitor.

[0062] Embodiment 48. The method of any one of embodiments 43-47, wherein the patient-matched stem cells are administered by transplant into the pancreas, liver, or fat pads via surgery, injection, or infusion of the patient.

[0063] Embodiment 49. A method of treating a patient with cancer comprising administering patient-matched stem cells of any one of embodiments 1-29, 34, or 35 or T cells of embodiment 36, to the patient.

[0064] Embodiment 50. The method of embodiment 49, wherein the patient has cancer and wherein the treating reduces growth of cancer cells.

[0065] Embodiment 51. A method of treating or preventing cancer in a patient, comprising (a) determining the HLA-A, HLA-B, and / or HLA-C genes endogenously expressed by the patient; (b) preparing a shuttle vector comprising a cassette comprising HLA-A, HLA-B, and / or HLA-C genes that match those endogenously expressed by the patient; (c) contacting the shuttle vector with modified stem cells of any one of embodiments 1-29, 34, or 35 in culture to allow insertion of the cassette comprising exogenous HLA-A, HLA-B, and / or HLA-C genes into of the genome of the modified stem cells, thereby preparing patient-matched stem cells; (d) selecting for patient-matched stem cells withinsertion of the cassette; (e) transplanting the patient-matched stem cells into the patient; and (f) allowing the transplanted stem cells to attack cancer cells in the patient.

[0066] Embodiment 52. The method of embodiment 51, wherein the stem cells are differentiated into T cells after step (d) and before step (e).

[0067] Embodiment 53. The method of embodiment 51 or 52, wherein the patient- matched stem cells are administered by infusion into the patient.

[0068] Embodiment 54. The method of any one of embodiments 43-53, further comprising making at least one gene edit before step (e) to introduce a suicide gene and / or a tumor suppressor gene.

[0069] Embodiment 55. The method of embodiment 54, wherein suicide gene encodes inducible caspase-9, cytosine deaminase, or herpes simplex virus thymidine kinase and / or the tumor suppressor gene encodes p53.

[0070] Embodiment 56. Use of a stem cell of any one of embodiments 1-31, 34, or 35 in the manufacture of a medicament to treat cancer, heart disease, a neurodegenerative disease, type I or type II diabetes, or a blood diseases in a human patient in need thereof.

[0071] Embodiment 57. A method of preparing a patient-matched stem cell comprising modifying a stem cell to lack expression of class I and class II human leukocyte antigen (HLA) genes; storing said modified stem cell; retrieving said modified stem cell after storage; and inserting HLA-A, HLA-B, and / or HLA-C genes matched to those of a patient in need thereof, thereby preparing a patient-matched stem cell; and optionally selecting for expression of the HLA-A, HLA-B, and / or HLA-C genes by the patient-matched stem cell, optionally wherein the selecting is by magnetic activated cell sorting using antibodies to HLA-A, HLA-B, and / or HLA-C.

[0072] Embodiment 58. A stem cell produced by the method of embodiment 57.

[0073] Embodiment 59. A patient-matched stem cell, wherein the stem cell is not autologous; lacks endogenous expression of class I and class II HLA genes; and comprises exogenous HLA-A, HLA-B, and / or HLA-C genes matched to those of a patient in need thereof.

[0074] Embodiment 60. The method of embodiment 57 or stem cell of embodiment58 or 59, wherein (a) the exogenous HLA-A, HLA-B, and / or HLA-C genes comprise a singleHLA-A, HLA-B, or HLA-C gene; (b) the exogenous HLA-A, HLA-B, and / or HLA-C genes comprise two HLA-A genes, two HLA-B genes, or two HLA-C genes; (c) the exogenous HLA-A, HLA-B, and / or HLA-C genes comprise a single HLA-A gene and a single HLA-B gene, a single HLA-A gene and a single HLA-C gene, or a single HLA-B gene and a HLA-C gene; (d) the exogenous HLA-A, HLA-B, and / or HLA-C genes comprise one allele or two alleles for each of HLA-A, HLA-B, and HLA-C genes, optionally wherein the exogenous HLA-A, HLA-B, and / or HLA-C genes comprise 4 total, 5 total, or 6 total different genes; (e) the stem cell does not trigger attack by natural killer cells; and / or (f) the HLA-A, HLA-B, and / or HLA-C genes are comprised in a cassette, wherein the cassette optionally further comprises (i) a promoter, optionally wherein the promoter is an HLA-A promoter or elongation factor 1 -alpha 1 promoter; (ii) a marker of insertion, optionally wherein the marker of insertion is green fluorescent protein; and / or (iii) a positive selection marker, optionally wherein the selection marker is neomycin.

[0075] Embodiment 61. The method or stem cell of embodiment 60, wherein the cassette (a) is inserted into the genome of the stem cell either (1) with a high-fidelity recombinase, optionally wherein the high-fidelity recombinase is Bxbl or (2) without introducing a double-stranded DNA break, optionally wherein inserting the cassette into the genome of the stem cell without introducing a double-stranded DNA break is performed via a recombinase-mediated cassette exchange (RMCE) element, transposon-mediated DNA insertion, or use of a CRISPR nickase and homology directed repair, optionally wherein the RMCE element is introduced into the cell using a CRISPR / Cas9 system; (b) is inserted into the genome of the stem cell at the adeno-associated virus integration site 1 (AAVS1) locus in intron 1 of the gene that encodes protein phosphatase 1 regulatory subunit 12C (PPP1R12C) or at the Pansio-1 locus; (c) further comprises a site-specific recombination site at a first end of the cassette and a different site-specific recombination site at the second end of the cassette, optionally wherein a site-specific recombination site comprises loxP, mutant loxP, flippase recognition target (FRT), or att; and / or (d) is prepared using Gibson assembly, InFusion cloning, Golden Gate assembly or restriction cloning.

[0076] Embodiment 62. The method or stem cell of any one of embodiments 57-61, wherein (a) the stem cell is an adult stem cell, pluripotent stem cell, embryonic stem cell, hematopoietic stem cell, bone marrow stromal stem cell, or mesenchymal stem cell, optionally wherein the stem cell is a human embryonic stem cell or induced pluripotent stemcell; (b) the beta 2-microglobulin gene is not modified in the stem cell; and / or (c) the major histocompatibility complex (MHC) Class II transactivator (CIITA) gene in the stem cell has been modified to block expression of class II HLA genes, optionally wherein the class I HLA genes and / or CIITA gene have been modified by gene editing.

[0077] Embodiment 63. The method or stem cell of embodiment 62, wherein (a) two flanking gRNAs are used to delete both copies of HLA-B and HLA-C simultaneously; (b) two flanking gRNAs are used to delete both copies of HLA- A; and / or (c) one gRNA is used to introduce null mutations in both copies of CIITA.

[0078] Embodiment 64. The method or stem cell of embodiment 63, wherein the null mutations are frameshift or indel mutations; and / or the one or more of the flanking gRNAs comprise SEQ ID NOs: 1-4, optionally wherein the flanking gRNAs each comprise one of SEQ ID NOs: 1-4 and / or the sequence of the one gRNA is used to introduce null mutations in both copies of CIITA is SEQ ID: 27.

[0079] Embodiment 65. The method or stem cell of any one of embodiments 57-64, wherein the method further comprises confirming deletion of class I and II HLA and / or CIITA by PCR and / or FACS analysis; and / or the stem cell further comprises at least one gene edit to inhibit expression of one or more of menin (SEQ ID No: 5); transcription factor HIVEP2 (SEQ ID No: 6); renalase (SEQ ID No: 7); lengsin (SEQ ID No: 8); eIF-2-alpha kinase activator GCN1 (SEQ ID No: 9); perilipin-4 (SEQ ID No: 10); mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 11); protein-glutamine gammaglutamyltransferase 6 (SEQ ID No: 12); zinc finger BED domain-containing protein 3 (SEQ ID No: 13); and metabotropic glutamate receptor 2 (SEQ ID No: 14).

[0080] Embodiment 66. The method or stem cell of embodiment 65, wherein the gene editing is performed using a CRISPR / Cas9 system, zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, or group one intron encoded endonucleases (GIIEEs), optionally wherein the gene editing is CRISPR gene editing; and / or the gene editing is within or outside of the coding region of the gene, and wherein no gene product is expressed, or wherein a non-functional gene product is produced.

[0081] Embodiment 67. The method or stem cell of any one of embodiments 57-66, wherein (a) the patient is a mammal, optionally wherein the mammal is a human; (b) the patient has cancer, heart disease, a neurodegenerative disease, type I or type II diabetes, or a blood diseases, optionally wherein the diabetes is type I diabetes; (c) the stem cell furthercomprises at least one gene edit to introduce a chimeric antigen receptor, optionally wherein the patient has cancer, optionally wherein the stem cell is differentiated into a T cell or natural killer cell; (d) the stem cell further comprises at least one gene edit to introduce one or more MHC class II proteins, optionally wherein the stem cell is differentiated into a dendritic cell, mononuclear phagocyte, endothelial cell, thymic epithelial cell, or B cell; and / or (e) the stem cell further comprises at least one gene edit to introduce a suicide gene and / or a tumor suppressor gene, optionally wherein the suicide gene encodes inducible caspase-9, cytosine deaminase, or herpes simplex virus thymidine kinase and / or the tumor suppressor gene encodes p53.

[0082] Embodiment 68. The method or stem cell of embodiment 67, wherein the patient-matched stem cell is differentiated so that it is capable of releasing insulin, optionally wherein the differentiated cell is a beta cell.

[0083] Embodiment 69. A method of treating a patient with type I or type II diabetes comprising administering stem cells of any one of embodiments 57-67 or cells capable of releasing insulin of embodiment 68 to the patient, optionally wherein the treating lowers blood glucose, increases insulin secretion in response to glucose, and / or ameliorates other symptoms of diabetes.

[0084] Embodiment 70. A method of treating or preventing diabetes in a patient, comprising (a) determining the HLA-A, HLA-B, and / or HLA-C genes endogenously expressed by the patient; (b) preparing a shuttle vector comprising a cassette comprising exogenous HLA-A, HLA-B, and / or HLA-C genes that match those endogenously expressed by the patient; (c) contacting the shuttle vector with modified stem cells of any one of embodiments 57-67 in culture to allow insertion of the cassette comprising HLA-A, HLA-B, and / or HLA-C genes into the genome of the modified stem cell, thereby preparing patient- matched stem cells; (d) selecting for patient-matched stem cells with insertion of the cassette; (e) transplanting the selected patient-matched stem cells into the patient; and (f) allowing the transplanted stem cells to release insulin in the patient in response to glucose.

[0085] Embodiment 71. The method of embodiment 70, wherein the method further comprises selecting for expression of the HLA-A, HLA-B, and / or HLA-C genes by the patient-matched stem cells after step (d) and before step (e), optionally wherein the selecting is by magnetic activated cell sorting using antibodies to HLA-A, HLA-B, and / or HLA-C; the method further comprises differentiating the stem cells into cells capable of releasing insulinin culture after step (d) and before step (e), optionally wherein the cells capable of releasing insulin are beta cells; the method is for use with an additional treatment, optionally wherein the additional treatment is insulin, a glucagon-like peptide analog or agonist, dipeptidyl peptidase-4 inhibitor, amylin analog, biguanide, thiazolidinedione, sulfonylurea, meglitinide, alpha-glucosidase inhibitor, or sodium / glucose transporter 2 inhibitor; and / or the transplanting is into the pancreas, liver, or fat pads via surgery, injection, or infusion.

[0086] Embodiment 72. A method of treating a patient with cancer comprising administering patient-matched stem cells of any one of embodiments 57-67, to the patient, optionally wherein the patient has cancer and wherein the treating reduces growth of cancer cells.

[0087] Embodiment 73. A method of treating or preventing cancer in a patient, comprising (a) determining the HLA-A, HLA-B, and / or HLA-C genes endogenously expressed by the patient; (b) preparing a shuttle vector comprising a cassette comprising HLA-A, HLA-B, and / or HLA-C genes that match those endogenously expressed by the patient; (c) contacting the shuttle vector with modified stem cells of any one of embodiments 57-67 in culture to allow insertion of the cassette comprising exogenous HLA-A, HLA-B, and / or HLA-C genes into of the genome of the modified stem cells, thereby preparing patient-matched stem cells; (d) selecting for patient-matched stem cells with insertion of the cassette; (e) transplanting the patient-matched stem cells into the patient; and (f) allowing the transplanted stem cells to attack cancer cells in the patient.

[0088] Embodiment 74. The method of embodiment 73, wherein the method further comprises selecting for expression of the HLA-A, HLA-B, and / or HLA-C genes by the patient-matched stem cells after step (d) and before step (e), optionally wherein the selecting is by magnetic activated cell sorting using antibodies to HLA-A, HLA-B, and / or HLA-C; the method further comprises differentiating the stem cells into T cells after step (d) and before step (e); and / or the transplanting is performed by infusion into the patient.

[0089] Embodiment 75. The method of any one of embodiments 69-74, further comprising making at least one gene edit before step (e) to introduce a suicide gene and / or a tumor suppressor gene, optionally wherein suicide gene encodes inducible caspase-9, cytosine deaminase, or herpes simplex virus thymidine kinase and / or the tumor suppressor gene encodes p53.

[0090] Embodiment 76. Use of a stem cell of any one of embodiments 57-68 in the manufacture of a medicament to treat cancer, heart disease, a neurodegenerative disease, type I or type II diabetes, or a blood diseases in a human patient in need thereof.

