Engineered cells for treating diabetes

Abstract

Provided herein are, inter alia, genetically engineered human pluripotent stem cells, beta cells derived from genetically engineered human pluripotent stem cells, compositions, and cell aggregates, and methods of treating diabetes. The human pluripotent stem cells and beta cells provided herein including embodiments thereof may be genetically modified to express exogenous genes and / or inactivate endogenous genes.

Description

ENGINEERED CELLS FOR TREATING DIABETESRELATED APPLICATION DATA

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application No. 63 / 719,060, filed on November 11, 2024, which is hereby incorporated by reference in its entirety and for all purposes.SEQUENCE LISTING

[0002] The material in the accompanying Sequence Listing is hereby incorporated by reference in its entirety. The accompanying file, named “048440-851001WO_SL_ST26. xml” was created on November 11, 2025, and is 29,849 bytes.BACKGROUND

[0003] Diabetes arises from dysfunction or death of islet beta cells. Cell replacement therapy seeks to overcome this by isolating human islets from donor pancreata and infusing them into recipient’s liver. While islet transplantation provides Type 1 diabetes (T1D) patients with an insulin-free life and resolves possibly fatal hypoglycemia, this approach is not a panacea. The major barrier for many qualified patients remains the limited donor source. In average, islets isolated from two donors are required for each recipient. Many individuals require multiple transplantations to achieve insulin independence and islet function still deteriorates over time. A majority of T1D individuals show a loss of islet function within five years of transplantation. Even with chronic immune suppression, only half of the transplanted patients remained insulin free at five years. This appears to be related to alloimmune rejection, autoimmune rejection and / or islet exhaustion as a result to their placement in the liver. Thus, major factors impacting the availability and success of islet cell replacement therapy include donor shortage, immune rejection and site of transplant. There is a significant need, therefore, to develop a source for unlimited supply of betalike cells from stem cells to address the issue of islet supply shortage, develop strategies for protection of these cells from the immune reaction, and exploring alternative sites for their placement outside the liver. At least one billion insulin-producing beta-like cells may be required for transplantation to restore glucose homeostasis in individuals with T1D. In addition, cell grafts that contain any residue undifferentiated or partially differentiated hPSCs could pose a risk for malignancy. Thus, there is a need in the art for safe and effective hPSCs to treat diabetes. ThePATENTAttorney Docket No. 048440-851001 WO disclosure is directed to this, as well as other, important ends.BRIEF SUMMARY

[0004] In an aspect is provided a human pluripotent stem cell including a mutant sr39 HSV-TK protein-encoding gene.

[0005] In another aspect is provided a human pluripotent stem cell including an inactivated TPH1 gene.

[0006] In another aspect is provided a human pluripotent stem cell including: (i) an inactivated B2M gene; and (ii) an inactivated CIITA gene.

[0007] In another aspect is provided a human pluripotent stem cell including: (i) an inactivated B2M gene; (ii) an HLA-E gene; and (ii) an inactivated CIITA gene.

[0008] In another aspect is provided a human pluripotent stem cell including: (i) a mutant sr39 HSV-TK protein-encoding gene; (ii) an inactivated B2M gene; (iii) an inactivated CIITA gene;(iv) an exogenous CD274 gene; and (v) an exogenous CD47 gene.

[0009] In another aspect is provided a composition including a plurality of the human pluripotent stem cells provided herein including embodiments thereof.

[0010] In another aspect is provided a beta cell including: (i) a mutant sr39 HSV-TK proteinencoding gene; (ii) an inactivated B2M gene; (iii) an inactivated CIITA gene; (iv) an exogenous CD274 gene; and (v) an exogenous CD47 gene.

[0011] In another aspect is provided a composition including a plurality of the beta cells provided herein including embodiments thereof.

[0012] In another aspect is provided a cell aggregate including a plurality of the human pluripotent stem cells provided herein including embodiments thereof.

[0013] In another aspect is provided a cell aggregate including a plurality of the beta cells provided herein including embodiments thereof.

[0014] In another aspect is provided a polymeric microcapsule including: (i) the human pluripotent stem cell provided herein including embodiments thereof; (ii) the beta cell providedPATENTAttorney Docket No. 048440-851001 WO herein including embodiments thereof; (iii) the composition provided herein including embodiments thereof; or (iv) the cell aggregate provided herein including embodiments thereof.

[0015] In another aspect is provided an implantable device including: (i) the human pluripotent stem cell provided herein including embodiments thereof; (ii) the beta cell provided herein including embodiments thereof; (iii) the composition provided herein including embodiments thereof; (iv) the cell aggregate provided herein including embodiments thereof; or (v) the polymeric microcapsule provided herein including embodiments thereof.

[0016] In another aspect is provided a method of treating diabetes in a patient in need thereof, the method including administering to the patient an effective amount of: (i) the human pluripotent stem cell provided herein including embodiments thereof; (ii) the beta cell provided herein including embodiments thereof; (iii) the composition provided herein including embodiments thereof; (iv) the cell aggregate provided herein including embodiments thereof; or (v) the polymeric microcapsule provided herein including embodiments thereof.

[0017] These and other embodiments of the disclosure are described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 shows a diagram of stepwise hPSC differentiation into the beta-like cells in culture of Hl cells.