[0091] Additional objects and advantages will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0092] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0093] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the description, serve to explain the principles described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1. Overview of the HLA gene editing strategy. In general, methods described herein start with a human embryonic stem cell (hESC) line or a human induced pluripotent stem cell (hiPSC) line that are well-characterized for specific cell lineage differentiation. CRISPR / Cas9 technology can be used to delete the stem cell’s class I and II HLA genes (Class II HLA deletion is done by deleting their master regulator, CIITA). In some methods, insertion of a RMCE landing-pad cassette into the genome at the AAVS1 locus is performed via CRISPR mediated homology directed repair / recombination (HDR). This HLA-null, RMCE landing-pad inserted hESC / hiPSC will be banked and used for introduction of patient-matched HLA genes via RMCE. Patient-matched HLA gene(s) will be assembled into shuttle vector and transfected together with plasmid that expresses recombinase (Cre, FLP, or other site-specific recombinases) into the banked cells, and then the HLAs will be inserted into the landing-pad cassette for expression.

[0095] Figures 2A and 2B. Strategy to delete Class I HLA genes and CIITA. (A) CRISPR / Cas9 technology is used to delete human class I HLA genes (HLA-A, -B and -C). Two single guide RNAs (sgRNAs) are used to delete the HLA-A gene (HLA-A-5’ sgRNA and HLA-A-53’ sgRNA), and then use another two sgRNA are used to delete HLA-B and C together (HLA-B-5’ sgRNA and HLA-C-3’ sgRNA). The sequence of the 4 sgRNA used inthe class I HLA deletion is listed (SEQ ID NOs: 1-4). (B) A representative method to delete the CIITA gene. The sequence of a sgRNA to delete CIITA is SEQ ID NO: 27.

[0096] Figures 3A-3C. Class I HLA gene deletion in human 293 cells. Proof-of- principle experiment in human 293 cells to show the efficacy of the sgRNAs to delete class I HLA genes. Plasmids carrying SpCas9 and sgRNA are co-transfected into 293 cells in two consequent steps (HLA-A first in (A) and then HLA-B / C in (B)). WT-293 cells or transfected 293 cells were analyzed by FACS with pan-HLA class I (HLA-A, -B, and -C) staining (C). A population of the Cas9 / sgRNA transfected 293 cells lost all their class I HLA expression (P4), and these cells were then sorted out and cloned as HLA-null cell line.

[0097] Figure 4. Landing-pad cassette insertion strategy. Shown is a strategy to insert the RMCE cassette into the genome at the human AAVS1 locus. An exemplary landing-pad cassette consists of a HLA-A promoter (this can also be replaced by other ubiquitous promoter, such as EFla promoter), followed by a loxP and FRT flanked cassette expressing eGFP, dTK, and neomycin. eGFP and neomycin can be used to identify cells with the landing-pad cassette and cells that failed to recombine after RMCE. dTK (delta thymidine kinase) can be used to specifically remove non-recombined cells from the culture by treatment with ganciclovir. The landing-pad cassette was cloned into pAAVSl-P-MCS vector with PacI and Sall, and the AAVS1 locus insert is facilitated by pXAT2 vector that expresses SpCas9 and AAVS1 -targeting sgRNA (T2). puroR = puromycin resistance gene; FRT = flip recombinase; pA = polyA terminator.

[0098] Figures 5A-5C. Landing-pad cassette insertion into 293 cells. Proof-of- principle experiment in human 293 cells to show feasibility of landing-pad cassette insertion. The HLA-null 293 cells were transfected with landing-pad cassette plasmid and pXAT2 (A), and FACS analysis shows a population of cells that are GFP positive (P4) (B). GFP+ cells were then single-cell cloned into different cell lines. (A2, D10 etc.) (C). Steps of the method include (1) introduction of a landing-pad cassette using Cas9 into cells lacking class I and class II HLA (in this case 293 cells), (2) cells are sorted and diluted to select single GFP- positive cells, and (3) a clone of interest is picked. The clone of interest would be chosen to have normal morphology and strong puromycin and / or blasticidin resistance.

[0099] Figure 6. Assembly of pHLA-shuttle vector. The pHLA empty shuttle vector is designed to have loxP and FRT flanked spacer, with two thosea asigna virus 2A “self-cleaving” protein (T2A) sequences (T2A1 and T2A4) on the two sides of the spacer.These T2A sequences are only for the ease of subsequence HLA cloning, which do not translate into protein. Patient-matched HLA genes were PCR amplified with two flanking T2A sequences (HL A- A with T2A1 and T2A2, HLA-B with T2A2 and T2A3, HLA-C with T2A3 and T2A4). All T2A sequences differ at the DNA sequence but encode the same protein sequence. The Golden Gate assembly technique (as described in Engler et al., PLOS ONE 3(1 l):e3647 (2008)) was used to clone three HLA genes into empty pHLA shuttle vector. The cloning efficiency is -80% when only one HLA gene was cloned, and the efficiencies are -63% and 30% when two or three HLA genes are simultaneously cloned. Optionally, up to 6 HLA genes can be assembled with additional T2A sequences.

[0100] Figure 7. Schematics of RMCE into landing-pad cassette into the genome at the AAVS1 locus. Using RMCE, the landing-pad in AAVS1 locus is replaced with the cassette in the shuttle vector by Cre / FLP recombinase, so that the HLA-A, -B and -C genes will be expressed under the HLA promoter (or other promoter such as EFla promoter).

[0101] Figures 8A-8D. Pilot testing: Landing-pad (LP) and shuttle vector (SV) Co-transfections (mCherry shuttle vector). Pilot test of RMCE efficiency using a shuttle vector with a mCherry gene. The landing-pad vector and mCherry shuttle vector are co-transfected into 293 cells with or without Cre / FLP expression vector (A). Cre recombinase can act at the loxP site, while FLP can act at the FRT site. FACS analysis shows -1.3% of the cells that are co-transfected with Cre / FLP starts to express mCherry protein (D), with LP- only (B) or LP+SV (without recombinase, C) acting as controls.

[0102] Figures 9A and 9B. LP / SV Co-transfections (HLA-A shuttle vector). Pilot test of RMCE efficiency using a shuttle vector with a HLA-A genes. The landing-pad vector and HLA-A shuttle vector are co-transfected into 293 cells with or without Cre / FLP expression vector (A). FACS analysis shows -1.58% of the cells that are co-transfected with Cre / FLP start to express HLA-A protein (B).

[0103] Figures 10A-10D. LP / SV Co-transfections (mCherry shuttle vector and a loxP-LoxPmut landing-pad). Pilot test of RMCE efficiency using a shuttle vector with a mCherry genes. A loxP-LoxPmut Landing-pad vector and mCherry shuttle vector are co-transfected into 293 cells with or without Cre expression vector (A). FACS analysis shows -0.7% of the cells that are co-transfected with Cre starts to express mCherry protein (D), with LP-only (B) or LP+SV (without recombinase, C) acting as controls. RMCE can beachieved by similar methods using (1) loxP+FRT, (2) loxP+loxPmut, or (3) other choices, such as att sites.

[0104] Figure 11. General strategy to generate patient-matched, HLA- matched, and autoimmune-protected hESC / hiPSC derived cells for administration to a patient. In an exemplary embodiment, the derived cells may be beta-like cells, the patient may suffer from T1D, and the autoimmune protection may be mutation of the RNLS gene. Other strategies may be used to prepare patient-matched stem cells for other uses with methods described herein.

[0105] Figure 12. Landing pad design with Bxbl recombinase. In an exemplary embodiment, a landing pad uses Bxbl recombinase to insert a attP flanked shuttle vector into a attB flanked landing pad. In this landing pad schematic, attP (CT) refers to a 5’ attP site comprising a CT dinucleotide, and attP (G) refers to a 3’ attP site comprising a GT dinucleotide.

[0106] Figure 13. Single HLA shuttle vector design. In an exemplary embodiment, a shuttle vector is designed for any one of the HLA-A, HLA-B, or HLA-C gene alleles. In this shuttle vector schematic, attB (CT) refers to a 5’ attB site comprising a CT dinucleotide, and attB (G) refers to a 3’ attB site comprising a GT dinucleotide.

[0107] Figure 14. Bicistronic HLA shuttle vector design. In an exemplary embodiment, a shuttle vector is bicistronic and designed for two HLA alleles selected from the HLA-A, HLA-B, and HLA-C gene alleles.

[0108] Figure 15. Tricistronic HLA shuttle vector design. In an exemplary embodiment, a shuttle vector is tricistronic and designed for three HLA alleles with one each of HLA-A, HLA-B, and HLA-C gene alleles.

[0109] Figure 16. Hexacistronic HLA shuttle vector design. In an exemplary embodiment, a shuttle vector is hexacistronic and designed for six HLA alleles with two each of HLA-A, HLA-B, and HLA-C gene alleles. This shuttle vector would be designed for shuttling each matched HLA gene of a given patient (i.e., the two HLA-A, two HLA-B, and two HLA-C alleles corresponding to those of the patient).

[0110] Figure 17. Transfection efficiency of landing pads. Genotyping showed successful incorporation of landing pads into heterozygous or homozygous cells.

[0111] Figure 18. Transfection efficiency of shuttle vector transfection. Data shows efficiency of Clover reporter shuttle vector transfection using Lipofectamine™Stem Transfection Reagent (Thermo Fisher), into heterozygous landing pad stem cells. Successful transfection as measured by shuttle vector integration was seen across different DNA mass conditions (various amounts of Clover shuttle vector + Bxbl integrase).

[0112] Figure 19. Magnetic enrichment of HLA+ cells. Schematic shows how magnetic activated cell sorting (MACS) using biotinylated HLA-A / B / C antibodies over a MS column (Miletnyi Biotec) can lead to enrichment of HLA+ cells.

[0113] Figure 20. Data on enrichment using MACS. MACS enrichment protocols led to a population of cells wherein 99% of cells are HLA+. SSC = side scatter.

[0114] Figure 21A and 21B. Data on endothelial cell markers and HLA expression in endothelial cells. When endothelial cells were subjected to insertion of exogenous HLA and MACS enrichment, cells retained endothelial markers CD144 and CD31 (A) and differentiated cells retained HLA expression similar to wildtype (WT) cells.DESCRIPTION OF THE SEQUENCES

[0115] Table 1 provides a listing of certain sequences referenced herein.DESCRIPTION OF THE EMBODIMENTSI. Modified stem cells

[0116] A goal of stem cell therapy is to prepare HLA-matched cells. Further, gene edits that improve cell survival would help to avoid immune killing of transplanted cells. Described herein is a method to generate patient-matched HLA hESC / hiPSC cells for differentiation and administration to a patient. As illustrated in Figure 1, instead of generating hiPSC for each patient, a well-characterized hESC / hiPSC cell line with broad differentiationpotential are the starting point. These cells have the capability to differentiate into pancreatic beta cells, T-cell, or any of a variety of other cells based on the user’s goals.

[0117] In some embodiments, a patient-matched stem cell lacks endogenous expression of class I and class II human leukocyte antigen (HLA) genes and instead comprises exogenous HLA-A, HLA-B, and / or HLA-C genes matched to those of a patient in need thereof.

[0118] In some embodiments, the class I (HLA-A, -B, and -C) and class II HLA in a stem cell will be deleted. In some embodiments, this deletion may be performed using CRISPR technology. In some embodiments, an RMCE (Recombinase-Mediated Cassette Exchange) landing-pad cassette, or one of a number of other means of introducing genes some of which are described herein, will be introduced into the cells to allow for expression of exogenous genes. In some embodiments, a HLA- / -, RMCE-ready hESC / hiPSC line is provided, and can be used to genetically edit autoimmune protective gene(s), as described below.

[0119] In some embodiments, for any individual patient, the class I HLA will be typed, and then their HLA-A, B and C alleles (1 or 2) will be cloned into a single shuttle vector. In some embodiments, the cloning is performed using Gibson Assembly (as described in Gibson et al., Nature Methods 6(5):343-347 (2009)), although a shuttle vector can be prepared using a variety of different well-known molecular biology techniques, such as cloning using restriction enzymes or with commercial reagents (e.g., In-Fusion® seamless cloning from Takara). In some embodiments, these HLA alleles can be incorporated into the autoimmune protective, HLA- / -, RMCE-ready hESC / hiPSC line. The patient-matched HLA- matched hESC / hiPSC cells can then be used for administration to the patient, such as for administering pancreatic beta-like cells for treatment of Type 1 diabetes.

[0120] In some embodiments, the present system banks double-stranded DNA encoding different HLA alleles along with stem cells. In this way, banked stem cells can be used to prepare differentiated cells for multiple different patients, which greatly reduced the cost of storage and maintenance.

[0121] In some embodiments, if the autoimmune protective gene editing strategy needs to be changed depending on the disease to be treated in the patient, only the HLA- / - stem cell line requires editing rather than all individual patient-matched stem cells.Therefore, the present patient-matched stem cells provide time-saving and great reductions in the overall cost of stem cell preparation.