[0019] FIGS. 2A-2G show selective eradication of undifferentiated H l cells by the iC9 suicide system. FIG. 2A: Diagram of gene editing to insert the iC9 gene into the SOX2 locus via homology directed recombination (HDR). The puromycin selectable marker is removed by the CRE after the gene edited clone is identified. FIG. 2B: Eradication of undifferentiated Hl-iC9 cells with increasing concentrations of AP1903. The viability of the parental Hl cells is not affected by the drug. FIGS. 2C-2D: Cell viability is not affected by API 903 upon differentiation of Hl and Hl-iC9 into the beta-like cells (FIG. 2C). The production of the beta-like (INS+ / MAFA+) cells is not affected by the iC9 gene insertion (FIG. 2D). FIG. 2E: AP1903 selectively eliminated undifferentiated Hl-iC9 cells in a cell mixture of undifferentiated Hl-iC9 cells and the Hl-iC9-derived beta-like cells. Undifferentiated Hl-iC9 cells are identified by the expression of the stem cell marker OCT4, a stem cell-specific marker. SO denotes undifferentiated Hl-iC9 cells whereas S7 denotes Hl-iC9-derived SC-islets. AP: AP1903. FIG. 2F: ThePATENTAttorney Docket No. 048440-851001 WO production of the beta-like (INS+ / NKX6.1+) cells is not affected by the IC9 gene insertion. FIG. 2G: In vitro cell killing with different concentrations of API 903.

[0020] FIG. 3 shows GCV-mediated killing of undifferentiated H1-CDK1-SR39 cells. The viability of the parental Hl cells is not affected by the increasing concentration of GCV.

[0021] FIG. 4 shows serotonin production from Hl -derived beta-like cells. KC1 treatment of the Hl-derived beta-like cells stimulates serotonin secretion into the culture media.

[0022] FIGS. 5A-5C show Hl -iC9-ATPHl -derived beta-like cells rescue glycemic control in diabetic mice. FIGS. 5A-5B: Inactivation of TPH1 gene did not disrupt function of Hl-iC9- ATPHl-derived beta-like cells. FIG. 5A: Clusters from Hl-iC9 and Hl-iC9-ATPHl-derived betalike cells were mock stimulated or stimulated with KC1 followed by measuring serotonin in the culture media. Reduced serotonin secretion was observed from Hl -iC9-ATPHl -derived beta-like cells (right box) compared to Hl-iC9 control cells (left box). FIG. 5B: The GSIS activity of SC- islets differentiated from Hl-iC9 and Hl-iC9-ATPHl cells was measured. The cells were exposed to 3 mM (low) and 17 mM (high) glucose followed by measuring C-peptide in the culture media. The fold of glucose stimulation was calculated and showed no difference between Hl-iC9 and Hl- iC9-ATPHl. FIG. 5C: Restoration of glycemic control in diabetic NODscid mice transplanted with SC-islets derived from either Hl-iC9 or Hl-iC9-ATPHl cells.

[0023] FIGS. 6A-6B show Hl clone with B2M gene knockout and HLA-E trimer (Etrimer) expression. Cell surface expression of HLA-A,B,C and HLA-E trimer in the undifferentiated (FIG. 6A) and in the differentiated beta-like cells (FIG. 6B). The - / Etrimer clone with the B2M gene knockout and HLA-E trimer expression was derived from Hl cells.

[0024] FIG. 7: Syngenic transplant into the omentum site of a rat. The omentum transplant reversed diabetes in 60% (3 / 5) rats.

[0025] FIG. 8 shows results from three islet transplant clinical trials. Insulin independence rates and islet doses for participants in three islet transplant trials. Typical islet dose to achieve insulin independence is >10,000 lEQ / kg. ITA: n=17 / 40; Participants received up to 3 islet transplants combined with Edmonton-based immunosuppression. TCD: n=7 / 13; Participants received up to 3 islet transplants combined with T-cell depleting immunosuppression inductions. Gastrin: n=7 / 7; Participants received only one islet transplant with T-cell depleting immunosuppression inductionPATENTAttorney Docket No. 048440-851001 WO and gastrin treatment. Baseline characteristics of participants in the three islet transplant clinical trials are described in Table 1.

[0026] FIG. 9 shows a comparison of rate of reduction in daily insulin requirements between TCD and Gastrin trial groups.

[0027] FIG. 10 shows a comparison of rate of engraftment in islet transplantation between TCD and Gastrin (Gas) trial groups. Gastrin treatment improved the rate of engraftment. In addition, gastrin increased beta cell mass as measured by in vivo imaging.

[0028] FIG. 11 shows results from participant who received 5,133 lEQ / kg islet transplant dose and gastrin treatment.

[0029] FIG. 12 shows a comparison of percent time in range in insulin-free islet transplant recipients among clinical trial participants and normal, non-diabetics.

[0030] FIG. 13 shows gastrin treatment down regulates inflammation-related genes including GCSF, GMCSF, IL6, IL-lb, CXCL1, and IL8.

[0031] FIG. 14 shows gastrin treatment suppressed pro-inflammatory cytokine release, including GM-CSF, MIP-3a, IL-6, IL-21, IL-7, and IL-8.

[0032] FIGS. 15A-15C shows gastrin reduces inflammation and promotes insulin secretion and progenitor cell expansion. FIG. 19A: After 48-hr cytokine treatment, gastrin protected cell survival against cytokine-induced death (IL-lb 10 ng / ml, TNFa 10 ng / ml, and IFNr 100 ng / ml). FIG. 19B: Islets treated with gastrin (100 nM) for 2 weeks have higher insulin secretion capacity. FIG. 19C: Gastrin-treated islets have increased dual hormonal positive cells (insulin and somatostatin positive cells) as analyzed by single cell Western.

[0033] FIG. 16 shows EDU incorporation increased in high Ale islets after 2 weeks of gastrin treatment.

[0034] FIGS. 17A-17B shows gastrin reduced incidence of autoimmune diabetes and insulitis in NOD mice. Nomoglycemic NOD mice were treated with GAST-17 for 14 weeks (weeks 8-22). FIG. 17A: 78% of NOD mice remained normoglycemic. FIG. 17B: NOD mice treated with gastrin had reduced islet insulitis.PATENTAttorney Docket No. 048440-851001 WO

[0035] FIG. 18 shows gastrin treatment increased number of islets in normoglycemic NOD mice.

[0036] FIG. 19 shows a comparison of the ratio of Insulin:Glucagon staining in mice treated with Gastrin vs control.

[0037] FIG. 20 shows the effect of gastrin treatment on islet distance to duct.

[0038] FIGS. 21A-21E: Mesenchymal stem cell extracellular vesicles (MSC-EVs) reduced adoptive transfer of diabetes.

[0039] FIGS. 22A-22B: MSC-EVs increased frequency of regulatory T cells (FIG. 22A) and reduced activated CD8+T cell populations (FIG. 22B) in peripheral blood mononuclear cells (PMBC).

[0040] FIGS. 23A-23B: Regulatory T cells (Tregs) blocked graft-versus-host disease (GvHD)(FIG. 23A) and blocked development of Type 1 diabetes (FIG. 23B) in mice.

[0041] FIG. 24: Antigen-specific regulator T cells (aTregs) showed superior suppression of CD4+and CD8+cells compared to polyclonal regulatory T cells (nTreg).

[0042] FIGS. 25A-25G: Comparison of the concentration of cytokines produced by nTregs and aTregs. FIG. 25A: IFN-g; FIG. 25B: IL-10; FIG. 25C: TGF-beta; FIG. 25D: IL-17A; FIG. 25E: TNF-a; FIG. 25F: IL-6; FIG. 25G: IL-4.

[0043] FIG. 26: Transplantation of stem cell-derived synthetic islets (SC-SI) restored normoglycemia in non-fasting rats.

[0044] FIGS. 27A-27C: GCV eliminated proliferating Hl cells without affecting the viability of SC-islets. FIG. 27A: GCV administration selectively killed dividing Hl-TK and H1-SR39 cells without affecting the viability of the parental Hl cells. FIG. 27B: GCV had no effect on the viability of SC-islets derived from Hl, Hl-TK or H1-SR39 cells. FIG. 27C: Schematic of the insertion of the TK or the SR39 gene into the CDK1 locus in Hl cells.

[0045] FIGS. 28A-28C: TPH1 knockout did not affect dynamic glucose-induced insulin secretion (GSIS). TPH1 homozygous gene knockout did not affect SC-SI differentiation and the response of SC-SI to secrete insulin upon glucose stimulation remained normal.PATENTAttorney Docket No. 048440-851001 WO

[0046] FIG. 29: Schematic for manufacturing process for human pluripotent stem cell-derived synthetic islets (SC-SI).

[0047] FIG. 30: Schematic for manufacturing process for human cadaveric islet-derived synthetic islets (CC-SI).

[0048] FIGS. 31A-31B: NK cell- mediated killing of SC-islets derived from H1(AB2M) differentiation. SC-islets were labeled with CFSE, followed by coculture with human NK cells for 4 hours. The resulting cell population was monitored for cell survival by FACS analysis of CFSE+ live cells (A) and PI staining for dying cells in the CFSE+ cell population.

[0049] FIG. 32: Schema to establish the GM-H1 line to improve the biosafety and hypoimmunogenicity of derived SC-islets.

[0050] FIG. 33: FACS analysis of HLA class I expression in undifferentiated H1(SR39) parental and H1(SR39;AB2M) cells demonstrating the absence of HLA class 1 expression on the cell surface of Hl (SR39;AB2M) clone.

[0051] FIG. 34: The H1(SR39;AB2M;ACIITA) clone was first transduced with the PD-L1 vector (top schematic) using lentiviral transduction. Immunofluorescence detection of INS+ and NKX6.1+ cells derived from the differentiation of parental H1(SR39) and H1(SR39; AB2M) cells (bottom panels). The cells expressing high levels of mCherry were pooled and named H 1 (SR39; AB2M; ACIIT A;PD-L 1 ).

[0052] FIG. 35: The H1(SR39;AB2M;ACIITA;PD-L1) cells were pooled and transduced by the CD47 vector (top schematic) using lentiviral transduction. Cells expressing high level of mCherry and GFP were pooled and named GM-H1 cells (e.g., H1(SR39;AB2M;ACIITA;PD-L1;CD47)).

[0053] FIG. 36: FACS analysis of GM-H1 cells for cell surface expression of PD-L1 and CD47. The H1(SR39) cells were used as the control parental cells. Mean fluorescence intensity (MFI) was shown to indicate the staining intensity.