[0122] A variety of different stem cells may be used to generate modified stem cells (i.e., the HLA -I- “off the shelf’ stem cell). In some embodiments, the stem cell is an adult stem cell, pluripotent stem cell, embryonic stem cell, hematopoietic stem cell, bone marrow stromal stem cell, or mesenchymal stem cell. In some embodiments, the stem cell is a human embryonic stem cell or induced pluripotent stem cell.A. HLA and CIITA

[0123] MHC class I molecules consist of an alpha heavy chain bound to a beta-2 microglobulin molecule. The heavy chain consists of 2 peptide-binding domains, an immunoglobulin (Ig)-like domain, and a transmembrane region with a cytoplasmic tail. This heavy chain of the class I molecule is encoded by genes at HLA-A, HLA-B, and HLA-C loci. T cells that express CD8 molecules can then react with class I MHC molecules, including cytotoxic function capable of recognizing an infected cell. As used herein, HLA-A / B / C coding sequence (CDS, as shown in Figures 13-16) can refer to any DNA coding sequence for any HLA-A, HLA-B, or HLA-C gene alleles.

[0124] Further, some class I MHC genes encode nonclassical MHC molecules, such as HLA-G (which may play a role in protecting the fetus from an immune response by the mother) and HLA-E (which presents peptides to certain receptors on natural killer (NK) cells). In some embodiments, the present methods use gene editing at the HLA-A, HLA-B, and / or HLA-C loci to inhibit expression of class I HLA in “off-the-shelf’ stem cells. In this way, these hypoimmunogenic stem cells have less recognition by T cells. These cells can then be used for introduction of one or more patient-matched allele of HLA-A, HLA-B, and / or HLA-C and differentiation before administration to a patient.

[0125] In some embodiments, endogenous class I HLA genes in the stem cell have been modified to block expression of class I HLA genes in stem cells. In some embodiments, the endogenous major histocompatibility complex (MHC) Class II transactivator (CIITA) gene in the stem cell has been modified to block expression of class II HLA genes. In some embodiments, endogenous class I HLA genes in the stem cell have been modified to block expression of class I HLA genes and the major histocompatibility complex (MHC) Class II transactivator (CIITA) gene has been modified to block expression of class II HLA genes.

[0126] In some embodiments, the endogenous class I HLA genes and / or CIITA gene have been modified by gene editing.

[0127] In some embodiments, the present stem cells retain nonclassical MHC molecules (for example HLA-G and HLA-E). While some methods in the art use deletion of the beta-2 microglobulin molecule to make hypoimmunogenic stem cells (such as US 2021 / 0024884), that deletion approach will also cause loss of nonclassical MHC molecules and thus could interfere with normal functions such as surveillance of transplanted cells by NK cells. In some embodiments, the beta 2-microglobulin gene is not modified by gene editing in the present modified stem cells.

[0128] As described herein, the present cells retain nonclassical MHC molecules as beta-2 microglobulin molecules and instead HLA- A, HLA-B, and HLA-C are deleted from the off-the-shelf stem cells that can be used to prepare patient-matched stem cells by introduction of exogenous HLA-A, HLA-B, and / or HLA-C. In some embodiments, the present patient-matched stem cell does not trigger attack by natural killer cells.

[0129] In some embodiments, the exogenous HLA-A, HLA-B, and / or HLA-C genes of the patient-matched stem cell comprise one allele for each gene. In some embodiments, one copy of each of the HLA genes is sufficient for cells to be recognized appropriately be the immune system. The fact that individuals may naturally be homozygous for a given HLA gene and have appropriate immune responses indicates that one allele of each of the HLA-A, HLA-B, and / or HLA-C genes may produce cells that are appropriately recognized by a patient’s immune system. In this way, one copy of a patient’s HLA may allow for acceptance of the stem cells by a patient’s immune system, even if the patient is heterozygous at one or more HLA gene (i.e., expressing two different alleles of HLA-A, HLA-B, and / or HLA-C genes). In some embodiments, a patient is homozygous for one of more of HLA-A, HLA-B, and HLA-C genes, and the patient-matched stem cells comprise one allele that matches the patient.

[0130] In some embodiments, the patient-matched stem cell has only one HLA-A allele. In some embodiments, the patient-matched stem cell has only one HLA-B allele. In some embodiments, the patient-matched stem cell has only one HLA-C allele. In some embodiments, the patient has only one allele of each of HLA-A, HLA-B, and HLA-C, and the patient-matched stem cells also only have one allele of each of HLA-A, HLA-B, and HLA-C.

[0131] In some embodiments, the patient-matched stem cells may match the patient’s profile of being heterozygous at one or more HL A genes. In some embodiments, the patient-matched stem cells comprise one or two alleles for each gene of HLA-A, HLA-B, and HLA-C. In some embodiments, the patient-matched stem cell has two different HLA-A alleles. In some embodiments, the patient-matched stem cell has two different HLA-B alleles. In some embodiments, the patient-matched stem cell has two different HLA-C alleles.

[0132] In some embodiments, the exogenous HLA-A, HLA-B, and HLA-C genes of the patient-matched stem cell comprise two alleles for each gene.B. Differentiation of stem cells

[0133] In some embodiments, a stem cell is differentiated. In some embodiments, a stem cell is differentiated before it is administered to a patient. In some embodiments, a stem cell is not differentiated before it is administered to a patient.

[0134] Methods of differentiating stem cells are well-known in the literature, and one skilled in the art could choose an appropriate methods of differentiating stem cells based on how the cell should function after being administered to a patient. In some embodiments, the differentiated cells function as insulin-releasing cells (such as beta cells of the pancreas or beta-like cells with similar properties to beta cells), T cells, dendritic cells, mononuclear phagocytes, endothelial cells, thymic epithelial cells, or B cells. In some embodiments, the cell capable of releasing insulin is a beta cell.

[0135] In some embodiments, the stem cells are differentiated into beta cells. Such cells may be referred to as “stem-cell derived beta cells.” Stem-cell derived beta cells can provide a renewable supply of insulin-producing cells for cellular therapy of diabetes. Hogrebe et al. Nature Protocols 16(9): 4109-4143 (2021) describes an exemplary protocol for differentiating stem cells into beta cells. One skilled in the art could use differentiation protocols of interest if they wish to differentiate stem cells into a different cell type.C. Cassettes comprising HLA genes

[0136] A number of different methods can be used to insert the HLA genes. In some embodiments, a cassette comprising HLA genes is inserted into the genome of a stem cell. In some embodiments, a cassette comprising HLA genes is inserted into the genome of a stem cell, wherein the stem cell lacks endogenous expression of HLA-A, HLA-B, and HLA- C. In some embodiments, a cassette comprising HLA genes is inserted into the genome of a stem cell, wherein the stem cell also lacks endogenous expression of MHC class II molecules.

[0137] In some embodiments, a cassette comprising HLA genes further comprises additional components. Figures 7 shows a representative cassette comprising HLA genes. In some embodiments, the cassette comprising HLA genes is comprising in a shuttle vector. As used herein, a shuttle vector is any type of vector capable of comprising the cassette. In some embodiments, a shuttle vector comprises LoxP / FRT or other pairs of recombination sites, as well as the HLA genes.1. Promoter

[0138] In some embodiments, the HLA- A, HLA-B, and / or HLA-C genes are comprised in a cassette further comprising a promoter. In some embodiments, the promoter is an HLA-A promoter or elongation factor 1 -alpha 1 promoter. In some embodiments, the promoter drives expression of the HLA-A, HLA-B, and / or HLA-C genes.

[0139] In some embodiments, inclusion of a promoter in the cassette drives higher expression of the HLA-A, HLA-B, and / or HLA-C genes as compared to a cassette lacking a promoter.2. T2A sites

[0140] In some embodiments, a cassette comprising HLA genes further comprises 2A peptide sites, such as T2A sites derived from thosea asigna virus 2A. In some embodiments, polyA terminators (pA) are used at the end of cassettes to end the region under control of the cassette’s promoter. In some embodiments, pA’s allow for separate expression of HLA-A, HLA-B, and / or HLA-C proteins from the same cassette. SEQ ID NOs: 28-32 correspond to representative T2A sequences that may be comprised in shuttle vectors.3. Resistance marker of cassette insertion

[0141] In some embodiments, a cassette comprising HLA genes further comprises a positive selection marker. In some embodiments, the selection marker is neomycin (i.e., cells with the cassette have neomycin resistance). In some embodiments, neomycin resistance allows for cells with the selection marker to be identified when culturing in media comprising neomycin. In some embodiments, cells that lack the cassette (and thus lack neomycin resistance) will not survive under culture conditions with media comprising neomycin. In some embodiments, the neomycin resistance neoR gene, which encodes for neomycin phosphotransferase.4. Marker of cassette insertion

[0142] In some embodiments, the exogenous HLA-A, HLA-B, and / or HLA-C genes are comprised in a cassette further comprising a marker of insertion. In some embodiments, the marker of insertion allows for a user to identify cells comprising the cassette comprising exogenous HLA-A, HLA-B, and / or HLA-C genes. In some embodiments, the marker of insertion is a fluorescent marker, which can be easily identified using a fluorescent microscope or plate reader. In some embodiments, the presence of a fluorescent marker indicates that a cassette has been successfully introduced. In some embodiments, the marker of insertion is green fluorescent protein.D. Safe harbor locus

[0143] In some embodiments, DNA is introduced into stem cells at a safe harbor locus. In some embodiments, a cassette comprising HLA-A, -B, and -C is introduced into a safe harbor locus. As used herein, a “safe harbor locus” refers to a site in the genome where disruption does not have adverse effects on the cell. In other words, an edit at a safe harbor locus does not have deleterious effects. A wide variety of safe harbor locations are known in the literature, and a user could choose from any of these well-known locations.

[0144] Use of a safe harbor locus helps to avoid potential unwanted effects by random editing at various locations in the genome (such as inadvertent mutation of a tumor suppressor gene). In some embodiments, a safe harbor locus has robust transcription that allows for expression of an exogenously inserted gene. In some embodiments, the safe harbor locus is the adeno-associated virus integration site 1 (AAVS1) locus or Pansio-1 locus. In some embodiments, an RMCE cassette is introduced into the genome at the AAVS1 locus. In some embodiments, an RMCE cassette is introduced into the genome at the Pansio-1 locus (see, Autio et al. eLife 2024;13:e79592).E. Protective gene edits

[0145] In some embodiments, one or more gene edit that protects the patient- matched stem cell from autoimmune attack is made. For example, patients with type I diabetes may have factors that promote autoimmune attack of stem cells that are differentiated into beta cells or other cells capable of secreting insulin. U.S. Patent No. 11,708,561, which is incorporated herein in its entirety, describes a variety of different protective gene edits.

[0146] In some embodiments, modified stem cells further comprise at least one gene edit to inhibit expression of one or more of (a) menin (SEQ ID No: 5); (b) transcription factor HIVEP2 (SEQ ID No: 6); (c) renalase (SEQ ID No: 7); (d) lengsin (SEQ ID No: 8); (e) eIF-2-alpha kinase activator GCN1 (SEQ ID No: 9); (f) perilipin-4 (SEQ ID No: 10); (g) mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 11); (h) protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 12); (i) zinc finger BED domain-containing protein 3 (SEQ ID No: 13); and (j) metabotropic glutamate receptor 2 (SEQ ID No: 14). SEQ ID NOs: 15-26 provide amino acid sequences of proteins associated with these genes.

[0147] In some embodiments, the patient has type I or type II diabetes. In some embodiments, the patient has type I or type II diabetes, and one or more gene edits decrease immune attack on the patient-matched stem cell or on a cell differentiated from the patient-matched stem cell.

[0148] Patient-matched stem cells may be prepared as a treatment for diabetes. In some embodiments, the treatment is for type I diabetes. In some embodiments, the treatment for type I diabetes includes administration of patient-matched stem cells or beta cells prepared from patient-matched stem cells to a patient.F. Gene edits to introduce a chimeric antigen receptor

[0149] In some embodiments, patient-matched stem cells are prepared as a treatment for cancer. In some embodiments, the cancer is a leukemia or lymphoma. In some embodiments, patient-matched stem cells are differentiated into T-cells for chimeric antigen receptor (CAR) T-cell therapy. While standard CAR T-cell therapy requires that cells be prepared unique to each patient, the present modified stem cells allow for “off-the-shelf’ generation of modified stem cells with a specific CAR, wherein these cells could be edited to express HLA-A, HLA-B, and / or HLA-C genes that match the patient.

[0150] In some embodiments, the modified stem cell comprises at least one gene edit to introduce a chimeric antigen receptor.

[0151] In some embodiments, the patient has cancer, and the patient-matched stem cell comprises at least one gene edit to introduce a chimeric antigen receptor. In some embodiments, the patient-matched stem cell comprising a chimeric antigen receptor is differentiated into a T cell or natural killer cell for administration to a patient with cancer. Insome embodiments, administration of such T cells causes the immune system to attack a cancer cell comprising an antigen comprised in the chimeric antigen receptor.

[0152] In some embodiments, the CAR corresponds to a known CAR known to be effective for treatment of a cancer. In some embodiments, the CAR is directed against cluster of differentiation 19 (CD 19) or B-cell maturation antigen (BCMA).G. Gene edits to introduce MHC class II proteins

[0153] In some embodiments, a modified stem cell further comprises at least one gene edit to introduce one or more MHC class II proteins to generate a patient-matched stem cell. In this way, the patient-matched stem cells can be differentiated into a mature immune cell. In some embodiments, the stem cell is differentiated into a dendritic cell, mononuclear phagocyte, endothelial cell, thymic epithelial cell, or B cell.H. Gene edits to introduce a suicide gene

[0154] For any type of patient-matched stem cell, a user may wish to be able to limit the life of the stem cell after it is transplanted into a patient. For example, if a cancer patient being treated with a CAR T-cell therapy achieves a full remission, a clinician might want to stop the therapy. In addition, if a patient develops side effects, a clinician might want to limit the number of transplanted cells (for example, if a patient with diabetes were to develop persistent hypoglycemia after administration of beta cells differentiated from patient- matched stem cells).