[0054] FIG. 37: The GSIS activity of pooled GM-H1 cells. Individual clusters were collected and subjected to the GSIS assay. C-peptide production in media containing low (3 mM) and high (17 mM) glucose was determined by Elisa.PATENTAttorney Docket No. 048440-851001 WO

[0055] FIG. 38: Ectopic CD47 expression in GM-H1 cells suppressed NK cell-mediated cell killing. SC-islets derived from H1(SR39) (HLA+CD47 ), H1(SR39; AB2M;ACIITA) (HLA CD47’ ), or GM-H1 (HLA CD47+) cells were co-cultured with purified NK cells for 4 h. Killing of SC- islet cells was quantified by PI staining of dead cells.

[0056] FIG. 39: Restoration of glycemic control with SC-islets derived from GM-H1 cell pools. The graft size was shown. Glucose tolerance test (IPGTT) was carried out on day 69 after transplantation.

[0057] FIG. 40: Isolation of an independent clone, clone 11, from the GM-H1 cell pool. Expression of CD47 in clone 11 was determined by FACS and the mean fluorescence intensity was determined and shown. Immunofluorescence (IF) staining of clone 11 -derived SC-islets was carried out with antibodies against NKX6.1, INS and GCG, which demonstrated expression of NKX6.1, INS and GCG.

[0058] FIG. 41: Restoration of glycemic control with SC-islets derived from clone 11 (see fig. 40). The graft size is indicated in the figure (e.g., 18 mm, 30 mm, 27 mm, and 30 mm).

[0059] FIG. 42: GSIS of SC-islets derived from EUR and HlR-MafA in vitro.

[0060] FIG. 43: Ectopic MAFA expression in an iPSC line boosted the SC-islet function. Three episomal plasmids encoding the Yamanaka’s reprogramming factors were used to transfect primary human MSCs and an iPSC line was established. The undifferentiated line expressed stem cell markers, including TRA1-60 and TRA1-81. When differentiated, cells from the three germ layers including endoderm (alpha feta protein), mesoderm (alpha smooth muscle actin) and ectoderm (Nestin) were detected. The MAFA cDNA was then introduced into the PC SKI locus in the iPSC line (iPSC-MafA) and GSIS was carried out with the derived SC-islets.

[0061] FIGS. 44A-44B: Glucose stimulated insulin release results from islets cultured with Rock inhibitor (RI) and / or cholesterol lipid concentrate (CLC). FIG. 44A: Insulin release from islet cells cultured with RI alone or in combination with CLC. FIG. 44B: Islet cells cultured with both RI and CLC showed the highest stimulation index (SI).

[0062] FIGS. 45A-45B: Overexpression of a transcription factor such as MAFA from the INS locus led to dysregulation of stem cell differentiation and / or maturation of the resulting SC-islets. FIG. 45A: H1R cells were genetically engineered to ectopically express MAFA via insertion inPATENTAttorney Docket No. 048440-851001 WO the INS locus (Hl cells carrying a tdTomato gene). Ectopic expression of MAFA was significantly increased from the INS locus. Consistently, the direct downstream target of MAFA, GLUT-2, is also upregulated although expression of INS, another downstream target of MAFA, is downregulated. FIG. 45B: GSIS showed no simulation of C-peptide secretion from H1R-INS- MAFA-derived SC-islets upon glucose stimulation.DETAILED DESCRIPTIONDEFINITIONS

[0063] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this disclosure. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0064] The term “gene” means the segment of DNA involved in producing a protein; it may include regions preceding and following the coding region (leader and trailer) and may include intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that may be necessary during the transcription and the translation of a gene. Further, a “protein gene product” is a protein expressed from a particular gene.

[0065] “Gene expression” refers to a gene that is transcribed into a polynucleotide (e.g., as mRNA) and then translated into a protein, and also those that are transcribed into RNA but not translated into a protein (for example, transfer RNA, ribosomal RNA, non-coding RNA). Therefore, “gene expression” includes nucleic acid expression (e.g., mRNA) or protein expression (e.g., protein).

[0066] The term “HSV-TK protein” or “herpes simplex virus-1 thymidine kinase protein” is used herein according to its plain and ordinary meaning and refers to a protein that is substantially identical to the amino acid sequence identified by UniProt ID: Q9QNF7, or a variant or homologPATENTAttorney Docket No. 048440-851001 WO having substantial identity thereto. In embodiments, the HSV-TK protein is a variant or homolog thereof. In embodiments, the variants or homologs have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence compared to the HSV-TK protein sequence.

[0067] The term “mutant sr39 HSV-TK protein” or “mutant sr39 herpes simplex virus-1 thymidine kinase” is used herein according to its plain and ordinary meaning and refers to a protein that is substantially identical to the amino acid sequence identified by SEQ ID NO: 1, or a variant or homolog having substantial identity thereto. In embodiments, the HSV-TK protein is a variant or homolog thereof. In embodiments, the variants or homologs have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence compared to SEQ ID NO: 1 . In embodiments, a variant or homolog having less than 100% amino acid sequence identity to SEQ ID NO: 1 will have the same amino acid at the position corresponding to 1159, F 160, L161, Fl 68, and Ml 69 in SEQ ID NO: 1. In embodiments, a variant or homolog having less than 100% amino acid sequence identity to SEQ ID NO: 1 will have the same amino acid at the position corresponding to 1159, F160, L161, D162, R163, Hl 64, Fl 68, Ml 69, Cl 71, Y172, and Pl 73 in SEQ ID NO: 1.

[0068] The term “mutant sr39 HSV-TK protein-encoding gene” is used herein according to its plain and ordinary meaning and refers to a gene that encodes a mutant sr39 HSV-TK protein.