[0155] In some embodiments, a user can introduce one or more gene edits that guarantee safety of the stem cells after administration to a patient, wherein the one or more gene edits allows a clinician to selectively induce death of stem cells. In some embodiments, once a sufficient clinical response is seen after administration of stem cells described herein, a clinician can then selectively kill the administered stem cells.

[0156] In some embodiments, a stem cell further comprises at least one gene edit to introduce a suicide gene. As used herein, a “suicide gene” is a gene that encodes a protein capable of selectively inducing death of the cell expressing it. For example, a cell expressing a thymidine kinase can be susceptible to death in response to ganciclovir treatment, wherein a cell not expressing this kinase is not susceptible. In some embodiments, herpes simplex virus thymidine kinase (HSV-TK) converts ganciclovir (GCV) into a toxic product and allows selective elimination of TK+ cells in vitro and in vivo. Similar mechanisms are known for a variety of suicide genes.

[0157] In some embodiments, the suicide gene is inducible caspase-9, cytosine deaminase, or herpes simplex virus thymidine kinase.I. Additional exemplary gene edits

[0158] While the present disclosure describes a number of different types of gene edits that may be of interest for modified stem cells, the present disclosure is not limited to the particular gene edits described herein. For example, stem cells may comprise one or more gene edits to protect genetic integrity of transplanted stem cells. In some embodiments, a gene edit to protect genetic integrity of transplanted stem cells comprises introduction of additional copies of a tumor suppressor gene to the stem cells. In some embodiments, introducing additional copies of a tumor suppressor gene leads to increased expression of a protein that inhibits uncontrolled growth of the cell. In some embodiments, the tumor suppressor gene is TP53 (encoding the p53 protein). In some embodiments, transplanted stem cells with additional copies of TP53 have a lower risk of developing into tumors after transplantation into a patient.II. Methods of preparing modified stem cells

[0159] Described herein are methods of preparing modified stem cells. In some embodiments, a series of gene editing steps are performed to engineer stem cells (such as hESC / hiPSC) in a step-by-step fashion to result in a modified cell. In some embodiments, preparing modified stem cells comprises deleting the class I HLA genes (A, B, and C) by any method known in the art, such as, for example, CRISPR gene editing, and abolishing the class II HLA genes expression by deleting CIITF. In some embodiments, the prepared “off- the-shelf’ modified stem cells lacking class I and class II HLA genes are ready for insertion of patient-matched HLA genes. A broad overview of this method is shown in Figure 1.

[0160] In some embodiments, exogenous HLA- A, HLA-B, and / or HLA-C genes are introduced into the genome of the modified stem cells to prepare patient-matched stem cells. As used herein, “patient-matched stem cells” refers to cells that express HLA-A, HLA-B, and / or HLA-C proteins expressed from exogenous HLA-A, HLA-B, and / or HLA-C genes. In some embodiments, a patient-matched stem cell is not autologous, meaning that it is not derived from the patient. In some embodiments, the patient-matched stem cell lacks endogenous expression of class I and class II HLA genes and comprises exogenous HLA-A, HLA-B, and / or HLA-C genes matched to the patient.

[0161] In some embodiments, a method of preparing a patient-matched stem cell comprises (a) modifying a stem cell to lack expression of class I and class II HLA genes; (b) storing said modified stem cell; (c) retrieving said modified stem cell after storage; (d) inserting HLA-A, HLA-B, and / or HLA-C genes matched to those of a patient in need thereof, thereby preparing a patient-matched stem cell.

[0162] In some embodiments, modified stem cells are stored at low temperatures, such as in liquid nitrogen. In some embodiments, modified stem cells are cryopreserved, such that a user can easily access and thaw cells once a patient is identified in need of treatment. At that time, HLA-A, HLA-B, and / or HLA-C genes matched to those of the patient can be inserted into the genome of the modified stem cells. In some embodiments, this approach avoids use of autologous stem cells (i.e., cells harvested from the patient) and instead uses heterologous (i.e., harvested from a different patient) or stem cell lines in culture.

[0163] In some embodiments, patient-matched HLA genes are introduced (such as with an RMCE cassette into the genome at the AAVS1 locus or Pansio-1 locus) into stem cells that lack class I and II HLA genes. In some embodiments, further gene editing may be performed, such as editing to delete genes that are known to mediate autoimmune attack on cells (such as deletion or mutation to inhibit expression of renalase). In some embodiments, editing is performed to prepare chimeric antigen receptors (CARs) for preparing patient-matched stem cells to treat cancer. In some embodiments, editing is performed to introduce a suicide gene that can work to limit survival of patient-matched stem cells.

[0164] In some embodiments, a method of making a patient-matched stem cell comprises (a) modifying the HLA-A, HLA-B, and / or HLA-C genes comprised in the cell to block their endogenous expression; (b) modifying the CIITA gene comprised in the cell to block its endogenous expression; and (c) introducing HLA-A, HLA-B, and / or HLA-C genes with a high-fidelity integrase or without introducing a double-stranded DNA break.

[0165] In some embodiments, patient-matched HLA genes are expressed using a shuttle vector containing patient-matched HLA genes by Gibson Assembly and incorporating the matched HLA genes by RMCE. In some embodiments, a shuttle vector is prepared without using Gibson Assembly, wherein another molecular biology technique is utilized. In some embodiments, a shuttle vector containing patient-matched HLA genes can be prepared using restriction cloning or In-Fusion® seamless cloning system (Takara).

[0166] In some embodiments, HEK 293T cells are first used to test the engineering methodology, and then stem cells can be engineered using similar approaches.A. Preparation of stem cells lacking class I and class II human leukocyte antigen (HLA) genes

[0167] In some embodiments, the first step of a method prepares modified stem cells lacking class I HLA genes. In some embodiments, a method also comprises preparing modified stem cells lacking class II HLA genes. Figure 3 shows representative data showing the ability to generate a population of cells lacking expression class I HLA genes (the P4 population in these test 293 cells).

[0168] In some embodiments, after these steps to block expression of class I and class II HLA, exogenous HLA-A, HLA-B, and / or HLA-C genes may be introduced to the modified stem cell. These exogenous genes may be matched to a specific patient in need thereof.1. Blocking expression of class I HLA genes

[0169] Figure 2 A presents a representative strategy to delete class I HLA genes. This particular method uses 2 gRNA sequences to delete the HLA-A gene and 2 gRNA sequences to delete HLA-B and HLA-C together with Cas enzyme. In some embodiments, the 2 gRNA sequences to delete the HLA-A gene are SEQ ID NOs: 1 and 2, although the present method is not limited to use of these specific gRNAs. In some embodiments, the 2 gRNA sequences to delete the HLA-B and HLA-C genes are SEQ ID NOs: 3 and 4, although the present method is not limited to use of these specific gRNAs.2. Blocking expression of class II HLA genes

[0170] Figure 2B shows how 2 gRNAs together with Cas enzyme could be used to make a targeted mutation in the CIITA gene. In some embodiments, this targeted mutation is an indel (insertion or deletion) in the CIITA gene that blocks proper expression of class II HLA proteins.3. Introducing HLA-A, HLA-B, and / or HLA-C genes

[0171] In some embodiments, methods to prepare patient-matched stem cells do not introduce a double-stranded (ds) DNA break when the HLA-A, HLA-B, and / or HLA- C genes are introduced. A dsDNA break in the genome of a patient-matched stem cell could have unpredictable effects; accordingly, avoiding such a dsDNA break avoids potential unexpected effects (such as an unwanted mutation in gene that is not being targeted).

[0172] In some embodiments, methods to prepare patient-matched stem cells use a high-fidelity integrase. As used herein, a “high-fidelity integrase” refers to an integrase that facilitates repair of a double-stranded break and / or that does not activate the DNA damage repair pathway and / or does not lead to apoptosis. For example, Bxbl is a high- fidelity integrase that incurs a double stranded break, but which has higher fidelity and specificity than other CRISPR DSB gene editing methods. With Bxbl, the double-stranded break is facilitated by the integrase and quickly ligated and repaired, and thus the doublestranded break does not activate the DNA damage repair pathway and lead to apoptosis. See, for example, Xu et al., BMC Biotechnology 2013, 13:87.)

[0173] In some embodiments, 1, 2, 3, 4, 5, or 6 HLA genes are introduced to the patient. For each of HLA-A, HLA-B, and / or HLA-C, a patient may naturally have the same gene for each of their two alleles (i.e., homozygous), or the patient may naturally have different genes for their two alleles (i.e., heterozygous). Accordingly, a given patient may express up to 6 total genes for HLA-A, HLA-B, and HLA-C. Any range and combination of 1-6 HLA genes (with each gene corresponding to HLA-A, HLA-B, or HLA-C) may be introduced to a patient.

[0174] In some embodiments, the exogenous HLA-A, HLA-B, and / or HLA-C genes comprise a single HLA-A, HLA-B, or HLA-C gene. In some embodiments, the exogenous HLA-A, HLA-B, and / or HLA-C genes comprise two HLA-A genes, two HLA-B genes, or two HLA-C genes. In some embodiments, the exogenous HLA-A, HLA-B, and / or HLA-C genes comprise a single HLA-A gene and a single HLA-B gene, a single HLA-A gene and a single HLA-C gene, or a single HLA-B gene and a HLA-C gene. In some embodiments, the exogenous HLA-A, HLA-B, and / or HLA-C genes comprise one allele or two alleles for each of HLA-A, HLA-B, and HLA-C. In some embodiments, the exogenous HLA-A, HLA-B, and / or HLA-C genes comprise 4 total, 5 total, or 6 total genes.

[0175] For each of HLA-A, HLA-B, and / or HLA-C, 1 or 2 HLA genes may be introduced in some embodiments, i.e., 1 or 2 HLA-A genes, and / or 1 or 2 HLA-B genes, and / or 1 or 2 HLA-C genes. As described below, shuttle vectors can be designed that enable the combination of one, two, three, or six (all) patient-matched HLA alleles into the shuttle vector.

[0176] In some embodiments, both HLA genes of the patient are matched for HLA-A, HLA-B, and / or HLA-C. In some embodiments, two HLA-A genes are introduced. Insome embodiments, two HLA-B genes are introduced. In some embodiments, two HLA-C genes are introduced.

[0177] While there is variability in each HLA gene, in some embodiments, a patient naturally has two copies of the same HLA- A, HLA-B, or HLA-C allele. In some embodiments, a patient has 2 copies the same HLA-A allele, and thus only 1 HLA-A gene may be introduced. In some embodiments, a patient has 2 copies the same HLA-B allele, and thus only 1 HLA-B gene may be introduced. In some embodiments, a patient has 2 copies the same HLA-C allele, and thus only 1 HLA-C gene may be introduced.

[0178] In some embodiments, a patient-matched stem cell is prepared by introducing one or more HLA allele that matches a HLA gene of the patient. In some embodiments, more than one HLA gene is introduced into a cell.

[0179] In some embodiments, a single HLA gene is introduced to prepare a patient-matched stem cell. An exemplary shuttle vector design for a single HLA gene is shown in Figure 13. In some embodiments, a single HLA-A gene is introduced. In some embodiments, a single HLA-B gene is introduced. In some embodiments, a single HLA-C gene is introduced.

[0180] In some embodiments, 2 HLA genes are introduced to prepare a patient-matched stem cell. An exemplary shuttle vector design for introducing 2 HLA genes is shown in Figure 14. In some embodiments, a shuttle vector for introducing 2 HLA genes is a bicistronic vector with 3 different genes corresponding to HLA-A / B / C. In some embodiments, two HLA-A genes are introduced. In some embodiments, two HLA-B genes are introduced. In some embodiments, two HLA-C genes are introduced. Alternatively, the 2 HLA genes may be (1) HLA-A and HLA-B, (2) HLA-A and HLA-C, or (3) HLA-B and HLA-C.

[0181] In some embodiments, 3 HLA genes are introduced to prepare a patient-matched stem cell. An exemplary shuttle vector design for introducing 3 HLA genes is shown in Figure 15. In some embodiments, a shuttle vector for introducing 3 HLA genes is a tricistronic vector with 3 different genes corresponding to HLA-A / B / C. In some embodiments, a tricistronic vector with 3 different genes comprises one allele each for HLA- A, HLA-B, and HLA-C.

[0182] In some embodiments, 4, 5, or 6 HLA genes are introduced to prepare a patient-matched stem cell. For example, a patient may be heterozygous for each of HLA-A,HLA-B, and HLA-C, and introduction of 6 HLA genes allows a stem cell to match the patient’s HLA genotype. In some embodiments, a patient is homozygous for 1 HLA gene, and introduction of 5 HLA genes allows a stem cell to match the patient’s HLA genotype. In some embodiments, a patient is homozygous for 2 HLA genes, and introduction of 4 HLA genes allows a stem cell to match the patient’s HLA genotype.

[0183] A variety of different means can be used to introduce a cassette into a stem cell, with a high-fidelity integrase in some embodiments, or in other embodiments, for example, without generating a double-stranded DNA break. Any one of these methods may be used in generation of the present stem cells. In some embodiments, the patient’s HLA- matched genes are inserted using an recombinase-mediated cassette exchange (RMCE) element that has been incorporated into the stem cells. In some embodiments, the patient’s HLA-matched genes are inserted using transposon-mediated DNA insertion, as described in Strecker J. et al. Science 365:48-53 (2019) and Lampe GD. et al. bioRxiv Preprint posted March 18, 2023. In some embodiments, the patient’s HLA-matched genes are inserted using CRISPR nickase and homology-directed repair. In some embodiments, a high-fidelity integrase (such as Bxbl recombinase) is used to introduce a cassette into a stem cell.

[0184] In some embodiments, methods of preparing patient-matched stem cells do not introduce a dsDNA break. In some embodiments, introducing the HLA-A, HLA- B, and / or HLA-C genes without introducing a double-stranded DNA break comprises using a RMCE element, transposon-mediated DNA insertion, or use of a CRISPR nickase and homology directed repair. In some embodiments, a RMCE element is introduced into the cell using a CRISPR / Cas9 system.

[0185] In some embodiments, the HLA-A, HLA-B, and / or HLA-C genes are introduced into a safe harbor locus. In some embodiments, the HLA-A, HLA-B, and / or HLA- C genes are inserted into the genome at the AAVS1 locus in intron 1 of the gene that encodes PPP1R12C. In some embodiments, the HLA-A, HLA-B, and / or HLA-C genes are inserted into the genome at the Pansio-1 locus.4. Evaluating patient-matched stem cells

[0186] Natural killer cells can attack cells that lack MHC genes. Normal, healthy cells express MHC class I molecules on their surface, which contribute to the selftolerance of NK cells. Cells infected with virus or tumor cells lose surface MHC class I expression, and this loss of MHC class I expression is a signal for attack by natural killercells. This is a drawback of “hypoallergenic” stem cells that lack MHC class I, as these stem cells are prone to attack by a patient’s immune system after administration.

[0187] In the present patient-matched stem cells, the MHC class I proteins encoded by the exogenously HLA-A, HLA-B, and / or HLA-C genes of the present cells avoids this effect by natural killer cells.B. HLA matching

[0188] In some embodiments, the HLA-A, HLA-B, and / or HLA-C genes exogenously introduced into the genome of the modified stem cells are matched to those of a patient in need thereof. Figure 11 shows a representative method. In this hypothetical, a patient (such as one in need of treatment for type I diabetes) is HLA typed, a cassette is prepared comprised HLA-A, HLA-B, and HLA-C genes matched to the patient, and the cassette is introduced to modified stem cells to prepare patient-matched stem cells. These patient-matched cells can be differentiated and transplanted into the patient. In some embodiments, HLA typing is performed using a blood test or a cheek swab.

[0189] The modified stem cells can also comprise one or more gene edits, such as to incorporate protective edits that reduce autoimmune attack on the transplanted cells.C. Methods of gene editing

[0190] A variety of different gene editing techniques may be used in the present methods. In some embodiments, one or more HLA gene and / or CIITA gene is gene edited. In some embodiments, one or more protective gene edit is made using gene editing. In some embodiments, a chimeric antigen receptor, tumor suppressor gene, or a suicide gene is introduced using gene editing.

[0191] In some embodiments, one or more HLA gene and / or CIITA gene is gene edited using two flanking gRNAs are used to delete both copies of HLA-B and HLA-C simultaneously; two flanking gRNAs are used to delete both copies of HLA-A; and / or one gRNA is used to introduce null mutations in both copies of CIITA. In some embodiments, the null mutations CIITA are the null mutations are frameshift or indel mutations. In some embodiments, the one or more of the flanking gRNAs to delete HLA-A and HLA-B / HLA-C comprise SEQ ID NOs: 1-4 and / or the sequence of the one gRNA is used to introduce null mutations in both copies of CIITA is SEQ ID: 27.

[0192] In some embodiments, deletion of class I and II HLA and / or CIITA is confirmed by PCR and / or FACS analysis. For example, FACS analysis can identify cells with deletion of class I HLA genes as shown in Figure 3.

[0193] In some embodiments, the gene editing is performed using a CRISPR / Cas9 system, zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, or group one intron encoded endonucleases (GIIEEs). In some embodiments, the gene editing is CRISPR gene editing. In some embodiments, a CRISPR / Cas9 system is used for gene editing. In some embodiments, zinc-finger nucleases are used for gene editing. In some embodiments, TALENs are used for gene editing. In some embodiments, a meganuclease is used for gene editing. In some embodiments, GIIEEs are used for gene editing. In some embodiments, the meganuclease or GHEE is I-Scel, LCre, I- Anil, I-Ceul, LChuI, LCpal, I-Cpall, I-Dmol, H-Drel, I-Hmul, I-HmuII, I-Llal, I-Msol, PL Pful, Pl-PkoII, I-Porl, I-Ppol, PI-PspI, LScal, PLScel, I-Scell, LSecIII, 1-SceIV, LSceV, I- SceVI, LSceVII, I-Ssp6803I, I-TevI, I-TevII, LTevIII, PI-Tlil, PI-Tlill, I-TspO61I, or I- Vdil41I.

[0194] In some embodiments, the gene editing is within or outside of the coding region of the gene, and wherein no gene product is expressed, or wherein a nonfunctional gene product is produced.

[0195] In some embodiments, additional gene editing is also performed. In some embodiments, a method further comprises gene editing to inhibit expression of one or more of:(a) menin (SEQ ID No: 5);(b) transcription factor HIVEP2 (SEQ ID No: 6);(c) renalase (SEQ ID No: 7);(d) lengsin (SEQ ID No: 8);(e) eIF-2-alpha kinase activator GCN1 (SEQ ID No: 9);(f) perilipin-4 (SEQ ID No: 10);(g) mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: 11);(h) protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 12);(i) zinc finger BED domain-containing protein 3 (SEQ ID No: 13); and(j) metabotropic glutamate receptor 2 (SEQ ID No: 14).

[0196] In some embodiments, additional gene editing protects patient-matched stem cells from autoimmune attack.D. Methods of inserting a cassette comprising HLA genes

[0197] In some embodiments, the modified “off the shelf’ stem cells lacking HLAI and II genes can be used to prepare stem cells for a wide variety of patients, wherein HLA genes specific to each patient are inserted. In some embodiments, the patient’s HLA- matched genes are inserted using a method that avoids double-stranded breaks in the DNA or using a method with a high-fidelity recombinase. In these ways, HLA- / - cells (optionally with one or more additional gene edits) can be fully genotyped, and then HLA genes matched to the patient can be introduced into the stem cell without the risk of a double-stranded DNA break that may introduce an unwanted mutation.

[0198] In some embodiments, the HLA- A, HLA-B, and / or HLA-C genes are comprised in a cassette that also comprises other components. In some embodiments, the cassette may comprise a promoter, optionally wherein the promoter is an HLA-A promoter or elongation factor 1 -alpha 1 promoter; a marker of insertion, optionally wherein the marker of insertion is green fluorescent protein; and / or a positive selection marker, optionally wherein the selection marker is neomycin.

[0199] In some embodiments, the cassette comprising HLA-A, HLA-B, and / or HLA-C genes is prepared using Gibson assembly (as described in Gibson et al., Nature Methods 6(5):343-347 (2009)). In some embodiments, the vector is prepared without using Gibson Assembly, wherein another molecular biology technique is utilized. For example, restriction cloning can be performed to prepare the cassette, such as with Nhel, PacI, Ncol, and / or BsrGI restriction enzymes. In some embodiments, cassettes are prepared using a commercial vector designed for preparing shuttle vectors, such as the In-Fusion® seamless cloning system (Takara) that allows directional cloning of PCR fragments into linearized vectors.E. Site-specific recombination for introduction of HLA genes

[0200] In some embodiments, site-specific recombination introduces the HLA-A, -B, and / or -C into the genome of the modified stem cell, optionally via a cassette. In some embodiments, the site-specific recombination is RMCE. In some embodiments, RMCE allows for a “swap” of a cassette comprising patient-matched HLA genes into a landing-pad cassette comprised in a modified stem cell. In this way, the modified stem cell comprising thelanding-pad cassette can be stored or grown in culture until the time that a patient in need of treatment is identified and HLA typed, and then RMCE is used to introduce patient-matched HLA exogenous genes into the genome of the modified stem cell. This procedure avoids a requirement for modified stem cells to be produced from the patient’s own cells, which is considerably more difficult and time-intensive. Further, use of RMCE avoids introduction of a double-stranded DNA break when incorporating a cassette comprising patient-matched HLA genes. Alternatively, a high-fidelity integrase may be used to incorporate a cassette from a shuttle vector into a landing-pad cassette.

[0201] RMCE is one exemplary procedure allowing targeted integration using site-specific recombination processes (as described in Phan et al., Scientific Reports 7: 17771 (2017)). In some embodiments, RMCE allows for exchange of a preexisting gene cassette for an analogous cassette carrying a gene of interest. In some embodiments, RMCE allows for precise placement of an inserted DNA sequence to avoid potential deleterious effects of random insertion of a DNA sequence.

[0202] In some embodiments, a cassette comprising HLA-A, HLA-B, and / or HLA-C genes is used, optionally further comprising a site-specific recombination site at a first end of the cassette and a different site-specific recombination site at the second end of the cassette. Such site-specific recombination sites can allow for ease of RMCE. In some embodiments, a site-specific recombination site comprises loxP, mutant loxP, flippase recognition target (FRT), or att. In some embodiments an “attP” site refers to a phage site and “attB” refers to a bacterial site, wherein the attP and attB comprise a pair of site-specific recombination sites. In another example, an exemplary cassette with attP sites with different central dinucleotides (CT or GT) is shown in Figure 12.

[0203] In some embodiments, a recombinase or a pair of recombinases is expressed in the modified stem cell to mediate the RMCE reaction. In some embodiments, a high-fidelity recombinase, such as Bxbl, is used to mediate the RMCE reaction.

[0204] In some embodiments, after the HLA-A, HLA-B, and / or HLA-C genes are introduced to the stem cell, enrichment is performed to select for a cell population expressing these genes.1. Use of a landing-pad cassette

[0205] In some embodiments, methods include incorporating a landing-pad cassette into modified stem cells. As used herein, a “landing-pad construct” or “landing-padcassette” refers to a DNA sequence that is inserted into a cell, wherein the DNA sequence comprises one or more sites that allow for the sequence to be substituted with a different sequence. In some embodiments, a stem cell line comprising a landing-pad cassette can be used to allow “swap” of cassettes comprising patient-matched HLA genes easily into stem cells to generate HLA-matched stem cells from banked stem cells with the landing-pad cassette. In some embodiments, the swap is mediated by site-specific recombination sites and their associated recombinases.

[0206] The purpose of the landing-pad cassette is to ensure that a cassette is incorporated into modified stem cells, after which further methods (such as RMCE) may be used to swap a cassette comprising class I MHC genes into the location of the landing-pad cassette. In some embodiments, the landing-pad cassette (as shown in Figure 4) may have a site-specific recombination site at one end of the cassette and a different site-specific recombination site at the other end of the cassette (loxP and FRT).

[0207] In some embodiments, both the landing-pad cassette and the shuttle vector have heterotypic recombination sites differing by their central dinucleotides (such as a 5’ attP site with a CT dinucleotide and a 3’ attP site with a GT dinucleotide, as shown in Figure 12). In some embodiments, a Bxbl recombinase inserts a attP flanked shuttle vector (Figure 12) into a attB flanked landing-pad cassette with a 5’ attB site with a CT dinucleotide and a 5’ attB site with a GT dinucleotide (Figure 13).

[0208] After insertion of the landing-pad cassette, positive cells can be identified using expression of eGFP (which is comprised in the landing-pad cassette). In addition, cells can be cultured in the presence of puromycin and / or blasticidin, which will select for cells containing the landing-pad cassette comprising a neomycin resistance gene.

[0209] Figure 5 shows steps for introduction of a landing-pad cassette into cells (in this case 293 cells). After insertion of the landing-pad cassette using Cas9 into cells lacking class I and class II HLA, cells are sorted to select GFP-positive cells. After dilution, single cells are sorted and a clone of interest is picked. The clone of interest would be chosen to have normal morphology and strong puromycin and / or blasticidin resistance.

[0210] In some embodiments, a cassette comprising HLA genes is introduced using RMCE with shuttle vector and a cell comprising a landing-pad cassette. In some embodiments, an RMCE is used to allow for insertion of a cassette encoding a specific set of patient-matched HLA-A, HLA-B, and / or HLA-C genes, along with other cassettecomponents such as those allowing for selection of cells with successful insertion of the cassette. Figure 6 shows a shuttle vector comprising HLA-A, HLA-B, and HLA-C, flanked by loxp and FRT. Figures 7, 8, and 9 show a variety of shuttle vectors that can be used to insert a cassette comprising HLA genes via a method using Cre / Flp recombinases into a landing-pad cassette comprised in a modified stem cell. Figure 10 shows an alternative approach using loxP and mutant loxP (loxpmut) to mediate the RMCE.2. Insertion of RMCE landing-pad cassette

[0211] In some embodiments, a landing-pad cassette may be prepared to allow for RMCE. In some embodiments, a landing-pad cassette comprises a genetically engineered sequence containing recombinase recognition sites. In some embodiments, the ends of the landing-pad cassette recombinase recognition sites. In some embodiments, this landing-pad cassette comprises one or more gene sequence that when expressed allows for identification of cells that have integrated the landing-pad cassette.