[0069] The term “ganciclovir” is used herein according to its plain and ordinary meaning and refers to a synthetic nucleoside analog of guanine that functions as an antiviral agent. In embodiments, ganciclovir is phosphorylated intracellularly to its active triphosphate form. In embodiments, the active triphosphate form of ganciclovir inhibits viral DNA polymerase and disrupts viral DNA synthesis. In embodiments, ganciclovir triphosphate is preferentially incorporated into viral DNA, resulting in premature chain termination. In embodiments, ganciclovir is used in combination with a herpes simplex virus thymidine kinase (HSV-TK) gene, which encodes an enzyme that selectively phosphorylates ganciclovir to its cytotoxic form. In embodiments, expression of HSV-TK in dividing cells enables selective conversion of ganciclovir to ganciclovir triphosphate, thereby inducing apoptosis. In embodiments, ganciclovir is administered intravenously, orally, or intravitreally. In embodiments, the ganciclovir is substantially similar to the ganciclovir described in DrugBank Accession No. DB01004. The usePATENTAttorney Docket No. 048440-851001 WO of a HSV-TK protein-encoding gene and ganciclovir is well known in the art and is described, for example, by Shen and Nemunaitis, Cancer Gene Therapy, 2006, 13:975-92, which is incorporated herein by reference in its entirety and for all purposes.

[0070] The term “TPH1 gene” is used herein according to its plain and ordinary meaning and refers to a gene that encodes a TPH1 protein. In embodiments, the TPH1 gene is substantially identical to the nucleic acid sequence identified by NCBI Gene ID: 7166, or a variant or homolog having substantial identity thereto. In embodiments, the TPH1 gene is a variant or homolog thereof. In embodiments, the variants or homologs have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence compared to the TPH1 gene sequence. In embodiments, the TPH1 gene encodes the tryptophan 5-hydroxylase 1 (TPH1) protein that catalyzes the biosynthesis of serotonin.

[0071] The term “inactivated TPH1 gene” is used herein according to its plain and ordinary meaning and refers to a TPH1 gene that does not encode a fully functional tryptophan 5- hydroxylase 1 (TPH1) protein. In embodiments, an inactivated TPH1 gene encodes a nonfunctional TPH1 protein or a partially functional TPH1 protein. In embodiments, an inactivated TPH1 gene encodes a truncated TPH1 protein, which is either non-functional or partially functional. In embodiments, an inactivated TPH1 gene does not encode a TPH1 protein. An inactivated TPH1 gene inhibits expression of serotonin.

[0072] The term “TPH1 protein” or “tryptophan 5-hydroxylase 1 protein” is used herein according to its plain and ordinary meaning and refers to a protein that is substantially identical to the amino acid sequence identified by UniProt ID: P17752, or a variant or homolog having substantial identity thereto. In embodiments, the TPH1 protein is a variant or homolog thereof. In embodiments, the variants or homologs have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence compared to the TPH1 protein sequence. In embodiments, the TPH1 protein includes the amino acid sequence of SEQ ID NO:2. In embodiments, the amino acid sequence of the TPH1 protein is set forth in SEQ ID NO:2.

[0073] The term “truncated TPH1 protein” is used herein according to its plain and ordinary meaning and refers to a TPH1 protein that is truncated. In embodiments, the truncated TPH1 protein is truncated at the C-terminus or the N-terminus. In embodiments, the truncated TPH1PATENTAttorney Docket No. 048440-851001 WO protein includes the amino acids at positions corresponding to positions 1-85 with reference to the full-length TPH1 protein identified as SEQ ID NO:2. In embodiments, the truncated TPH1 protein includes the amino acid sequence of SEQ ID NO: 12. In embodiments, the truncated TPH1 protein is the amino acid sequence of SEQ ID NO: 12. In embodiments, the truncated TPH1 protein does not include the amino acid residues corresponding to positions 86-444 of SEQ ID NO:2.

[0074] The term “B2M gene” is used herein according to its plain and ordinary meaning and refers to a gene that encodes a beta-2-microglobulin (B2M) protein. In embodiments, the B2M gene is substantially identical to the nucleic acid sequence identified by NCBI Gene ID: 567, or a variant or homolog having substantial identity thereto. In embodiments, the B2M gene is a variant or homolog thereof. In embodiments, the variants or homologs have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence compared to the B2M gene sequence. In embodiments, the B2M gene encodes a serum protein found in association with the major histocompatibility complex (MHC) class I heavy chain.

[0075] The term “inactivated B2M gene” is used herein according to its plain and ordinary meaning and refers to a B2M gene that does not encode a fully functional beta-2 microglubulin protein (B2M protein). In embodiments, an inactivated B2M gene encodes a non-functional B2M protein or a partially functional B2M protein. In embodiments, an inactivated B2M gene does not encode a B2M protein. In embodiments, an inactivated B2M gene inhibits expression of an HLA-I proteins. In embodiments described herein, an HLA-E trimer gene is inserted into exon 1 of the B2M gene, such that the full-length B2M gene may not be expressed due to the presence of the stop codon of the HLA-E trimer gene.