[0212] Such a landing-pad cassette is shown at the top of Figure 7. In this exemplary cassette, the landing-pad cassette begins with a loxp site and ends with a FRT site, but other combinations of recombinase sites can be used including loxp and loxpmut as shown in Figure 10. Specific vectors with cassettes targeted to a specific safe harbor locus are available in the art, such as the hAAVSl targeting vector (pAAVSl-P-MCS, Addgene #80488).3. Enrichment of stem cells expressing exogenous HLA

[0213] In some embodiments, after the HLA-A, HLA-B, and / or HLA-C genes have been introduced to the stem cells, enrichment is performed to select for cells with HLA expression. As shown in Figure 18, cells can be enriched using magnetic activated cell sorting (MACS) using biotinylated HLA-A, HLA-B, and / or HLA-C antibody. Multiple cycles of sorting and column enrichment can be performed. The effect of 4 rounds of MACS is shown in Figure 19, which leads to a population of cells that are 99% HLA+.III. Methods of treatment

[0214] Cell therapy can successfully treat a wide range of disorders, and differentiated stem cells as described herein can be used for any such treatment. Diseases that can be treated by cell therapy include heart disease by cardiomyocytes transplantation, neurodegenerative disease by transplantation of neurons, and various blood diseases by blood cell infusion.

[0215] While the present disclosure is not limited to treatment for a specific disease, described below are representative descriptions of methods of treating diabetes or cancer with the presently claimed stem cells. Using the present state of knowledge of cell therapy, one skilled in the art would understand how to use the present stem cells for any type of such therapy.A. Methods of treating diabetes

[0216] In some embodiments, a method of treating a patient with type I or type II diabetes comprises administering patient-matched stem cells or cells differentiated from patient-matched stem cells. In some embodiments, the patient has type I diabetes. In some embodiments, the patient has type I diabetes or type II diabetes and wherein the treating lowers blood glucose, increases insulin secretion in response to glucose, and / or ameliorates other symptoms of diabetes.

[0217] In some embodiments, a method of treating or preventing diabetes in a patient, comprises (a) determining the HLA-A, HLA-B, and / or HLA-C genes endogenously expressed by the patient; (b) preparing a shuttle vector comprising a cassette comprising HLA-A, HLA-B, and / or HLA-C genes that match those endogenously expressed by the patient; (c) contacting the shuttle vector with modified stem cells in culture to allow insertion of the cassette comprising HLA-A, HLA-B, and / or HLA-C genes into the stem cell; (d) selecting for modified stem cells with insertion of the cassette; (e) transplanting the selected patient-matched stem cells into the patient; and (f) allowing the transplanted patient-matched stem cells to release insulin in the patient in response to glucose. In some embodiments, the stem cells are differentiated into cells capable of releasing insulin in culture after step (d) and before step (e). In some embodiments, the cells capable of releasing insulin are beta cells. In this way, transplanted cells capable of releasing insulin in the patient can help to reduce hyperglycemia and mediate appropriate glucose-stimulated insulin release.

[0218] In some embodiments, cells capable of releasing insulin are administered together with an additional treatment. In some embodiments, the additional treatment is insulin, a glucagon-like peptide analog or agonist, dipeptidyl peptidase-4 inhibitor, amylin analog, biguanide, thiazolidinedione, sulfonylurea, meglitinide, alphaglucosidase inhibitor, or sodium / glucose transporter 2 inhibitor.

[0219] In some embodiments, the patient-matched stem cells are administered by transplanting into the pancreas, liver, or fat pads via surgery, injection, or infusion of thepatient. In some embodiments, the patient is a mammal. In some embodiments, the patient is a human.

[0220] In some embodiments, the method further comprises making at least one gene edit before step (e) to introduce a suicide gene and / or a tumor suppressor gene. In some embodiments, the suicide gene encodes inducible caspase-9, cytosine deaminase, or herpes simplex virus thymidine kinase and / or the tumor suppressor gene encodes p53. In some embodiments, treatment with patient-matched stem cells or T cells differentiated from them can be reduced or stopped by treatment with an agent acted upon by a protein encoded by a suicide gene.B. Methods of treating cancer

[0221] In some embodiments, the patient has cancer and treatment comprises administering patient-matched stem cells to reduce growth of cancer cells. In some embodiments, a method of treating a patient with cancer comprises administering patient- matched stem cells or T cells of any one of claims to the patient.

[0222] In some embodiments, a method of treating or preventing cancer in a patient, comprises (a) determining the HLA-A, HLA-B, and / or HLA-C genes endogenously expressed by the patient; (b) preparing a shuttle vector comprising a cassette comprising HLA-A, HLA-B, and / or HLA-C genes that match those endogenously expressed by the patient; (c) contacting the shuttle vector with modified stem cells in culture to allow insertion of the cassette comprising HLA-A, HLA-B, and / or HLA-C genes into each of the stem cells; (d) selecting for patient-matched stem cells with insertion of the cassette; (e) transplanting the selected patient-matched stem cells into the patient; and (f) allowing the transplanted patient- matched stem cells to attack cancer cells in the patient.

[0223] In some embodiments, the stem cells are differentiated into T cells or natural killer cells after step (d) and before step (e). In some embodiments, the patient- matched stem cells are administered by infusion into the patient. In some embodiments, the cancer is a cancer originating from a blood cell. In some embodiments, the cancer is a leukemia or a lymphoma. In some embodiments, the patient is a mammal. In some embodiments, the patient is a human.

[0224] In some embodiments, the method further comprises making at least one gene edit before step (e) to introduce a suicide gene and / or a tumor suppressor gene. In some embodiments, the suicide gene encodes inducible caspase-9, cytosine deaminase, orherpes simplex virus thymidine kinase and / or the tumor suppressor gene encodes p53. In some embodiments, treatment with patient-matched stem cells or T cells or natural killer cells differentiated from them can be reduced or stopped by treatment with an agent acted upon by a protein encoded by a suicide gene.EXAMPLESExample 1. Preparation of human modified stem cells

[0225] Experiments will be performed to generate modified stem cells that are patient-matched. Preliminary experiments and data with non-stem cells (such as HEK 293T cells) are presented below. While a number of different means of generating modified stem cells are described herein, this representative example uses CRISPR-mediated deletion of HLA-A, B, and C genes and gene editing of RNLS to produce beta cells protected from autoimmunity, after which patient-matched HLA genes are introduced. An overview of this method is shown in Figure 1.

[0226] CRISPR technology will be used to delete HLA-A, B and C genes. The beta-2M gene will not be deleted, though many researchers do incorporate beta-2M deletion. Thus, the present method avoids deletion of beta-2B to retain functional HLA-E, F and G. Further, class II HLA genes will be removed by using CRISPR technology to delete CIITF, a master transcription factor that controls class II HLA gene expression.

[0227] As illustrated in Figure 2, a representative method involves three steps of gene editing. First, 2 flanking gRNAs will be used to delete both copies of HLA-B and C simultaneously. Then, 2 flanking gRNAs will be used to delete both copies of the HLA-A gene (Figure 2A). Finally, one single gRNA that targets the first exon of human CIITA will be used to introduce indel (i.e., insertion or deletion) and / or frameshift mutations to generate null mutation on both copies (Figure 2B). The gRNAs to edit human HLA loci have been designed and validated (3), and these well-characterized gRNAs can be used for HLA locus editing.

[0228] The successful deletion of class I and II HLA will be confirmed by genome PCR and HLA expression loss using FACS analysis. As discussed below, preliminary experiments with these gRNAs were performed using 293T cells to provide proof-of-concept (Figure 3A).

[0229] A RMCE landing-pad cassette can then be introduced into one of human safe harbor loci, such as the human adeno-associated virus integration site 1 (AAVS1)locus. As shown in Figure 4 (providing a representative example of a cassette for use with RMCE), the splicing acceptor (SA)-T2A-puroR comes from the hAAVSl targeting vector (pAAVSl-P-MCS, Addgene #80488). The thosea asigna virus 2A (T2A) “self-cleaving” peptide sites mediate “cleavage” of polypeptides during translation in eukaryotic cells and allows for expression of separate polypeptides of HLA-A, HLA-B, and HLA-C using TSA1, TSA2, TSA3, and TSA4(as described in Liu et al. Scientific Reports 7: 2193 (2017)).

[0230] In this targeting vector, a human HLA-A promoter (Ikb) will be followed by a single loxP site. After the loxP, cloning will include a CMV promoter controlling EGFP, T2A and dTK-neoR genes, followed by an FRT site, three stop codons in different reading frames, and a polyA terminator. The neoR gene (encoding neomycin resistance) will be used for positive selection, and the dTK thymidine kinase gene will be used for negative selection. The EGFP gene serves as an indicator of successful insertion into the genome at the AAVS1 locus . Also, loss of EGFP indicates a successful RMCE where the DNA region between loxP and FRT is replaced by HLA alleles from the cassette. The assembled pAAVSl targeting vector will be used to inserted into the human genome at the AAVS1 locus using CRISPR technology, as described below. The successful incorporation of the RMCE cassette will be confirmed by genome PCR, puromycin and neomycin resistance, and EGFP imaging. This targeting vector will be tested in 293T cells first, and then applied to hESC / hiPSC.

[0231] The next step will be to introduce gene edits into the genome of the modified stem cells that can protect from autoimmunity. In the case of stem cells for preparing beta cells, gene editing can comprise mutation to inhibit expression of renalase (encoded by RNLS). A first gene editing target will be RNLS in HLA7', RMCE-ready hESC / hiPSC. Two gRNAs were designed to be used to delete the exon 2 of RNLS, which results in a complete loss-of-function of RNLS gene. Deletion of both copies of RNLS will be confirmed by genome PCR, sequencing and western blot of renalase protein. Alternatively a RNLS- / - hiPSC cell line (4) may be used followed by HLA-A, HLA-B, and HLA-C deletion.

[0232] Most people have two different alleles of HLA-A, B, and C to present a greater variety of peptide epitopes on the class I HLA molecules for maximum immune protection. Assembly of 6 different HLA genes in the shuttle vector might be technically difficult and inefficient. However, some people are homozygote for class I HLA genes, suggesting a single copy of HLA-A, -B and -C already provides sufficient level of immuneprotection. Therefore, experiments can begin by assembling only one copy of patient- matched HLA-A, -B and -C gene in the shuttle vector. A shuttle vector can be used containing a loxP site at its 5’ end and an FRT site at its 3’ end, with two distinct synonymous DNA fragment encoding T2A peptide (T2A1 and T2A4) in between. For a certain group of HLA-A, B and C alleles matching those of a patient, synthesis and PCR amplification will be performed of each allele with two flanking synonymous T2A DNA sequences (as shown in Figure 6, T2A1 and T2A2 flanking HLA-A, T2A2 and T2A3 flanking HLA-B, and T2A3 and T2A4 flanking HLA-C). The empty shuttle vector will be linearized with two BsmBI enzyme digestion to expose T2A1 and T2A4, and Gibson Assembly will be used to construct the final shuttle vector containing the HLA-A, B, and C genes connected by T2A peptide sequence, flanked by loxP and FRT sites. Cre recombinase recognizes the loxP site, whereas FLP recombinase recognizes the distinct FRT site. As a pilot experiment, donor PBMCs from control peripheral blood mononuclear cells (PBMC) were obtained with HLA type of A*03:01, A*31:01, B*15:01, B*51:01, C*07:04, and C* 15:02. Various shuttle vectors containing different combinations of these HLA-A, B, and C alleles will be assembled for later testing.

[0233] The fully assembled shuttle vector will be linearized by restriction enzyme digestion and transfected or electroporated into the HLA- / -, RMCE-ready human cells together with a plasmid expressing both Cre and FLP (e.g. pDIRE, Addgene #26745 (Osterwalder M et al., Nat Methods. 2010;7(l 1): 893-5)). The HLA- / -, RMCE-ready 293T cells will be used for an initial test and then further experiments will be performed with the hESC / hiPSC lines with or without the RNLS deletion. After transfection, the cells will be selected for loss of dTK cassette by ganciclovir treatment and also confirmed by loss of EGFP expression. Genome PCR evaluation will also be used to make sure the transgenes assembly and orientation is correct as designed.

[0234] For all the hESC / hiPSC lines, genome integrity will be determined to be intact and that there is no unwanted DNA mutation introduced by CRISPR gene editing. For this purpose, the HLA- / - line; the HLA- / -, RMCE-ready line; and the HLA- / -, RMCE- ready, RNLS- / - line with or without HLA gene insertion will be subjected to karyotyping and genome sequencing of potential off-targeting loci by the gRNAs used in the engineering. Whole genome DNA sequencing will also be performed to ensure no excessive unwanted mutations exist in these cell lines.