[0076] The term “CIITA gene” is used herein according to its plain and ordinary meaning and refers to a gene that encodes a class II major histocompatibility complex transactivator (CIITA) protein. In embodiments, the CIITA gene is substantially identical to the nucleic acid sequence identified by NCBI Gene ID: 4261 or a variant or homolog having substantial identity thereto. In embodiments, the CIITA gene is a variant or homolog thereof. In embodiments, the variants or homologs have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence compared to the CIITA gene sequence. In embodiments, the CIITA gene encodes a protein with an acidic transcriptionalPATENTAttorney Docket No. 048440-851001 WO activation domain, 4 LRRs (leucine-rich repeats) and a GTP binding domain. In embodiments, the protein is located in the nucleus and acts as a positive regulator of class II major histocompatibility complex gene transcription

[0077] The term “inactivated CIITA gene” is used herein according to its plain and ordinary meaning and refers to a CIITA gene that does not encode a fully functional class II major histocompatibility complex transactivator (CIITA) protein. In embodiments, an inactivated CIITA gene encodes a non-functional CIITA protein or a partially functional CIITA protein. In embodiments, an inactivated CIITA gene does not encode a CIITA protein. In embodiments, an inactivated CIITA gene inhibits expression of HLA-II proteins. In embodiments, a frame-shift mutation is introduced into the first exon of the CIITA gene, such that a CIITA protein will likely not be produced.

[0078] The term “CD47 gene” is used herein according to its plain and ordinary meaning and refers to a gene encoding a cluster of differentiation 47 (CD47) protein. In embodiments, the CD47 protein is involved in regulation of innate immune responses. In embodiments, the CD47 protein inhibits macrophages and / or natural killer (NK) cells. In embodiments, the CD47 protein inhibits macrophage-mediated phagocytosis by interacting with signal regulatory protein alpha (SIRPa). In embodiments, the CD47 protein is involved in modulation of inflammation, apoptosis, and cellular adhesion. In embodiments, the CD47 gene is substantially identical to the nucleic acid sequence identified by NCBI Gene ID: 961, or a variant or homolog having substantial identity thereto. In embodiments, the variants or homologs have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 continuous nucleotide portion) compared to a naturally occurring CD47 gene sequence. In embodiments, the CD47 gene encodes a protein that retains CD47 activity (e.g., a CD47 protein). In embodiments, the CD47 protein is capable of binding to SIRPa. In embodiments, the CD47 amino acid sequence is substantially identical to the amino acid sequence identified by the UniProt reference number Q08722 or a variant or homolog having substantial identity thereto.

[0079] The term “CD274 gene” is used herein according to its plain and ordinary meaning and refers to a gene encoding a cluster of differentiation 274 (CD274) protein, also known as programmed death-ligand 1 (PD-L1). In embodiments, the CD274 protein is involved inPATENTAttorney Docket No. 048440-851001 WO regulation of adaptive immune responses. In embodiments, the CD274 protein inhibits T cell activation. In embodiments, the CD274 protein promotes immune tolerance by binding to the PD- 1 receptor on T cells. In embodiments, the CD274 gene is substantially identical to the nucleic acid sequence identified by NCBI Gene ID: 29126, or a variant or homolog having substantial identity thereto. In embodiments, the variants or homologs have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 continuous nucleotide portion) compared to a naturally occurring CD274 gene sequence. In embodiments, the CD274 gene encodes a protein that retains CD274 activity (e g., a CD274 protein). In embodiments, the CD274 protein is capable of binding to a PD-1 receptor. In embodiments, the CD274 amino acid sequence is substantially identical to the amino acid sequence identified by the UniProt reference number Q9NZQ7 or a variant or homolog having substantial identity thereto. The terms “CD247” and “PD-L1” are used interchangeably herein.

[0080] The term “HLA-E gene” is used herein according to its plain and ordinary meaning and refers to a gene encoding a major histocompatibility complex, class I, E protein. In embodiments, the HLA-E protein is involved in innate immune regulation. In embodiments, the HLA-E protein is involved in modulation of natural killer (NK) cell activity. In embodiments, the HLA-E gene is substantially identical to the nucleic acid sequence identified by NCBI Gene ID: 3133, or a variant or homolog having substantial identity thereto. In embodiments, the variants or homologs have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 continuous nucleotide portion) compared to a naturally occurring HLA-E gene sequence. In embodiments, the HLA-E gene encodes a protein that retains HLA-E activity (e.g., an HLA-E protein). In embodiments, the HLA-E protein is capable of binding to a CD94 / NKG2 receptor. In embodiments, the HLA-E protein is capable of presenting peptide antigens. In embodiments, the HLA-E amino acid sequence is substantially identical to the amino acid sequence identified by the UniProt reference number Pl 3747 or a variant or homolog having substantial identity thereto.

[0081] The term “HLA-G gene” is used herein according to its plain and ordinary meaning and refers to a gene encoding a major histocompatibility complex, class I, G protein. In embodiments, the HLA-G protein is involved in immune tolerance. In embodiments, the HLA-G protein isPATENTAttorney Docket No. 048440-851001 WO involved in modulation of matemal-fetal immune responses and inhibition of natural killer (NK) cell activity. In embodiments, the HLA-G gene is substantially identical to the nucleic acid sequence identified by NCBI Gene ID: 3135, or a variant or homolog having substantial identity thereto. In embodiments, the variants or homologs have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 continuous nucleotide portion) compared to a naturally occurring HLA-G gene sequence. In embodiments, the HLA-G gene encodes a protein that retains HLA-G activity (e.g., an HLA-G protein). In embodiments, the HLA-G protein is capable of binding to an inhibitory receptor (e.g., LILRB1, LILRB2, or KIR2DL4). In embodiments, the HLA-G protein is capable of presenting peptide antigens. In embodiments, the HLA-G amino acid sequence is substantially identical to the amino acid sequence identified by the UniProt reference number Pl 7693 or a variant or homolog having substantial identity thereto.

[0082] The term “CD59 gene” is used herein according to its plain and ordinary meaning and refers to a gene encoding a cluster of differentiation 59 (CD59) protein. In embodiments, the CD59 protein is involved in regulation of innate immune responses. In embodiments, the CD59 protein inhibits formation of the membrane attack complex (MAC) and protects host cells from complement-mediated lysis. In embodiments, the CD59 protein is involved in signal transduction during T cell activation. In embodiments, the CD59 gene is substantially identical to the nucleic acid sequence identified by NCBI Gene ID: 966, or a variant or homolog having substantial identity thereto. In embodiments, the variants or homologs have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 continuous nucleotide portion) compared to a naturally occurring CD59 gene sequence. In embodiments, the CD59 gene encodes a protein that retains CD59 activity (e.g., a CD59 protein). In embodiments, the CD59 protein is capable of binding complement components C8 and / or C9. In embodiments, the CD59 amino acid sequence is substantially identical to the amino acid sequence identified by the UniProt reference number P13987 or a variant or homolog having substantial identity thereto.

[0083] The term “MAFA gene” is used herein according to its plain and ordinary meaning and refers to a gene encoding a MAF bZIP transcription factor A (MAFA) protein. In embodiments,PATENTAttorney Docket No. 048440-851001 WO the MAFA protein is involved in regulation of glucose-responsive insulin gene transcription. In embodiments, the MAFA protein promotes the maturation and function of pancreatic P-cells. In embodiments, the MAFA gene is substantially identical to the nucleic acid sequence identified by NCBI Gene ID: 389692, or a variant or homolog having substantial identity thereto. In embodiments, the variants or homologs have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 continuous nucleotide portion) compared to a naturally occurring MAFA gene sequence. In embodiments, the MAFA gene encodes a protein that retains MAFA activity (e.g., a MAFA protein). In embodiments, the MAFA protein is capable of binding to the insulin enhancer Cl / RIPE3b element. In embodiments, the MAFA protein acts synergistically with other transcription factors (e.g., PDX1, NEUR.OD1) to activate insulin gene expression. In embodiments, the MAFA amino acid sequence is substantially identical to the amino acid sequence identified by the UniProt reference number Q8NHW3 or a variant or homolog having substantial identity thereto.

[0084] The term “PCSK1 gene” is used herein according to its plain and ordinary meaning and refers to a gene encoding a proprotein convertase subtilisin / kexin type 1 (PCSK1) protein. In embodiments, the PCSK1 protein is involved in endocrine regulation. In embodiments, the PCSK1 protein is involved in proteolytic processing of prohormones, including proinsulin, into biologically active hormones. In embodiments, the PCSK1 protein contributes to glucose homeostasis and insulin maturation in pancreatic P-cells. In embodiments, the PCSK1 gene is substantially identical to the nucleic acid sequence identified by NCBI Gene ID: 5122, or a variant or homolog having substantial identity thereto. In embodiments, the variants or homologs have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 continuous nucleotide portion) compared to a naturally occurring PCSK1 gene sequence. In embodiments, the PCSK1 gene encodes a protein that retains PCSK1 activity (e.g., a PCSK1 protein). In embodiments, the PCSK1 protein is capable of cleaving substrates at paired basic amino acid residues. In embodiments, the PCSK1 amino acid sequence is substantially identical to the amino acid sequence identified by the UniProt reference number P29120 or a variant or homolog having substantial identity thereto.PATENTAttorney Docket No. 048440-851001 WO

[0085] The term “CD46 gene” is used herein according to its plain and ordinary meaning and refers to a gene encoding a cluster of differentiation 46 (CD46) protein, also known as membrane cofactor protein (MCP). In embodiments, the CD46 protein is involved in regulation of innate and adaptive immune responses. In embodiments, the CD46 protein inhibits complement activation by serving as a cofactor for the inactivation of complement components C3b and C4b. In embodiments, the CD46 protein modulates T cell activation and promotes differentiation of regulatory T cells. In embodiments, the CD46 gene is substantially identical to the nucleic acid sequence identified by NCBI Gene ID: 4179, or a variant or homolog having substantial identity thereto. In embodiments, the variants or homologs have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 continuous nucleotide portion) compared to a naturally occurring CD46 gene sequence. In embodiments, the CD46 gene encodes a protein that retains CD46 activity (e g., a CD46 protein). In embodiments, the CD46 protein is capable of binding complement components C3b and / or C4b. In embodiments, the CD46 amino acid sequence is substantially identical to the amino acid sequence identified by the UniProt reference number P15529 or a variant or homolog having substantial identity thereto.

[0086] The term “CD55 gene” is used herein according to its plain and ordinary meaning and refers to a gene encoding a cluster of differentiation 55 (CD55) protein, also known as decayaccelerating factor (DAF). In embodiments, the CD55 protein is involved in regulation of innate immune responses. In embodiments, the CD55 protein inhibits complement activation by accelerating the decay of C3 and C5 convertases, thereby preventing formation of the membrane attack complex (MAC). In embodiments, the CD55 protein protects host cells from complement- mediated damage. In embodiments, the CD55 gene is substantially identical to the nucleic acid sequence identified by NCBI Gene ID: 1604, or a variant or homolog having substantial identity thereto. In embodiments, the variants or homologs have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 continuous nucleotide portion) compared to a naturally occurring CD55 gene sequence. In embodiments, the CD55 gene encodes a protein that retains CD55 activity (e.g., a CD55 protein). In embodiments, the CD55 protein is capable of binding complement components C3b and / or C4b. In embodiments, thePATENTAttorney Docket No. 048440-851001 WOCD55 amino acid sequence is substantially identical to the amino acid sequence identified by the UniProt reference number P08174 or a variant or homolog having substantial identity thereto.