[0235] For all the engineered hESC / hiPSC lines, their embryonic stem cell identity and pluripotency will be verified to ensure that the cells still have full potential of differentiation into different cell lineages. To evaluate the embryonic stem cell identity, the HLA- / - line; HLA- / -, RMCE-ready line; and HLA- / -, RMCE-ready, RNLS- / - with or without HLA gene insertion hESC / hiPSC line will be examined for the expression of embryonic stem cell (ESC) markers including NANOG, OCT4, SSEA3 / 4 and TRA-1-60 by immunostaining. To check the pluripotency and differentiation potential of the cell lines, these cell lines also will be differentiated into three germ layers: endoderm, mesoderm and ectoderm lineages, and the differentiation will be evaluated by quantification of lineage specific markers (e.g. AFP and Soxl7 for endoderm lineage, Brachyury and Flkl for mesoderm lineage, and Map2 and Pax6 for ectoderm lineage).

[0236] The allo-immunogenicity of the engineered hESC / hiPSC lines with patient-matched HLA insertion will be measured (Xu H et al., Cell Stem Cell. 2019;24(4):566-78 e7). Basically, the patient-matched HLA-matched hESC / hiPSC (HLA matched to the donor PBMC) will be differentiated into CD43+blood cells, and then stimulated by IFN-y. CD8+T cells will be purified from the donor PBMCs, co-cultured with the CD43+blood cells, and treated with IL-2. The allo-immunogenic CD8+T cells will be primed at this step and start to proliferate. A cell proliferation by flow cytometry assay (such as one using carboxyfluorescein succinimidyl ester (CFSE)) will be used to quantify CD8+T cell proliferation, which reflects the activity of allo-immunity. The activated CD8+T cells will then be purified by FACS sorting and co-cultured with IFN-y stimulated CD43+blood cells differentiated from the engineered hiPSC.51Cr release assay will be used to quantify the CD8+T cells cytotoxicity. In these experiments, unedited hiPSC (HLA mismatched with the donor PBMCs) and HLA ' hiPSC will be used as positive and negative controls, respectively.

[0237] Natural killer (NK) cells react to cells with low or no class I HLA molecules. To examine whether or not HLA-inserted hiPSC line will trigger NK cell response, NK cells will be purified from the donor PBMCs. These NK cells will be incubated with CD43+blood cells differentiated from the HLA matched hiPSC. The activation of NK cells will be quantified by CD107a expression by FACS analysis. In addition,51Cr release assay will also be performed to evaluate the cytotoxicity of NK cells against the hiPSC derived CD43+blood cells.

[0238] Finally, gene editing (e.g. RNLS deletion) will be evaluated as a means to protect hiPSC derived beta-like cells from autoimmune destruction. To do this for renalase, T1D patients PBMCs or established autoimmune T cell lines see, for example, Babon JA et al., Nat Med. 2016;22(12): 1482-7 and Kent SC et al. Nature. 2005;435(7039):224-8) can be used. The flexibility of the present HLA engineering strategy makes this type of testing possible and easier because there is no need to derive individual hiPSC lines for each donor PBSCs or T cell lines. The class I HLA alleles for the donor PBSCs or T cell lines can be typed and then the hiPSC can be engineered with matched HLA. The HLA-matched RNLS' ' hiPSC will be differentiated into beta-like cells. If donor PBMCs will be used for the assay, the autoimmune CD8+T cells activation will be evaluated by ELISPOT assay for IFN-y expression. Autoimmune T cell lines activation and cytotoxicity will be examined by ELISPOT assay and51Cr release assay. The HLA' ' hiPSC derived beta-like cells will be used as control in these experiments.

[0239] Figure 11 shows an overview of this general strategy, from HLA typing of a patient in need of treatment, preparation of stem cells patient-matched HLA genes using a bank of stem cells lacking HLA-A, -B, and -C, and administration of these stem cells to a patient. Depending upon the type of treatment desired, additional gene edits may be performed on the banked stem cells. For example, for treatment of T1D, autoimmune protective gene editing may also be performed on the cells. Alternatively, banked stem cells may have editing to express chimeric antigen receptors, if the patient is in need of treatment for cancer.

[0240] In any of the methods described herein, gene editing may also introduce a suicide gene, such as a tyrosine kinase. In this embodiment, transplanted stem cells can be killed by administration of an agent that yields a toxic metabolite on exposure to a tyrosine kinase, allowing for selective killing of the patient-matched stem cells after administration to a patient. Such an approach with a suicide gene could be a measure to control the duration of therapy with patient-matched stem cells.Example 2. Cell line experiments

[0241] A number of pilot experiments were performed to show proof-of- concept of the present methods in cell lines. Using the strategy and single-guide RNAs described in Figure 2A, experiments were performed to delete Class I HLA genes in human HEK 293 cells. Two sgRNAs were used to delete the HLA-A gene (HLA-A-5’ sgRNA andHLA-A-53’ sgRNA, SEQ ID NOs: 1 and 2, respectively), and then another two sgRNA were used to delete HLA-B and C together (HLA-B-5’ sgRNA and HLA-C-3’ sgRNA, SEQ ID NOs: 3 and 4, respectively).

[0242] This procedure is shown in Figure 5, wherein a plasmid carrying SpCas9 and sgRNA was co-transfected into 293 cells in two consequent steps (HLA-A first and then HLA-B / C). WT-293 cells or transfected 293 cells were then analyzed by FACS with pan-HLA class I (recognizing HLA-A, B and C) staining. A population of the Cas9 / sgRNA transfected 293 cells lost all their class I HLA expression (P4, shown in the boxed region), and these cells were then sorted out and cloned as HLA-null cell line. These data indicate that gRNAs described herein can be used to delete Class I HLA genes. A similar approach can be used to delete CIITA, as shown in Figure 2B.

[0243] Next, a strategy was designed to insert the RMCE cassette into the human genome at the AAVS1 locus using a landing-pad cassette, such as the cassette as shown in Figure 6. This representative landing-pad cassette includes a HLA-A promoter (this can also be replaced by other ubiquitous promoter, such as EFla promoter), followed by a loxP and FRT flanked cassette expressing eGFP, dTK and neomycin. Alternatively, other ubiquitous promoters, such as the EFla promoter, can be used. The loxP and FRT (flippase recognition target) sites allow for site-directed recombination later in methods, to allow insertion of a patient-matched cassette with HLA genes without a need for generating a random break in the DNA.

[0244] eGFP and neomycin can be used to identify cells with successful introduction of the landing-pad cassette. Later in methods, GFP -positive cells can also be identified as cells that failed to recombine after RMCE. Further, dTK (delta Thymidine Kinase) can be used to specifically remove non-recombined cells from the culture by treatment with ganciclovir. The p2A site allows for expression of separate GFP and dTK proteins.

[0245] The landing-pad cassette was cloned into the pAAVSl-P-MCS vector using PacI and Sall, and the AAVS1 locus insertion is facilitated by use of the pXAT2 vector expressing SpCas9 and AAVS1 -targeting sgRNA (T2) to coordinate introduction of the landing-pad cassette into this safe harbor locus. Alternatively, other safe harbor loci may be used, as generally any loci in the human genome that supports safe and efficient transgene expression, without detriment to cellular functions, may be used.

[0246] Successful insertion of the landing-pad (LP) cassette is shown in Figure 5. The HLA-null 293 cells were transfected with landing-pad cassette plasmid and pXAT2, and FACS analysis was used to identify a population of cells that are GFP positive (P4). GFP+ cells were sorted out of the population, and then single GFP+ cells were sorted via dilution. GFP+ cells were then single-cell cloned into different cell lines. (A2, DIO etc.). A range of different GFP+ clones are shown in Figure 5. Cells for further experiments were selected based on having normal morphology and strong puromycin and / or blasticidin resistance.

[0247] Next, a shuttle vector was prepared with plasmid comprising patient- matched HLA genes (pHLA). The pHLA empty shuttle vector has a loxP and FRT flanked spacer, with two T2A sequence (T2A1 and T2A4) each side of the spacer. The T2A sequences do not translate protein and instead allow for easier HLA cloning. Patient-matched HLA genes were PCR amplified with two flanking T2A sequences (HLA-A is flanked by T2A1 and T2A2, HLA-B with is flanked by T2A2 and T2A3, and HLA-C is flanked by T2A3 and T2A4). While the different T2A sequences differ at the DNA sequence, they each encode the same protein sequence.

[0248] The Golden Gate assembly technique (as described in Engler et al., PLOS ONE 3(1 l):e3647 (2008)) was used to clone three HLA genes into empty pHLA shuttle vector, as shown in Figure 6. Experiments showed that cloning efficiency decreased as the number of alleles in the shuttle vector increased, as expected. The cloning efficiency is -80% when only one HLA gene was cloned, while rates dropped to -63% and 30%, respectively, when two or three HLA genes were simultaneously cloned. A given patient may be homozygous for any of HLA-A, -B, and -C (i.e., expressing 2 copies of the same alleles for HLA-A, -B, and -C) or heterozygous for any of HLA-A, B, and C (i.e., expressing 2 different alleles of HLA-A, B, and C, for a total of 6 different HLAs). Up to 6 HLA genes may be cloned into the shuttle vector using additional T2A sequences.

[0249] Figure 7 shows a schematic of how RMCE may be used to insert the cassette sequence in the shuttle vector into the landing-pad cassette in cells. The RMCE can be mediated by Cre / FLP enzymes, using the loxp and FRT sites in the landing-pad cassette and shuttle vector, or other pairs of site-specific recombination sites may be used. By this process, the landing-pad located in the AAVS1 locus in the cell is replaced with the cassette in the shuttle vector using Cre / FLP recombinase. In this way, the HLA-A, -B and -C genesmatched to a given patient are expressed under control of the HLA promoter. Alternatively, a different promoter such as an EFla promoter may be used to control expression of the HLA proteins.

[0250] Pilot experiments were performed using an mCherry shuttle vector to allow for easy identification of cells with incorporation of the cassette from the shuttle vector (Figure 8). The landing-pad vector and the mCherry shuttle vector were co-transfected into 293 cells with or without Cre / FLP expression vector, without the cells without Cre / FLP serving as a negative control. FACS analysis showed that approximately 1.3% of the cells subjected to this co-transfection with Cre / FLP expression start to express mCherry protein. Cells only subjected to the LP showed 0% mCherry positive cells, and cells subjected to the LP plus SV (without Flp / Cre) showed 0.2% mCherry positive cells. These results indicate that the use of the landing-pad and shuttle vector constructs can lead to successful integration of a desired cassette (in this case one encoding for mCherry) into the genome of cells.

[0251] Next, experiments were performed to assess the ability to introduce HLA-A into HEK cells using the LP / SV system with Cre / Flp. RMCE efficiency was assessed using a shuttle vector comprising a HLA-A gene (shown in Figure 9). The landing-pad vector and HLA-A shuttle vector were co-transfected into HEK 293 HLA-inserted (KO) cells with or without Cre / FLP expression vector. FACS analysis showed approximately 1.58% of the cells that are co-transfected with Cre / FLP expressed HLA-A protein (Double+ in the LP+SV+Cre / Flp group).

[0252] An alternative means of inserting a cassette from a shuttle vector using loxP-loxPmut was also tested, as shown in Figure 10. While Figure 9 showed data with Cre / Flp, a variety of different RMCE protocols could be performed using pairs of sitespecific recombination sites, such as loxP / FRT, loxP / loxPmut, or other choices such as att sites that can be used by SV1 integrase. A loxP-LoxPmut landing-pad vector and a shuttle vector with a cassette comprising mCherry were co-transfected into HEK293 cells with or without Cre expression vector. FACS analysis shows that approximately 0.7% of the cells that are co-transfected with Cre begin to express mCherry protein (LP+SV+Cre group).

[0253] These pilot experiments prove that HLA genes can be successfully inserted into cells after modification of endogenous HLA genes. These data support the present methods of preparing HLA genes matched to patients using a bank of stem cells lacking HLA-A, -B, and -C genes.Example 3. Additional landing pad and shuttle designs

[0254] Additional designs were developed for using Bxbl recombinase to insert an attP flanked shuttle vector into an attB flanked landing pad. Both the landing pad and shuttle vector have heterotypic sites differing by their central dinucleotides, with the 5’ site comprising a CT dinucleotide and the 3’ site comprising a GT dinucleotide.

[0255] A representative landing pad design is shown in Figure 12, with a 5’ attP site comprising CT and a 3’ attP site comprising GT. Such a landing pad can be used with a variety of different HLA shuttle vectors comprising a 5’ attB site comprising CT and a 3’ attB site comprising GT as shown in Figures 13-16. The different shuttle vectors may be selected based on the number of HLA genes (i.e., a HLA-A, HLA-B, or HLA-C coding sequence (CDS)) that a user wants to introduce to a patient, often wherein the user wants to introduce 1-6 HLA genes. In these representative designs, HLA-A / B / C CDS can refer to any DNA coding sequence for any HLA-A, HLA-B, or HLA-C gene alleles. These designs enable the combination of one, two, three, or six alleles of patient matched HLA into the shuttle vector. Six alleles can represent all the patient’s natural alleles for HLA-A, HLA-B, and HLA-C.

[0256] For example, the shuttle vectors of Figure 13 can be used to insert a single HLA CDS into the landing pad shown in Figure 12. Similarly, shuttle vectors can be used to insert 2 HLA CDS (Figure 14, a bicistronic design), 3 HLA CDS (Figure 15, a tricistronic design), or 6 HLA CDS (Figure 16, a hexacistronic design), though shuttle vectors for introducing any number of HLA genes may be designed.

[0257] In each of the shuttle vector designs of Figures 13-16, a TSA site is present between different HLA CDS to allow for them to be expressed as separate proteins. Any polyA terminator (pA) can be used for this purpose, including those represented by SEQ ID NOs: 28-32.

[0258] Figures 17 and 18 show representative data on transfection efficiency of the landing pad (Figure 17) and shuttle vector (Figure 18). Transfection efficiency of landing pad insertion was measured via genotyping, while transfection efficiency of the shuttle vector was measured by its integration. The Clover reporter gene (a constitutively fluorescent green / yellow fluorescent protein) was comprised in the shuttle vector construct to allow for assessment of integration. The shuttle vector was transfected together with the Bxbl integrase into heterologous stem cells using Lipofectamine Stem (Thermo Fisher) at thedifferent mass conditions shown in Figure 18. Efficient integration of the shuttle vector was seen over a range of different shuttle vector / Bxbl concentrations.Example 4. Enrichment of HLA+ stem cells

[0259] Enrichment protocols can be used to significantly increase the percentage of modified stem cells that express one or more HLA protein. As shown in Figure 19, a shuttle vector comprising one or more HLA CDS can be transfected with Lipofectamine Stem into stem cells lacking HLA-A, HLA-B, and HLA-C genes, and after 2 days approximately 1% of cells will be HLA+. Then two rounds of magnetic activated cell sorting (2X MACS) using biotinylated HLA-A / B / C antibody over a Miltenyi magnetic separation (MS) column can be performed, with the antibody chosen based on which HLA CDS was present in the shuttle vector. In a representative example, two rounds of sorting (one after 5 days and one after 3 weeks) can be used with both rounds enriched over two columns for higher purity as shown in Figure 19.

[0260] Figure 20 shows representative data wherein, HLA knockout (KO) landing pad (LP) cells had 0.69% HLA+ cells, which increased to 1.29% after transfection of the shuttle vector. The HLA+ percentage increased to 44.6% after an initial 2 rounds of MACS and then to 99% HLA+ after the full 4 rounds of MACS. Thus, the enrichment leads to a cell population highly enriched for expression of exogenous HLA genes. Figure 21A shows that endothelial cell expression markers CD144 and CD31 of the stem cells were retained after the enrichment for HLA expression. Figure 2 IB shows that expression of inserted HLA genes is still seen in these differentiated cell types when compared to A2 homozygous wild type cells or HLA knockout cells.EQUIVALENTS

[0261] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describe the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0262] As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., + / -5-10% of the recitedrange) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.

Claims

What is Claimed is:

1. A method of preparing a patient-matched stem cell comprising:(a) modifying a stem cell to lack expression of class I and class II human leukocyte antigen (HLA) genes;(b) storing said modified stem cell;(c) retrieving said modified stem cell after storage; and(d) inserting HLA-A, HLA-B, and / or HLA-C genes matched to those of a patient in need thereof, thereby preparing a patient-matched stem cell; and(e) optionally selecting for expression of the HLA-A, HLA-B, and / or HLA-C genes by the patient-matched stem cell, optionally wherein the selecting is by magnetic activated cell sorting using antibodies to HLA-A, HLA-B, and / or HLA-C.

2. A stem cell produced by the method of claim 1.

3. A patient-matched stem cell, wherein the stem cell:(a) is not autologous;(b) lacks endogenous expression of class I and class II HLA genes; and(c) comprises exogenous HLA-A, HLA-B, and / or HLA-C genes matched to those of a patient in need thereof.

4. The method of claim 1 or stem cell of claim 2 or 3, wherein:(a) the exogenous HLA-A, HLA-B, and / or HLA-C genes comprise a single HLA- A, HLA-B, or HLA-C gene;(b) the exogenous HLA-A, HLA-B, and / or HLA-C genes comprise two HLA-A genes, two HLA-B genes, or two HLA-C genes;(c) the exogenous HLA-A, HLA-B, and / or HLA-C genes comprise a single HLA- A gene and a single HLA-B gene, a single HLA-A gene and a single HLA-C gene, or a single HLA-B gene and a HLA-C gene;(d) the exogenous HLA-A, HLA-B, and / or HLA-C genes comprise one allele or two alleles for each of HLA-A, HLA-B, and HLA-C genes, optionally wherein the exogenous HLA-A, HLA-B, and / or HLA-C genes comprise 4 total, 5 total, or 6 total different genes;(e) the stem cell does not trigger attack by natural killer cells; and / or(f) the HLA-A, HLA-B, and / or HLA-C genes are comprised in a cassette, wherein the cassette optionally further comprises:(i) a promoter, optionally wherein the promoter is an HLA-A promoter or elongation factor 1 -alpha 1 promoter;(ii) a marker of insertion, optionally wherein the marker of insertion is green fluorescent protein; and / or(iii) a positive selection marker, optionally wherein the selection marker is neomycin.

5. The method or stem cell of claim 4, wherein the cassette:(a) is inserted into the genome of the stem cell either (1) with a high-fidelity recombinase, optionally wherein the high-fidelity recombinase is Bxbl or (2) without introducing a double-stranded DNA break, optionally wherein inserting the cassette into the genome of the stem cell without introducing a double-stranded DNA break is performed via a recombinase-mediated cassette exchange (RMCE) element, transposon-mediated DNA insertion, or use of a CRISPR nickase and homology directed repair, optionally wherein the RMCE element is introduced into the cell using a CRISPR / Cas9 system;(b) is inserted into the genome of the stem cell at the adeno-associated virus integration site 1 (AAVS1) locus in intron 1 of the gene that encodes protein phosphatase 1 regulatory subunit 12C (PPP1R12C) or at the Pansio-1 locus;(c) further comprises a site-specific recombination site at a first end of the cassette and a different site-specific recombination site at the second end of the cassette, optionally wherein a site-specific recombination site comprises loxP, mutant loxP, flippase recognition target (FRT), or at and / or(d) is prepared using Gibson assembly, In-Fusion cloning, Golden Gate assembly or restriction cloning.

6. The method or stem cell of any one of claims 1-5, wherein:(a) the stem cell is an adult stem cell, pluripotent stem cell, embryonic stem cell, hematopoietic stem cell, bone marrow stromal stem cell, or mesenchymal stem cell, optionally wherein the stem cell is a human embryonic stem cell or induced pluripotent stem cell;(b) the beta 2-microglobulin gene is not modified in the stem cell; and / or(c) the major histocompatibility complex (MHC) Class II transactivator (CIITA) gene in the stem cell has been modified to block expression of class II HLA genes, optionally wherein the class I HLA genes and / or CIITA gene have been modified by gene editing.

7. The method or stem cell of claim 6, wherein:(a) two flanking gRNAs are used to delete both copies of HLA-B and HLA-C simultaneously;(b) two flanking gRNAs are used to delete both copies of HLA- A; and / or(c) one gRNA is used to introduce null mutations in both copies of CIITA.

8. The method or stem cell of claim 7, wherein:(a) the null mutations are frameshift or indel mutations; and / or(b) the one or more of the flanking gRNAs comprise SEQ ID NOs: 1-4, optionally wherein the flanking gRNAs each comprise one of SEQ ID NOs: 1-4 and / or the sequence of the one gRNA is used to introduce null mutations in both copies of CIITA is SEQ ID: 27.

9. The method or stem cell of any one of claims 1-8, wherein:(a) the method further comprises confirming deletion of class I and II HLA and / or CIITA by PCR and / or FACS analysis; and / or(b) the stem cell further comprises at least one gene edit to inhibit expression of one or more of:(i) menin (SEQ ID No: 5);(ii) transcription factor HIVEP2 (SEQ ID No: 6);(iii) renalase (SEQ ID No: 7);(iv) lengsin (SEQ ID No: 8);(v) eIF-2-alpha kinase activator GCN1 (SEQ ID No: 9);(vi) perilipin-4 (SEQ ID No: 10);(vii) mediator of RNA polymerase II transcription subunit 11 (SEQ ID No: i i);(viii) protein-glutamine gamma-glutamyltransferase 6 (SEQ ID No: 12);(ix) zinc finger BED domain-containing protein 3 (SEQ ID No: 13); and(x) metabotropic glutamate receptor 2 (SEQ ID No: 14).

10. The method or stem cell of claim 9, wherein:(a) the gene editing is performed using a CRISPR / Cas9 system, zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, or group one intron encoded endonucleases (GIIEEs), optionally wherein the gene editing is CRISPR gene editing; and / or(b) the gene editing is within or outside of the coding region of the gene, and wherein no gene product is expressed, or wherein a non-functional gene product is produced.

11. The method or stem cell of any one of claims 1-10, wherein:(a) the patient is a mammal, optionally wherein the mammal is a human;(b) the patient has cancer, heart disease, a neurodegenerative disease, type I or type II diabetes, or a blood diseases, optionally wherein the diabetes is type I diabetes;(c) the stem cell further comprises at least one gene edit to introduce a chimeric antigen receptor, optionally wherein the patient has cancer, optionally wherein the stem cell is differentiated into a T cell or natural killer cell;(d) the stem cell further comprises at least one gene edit to introduce one or more MHC class II proteins, optionally wherein the stem cell is differentiated into a dendritic cell, mononuclear phagocyte, endothelial cell, thymic epithelial cell, or B cell; and / or(e) the stem cell further comprises at least one gene edit to introduce a suicide gene and / or a tumor suppressor gene, optionally wherein the suicide gene encodes inducible caspase-9, cytosine deaminase, or herpes simplex virus thymidine kinase and / or the tumor suppressor gene encodes p53.

12. The method or stem cell of claim 11, wherein the patient-matched stem cell is differentiated so that it is capable of releasing insulin, optionally wherein the differentiated cell is a beta cell.

13. A method of treating a patient with type I or type II diabetes comprising administering stem cells of any one of claims 1-11 or cells capable of releasing insulin of claim 12 to the patient, optionally wherein the treating lowers blood glucose, increases insulin secretion in response to glucose, and / or ameliorates other symptoms of diabetes.

14. A method of treating or preventing diabetes in a patient, comprising:(a) determining the HLA-A, HLA-B, and / or HLA-C genes endogenously expressed by the patient;(b) preparing a shuttle vector comprising a cassette comprising exogenous HLA- A, HLA-B, and / or HLA-C genes that match those endogenously expressed by the patient;(c) contacting the shuttle vector with modified stem cells of any one of claims 1- 11 in culture to allow insertion of the cassette comprising HLA-A, HLA-B, and / or HLA-C genes into the genome of the modified stem cell, thereby preparing patient- matched stem cells;(d) selecting for patient-matched stem cells with insertion of the cassette;(e) transplanting the selected patient-matched stem cells into the patient; and(f) allowing the transplanted stem cells to release insulin in the patient in response to glucose.

15. The method of claim 14, wherein:(a) the method further comprises selecting for expression of the HLA-A, HLA-B, and / or HLA-C genes by the patient-matched stem cells after step (d) and before step (e), optionally wherein the selecting is by magnetic activated cell sorting using antibodies to HLA-A, HLA-B, and / or HLA-C;(b) the method further comprises differentiating the stem cells into cells capable of releasing insulin in culture after step (d) and before step (e), optionally wherein the cells capable of releasing insulin are beta cells;(c) the method is for use with an additional treatment, optionally wherein the additional treatment is insulin, a glucagon-like peptide analog or agonist, dipeptidyl peptidase-4 inhibitor, amylin analog, biguanide, thiazolidinedione, sulfonylurea, meglitinide, alpha-glucosidase inhibitor, or sodium / glucose transporter 2 inhibitor; and / or(d) the transplanting is into the pancreas, liver, or fat pads via surgery, injection, or infusion.

16. A method of treating a patient with cancer comprising administering patient-matched stem cells of any one of claims 1-11, to the patient, optionally wherein the patient has cancer and wherein the treating reduces growth of cancer cells.

17. A method of treating or preventing cancer in a patient, comprising:(a) determining the HLA-A, HLA-B, and / or HLA-C genes endogenously expressed by the patient;(b) preparing a shuttle vector comprising a cassette comprising HLA-A, HLA-B, and / or HLA-C genes that match those endogenously expressed by the patient;(c) contacting the shuttle vector with modified stem cells of any one of claims 1- 11 in culture to allow insertion of the cassette comprising exogenous HLA-A, HLA- B, and / or HLA-C genes into of the genome of the modified stem cells, thereby preparing patient-matched stem cells;(d) selecting for patient-matched stem cells with insertion of the cassette;(e) transplanting the patient-matched stem cells into the patient; and(f) allowing the transplanted stem cells to attack cancer cells in the patient.

18. The method of claim 18, wherein:(a) the method further comprises selecting for expression of the HLA-A, HLA-B, and / or HLA-C genes by the patient-matched stem cells after step (d) and before step (e), optionally wherein the selecting is by magnetic activated cell sorting using antibodies to HLA-A, HLA-B, and / or HLA-C;(b) the method further comprises differentiating the stem cells into T cells after step (d) and before step (e); and / or(c) the transplanting is performed by infusion into the patient.

19. The method of any one of claims 13-18, further comprising making at least one gene edit before step (e) to introduce a suicide gene and / or a tumor suppressor gene, optionally wherein suicide gene encodes inducible caspase-9, cytosine deaminase, or herpes simplex virus thymidine kinase and / or the tumor suppressor gene encodes p53.

20. Use of a stem cell of any one of claims 1-12 in the manufacture of a medicament to treat cancer, heart disease, a neurodegenerative disease, type I or type II diabetes, or a blood diseases in a human patient in need thereof.

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