[0087] The term “CD64 gene” is used herein according to its plain and ordinary meaning and refers to a gene encoding a cluster of differentiation 64 (CD64) protein, also known as Fc gamma receptor I (FcyRI). In embodiments, the CD64 protein is an integral membrane glycoprotein. In embodiments, the CD64 protein is involved in regulation of innate and adaptive immune responses. In embodiments, the CD64 protein binds the Fc region of immunoglobulin G (IgG) antibodies with high affinity and mediates antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, and cytokine release. In embodiments, the CD64 gene is substantially identical to the nucleic acid sequence identified by NCBI Gene ID: 2209, or a variant or homolog having substantial identity thereto. In embodiments, the variants or homologs have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 continuous nucleotide portion) compared to a naturally occurring CD64 gene sequence. In embodiments, the CD64 gene encodes a protein that retains CD64 activity (e g., a CD64 protein). In embodiments, the CD64 protein is capable of binding monomeric IgG and triggering immune cell activation. In embodiments, the CD64 amino acid sequence is substantially identical to the amino acid sequence identified by the UniProt reference number Pl 2314 or a variant or homolog having substantial identity thereto. CD64 genes and proteins are well known in the art (See, e.g., Gravina et al. Nat Biotechnol, 2023;41(5):717-27, which is incorporated herein by reference in its entirety and for all purposes).

[0088] The term “tissue factor pathway inhibitor (TFPI) protein-encoding gene” is used herein according to its plain and ordinary meaning and refers to a gene encoding a tissue factor pathway inhibitor (TFPI) protein. In embodiments, the TFPI protein is involved in regulation of blood coagulation. In embodiments, the TFPI protein inhibits the tissue factor-factor Vila complex and activated factor X (FXa), thereby modulating the initiation of the extrinsic coagulation pathway. In embodiments, the TFPI protein exhibits anticoagulant activity and contributes to vascular hemostasis. In embodiments, the TFPI protein-encoding gene is substantially identical to the nucleic acid sequence identified by NCBI Gene ID: 7035, or a variant or homolog having substantial identity thereto. In embodiments, the variants or homologs have at least 85%, 90%,PATENTAttorney Docket No. 048440-851001 WO95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 continuous nucleotide portion) compared to a naturally occurring TFPI gene sequence. In embodiments, the TFPI gene encodes a protein that retains TFPI activity (e g., a TFPI protein). In embodiments, the TFPI protein includes one or more Kunitz-type domains. In embodiments, the TFPI amino acid sequence is substantially identical to the amino acid sequence identified by the UniProt reference number P l 0646 or a variant or homolog having substantial identity thereto.

[0089] The term “RNLS gene” is used herein according to its plain and ordinary meaning and refers to a gene encoding a renalase (RNLS) protein, also known as monoamine oxidase-C. In embodiments, the RNLS protein is involved in regulation of cellular metabolism. In embodiments, the RNLS protein is a secreted flavin adenine dinucleotide (FAD)-dependent amine oxidase. In embodiments, the RNLS protein is a cytosolic oxidase. In embodiments the RNLS protein modulates oxidative stress and metabolic flux. In embodiments, the RNLS protein contributes to the metabolic profile of pancreatic P-cells. In embodiments, the RNLS protein promotes immune cell activation and antigen presentation in the context of [3-cell autoimmunity. In embodiments, the RNLS protein catalyzes the oxidation of isomeric forms of NAD(P)H. In embodiments, the RNLS gene is substantially identical to the nucleic acid sequence identified by NCBI Gene ID: 55328, or a variant or homolog having substantial identity thereto. In embodiments, the variants or homologs have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 continuous nucleotide portion) compared to a naturally occurring RNLS gene sequence. In embodiments, the RNLS gene encodes a protein that retains RNLS activity (e.g., an RNLS protein). In embodiments, the RNLS amino acid sequence is substantially identical to the amino acid sequence identified by the UniProt reference number Q5VYX0 or a variant or homolog having substantial identity thereto.

[0090] The term “inactivated RNLS gene” is used herein according to its plain and ordinary meaning and refers to an RNLS gene that includes a modification which reduces or eliminates RNLS protein expression or activity. In embodiments, the inactivated RNLS gene includes a deletion, insertion, or substitution of at least one nucleotide. In embodiments, the inactivated RNLS gene includes a frameshift mutation, a nonsense mutation, or a targeted disruptionPATENTAttorney Docket No. 048440-851001 WO introduced by a gene editing system (e.g., CRISPR / Cas9). In embodiments, the inactivated RNLS gene does not encode a functional RNLS protein. In embodiments, the inactivation of the RNLS gene in pancreatic P-cells increases glycolytic activity and alters -cell metabolism. In embodiments, the inactivated RNLS gene modulates the local immune microenvironment by reducing antigen-presenting cell activity and promoting anti-inflammatory immune cell phenotypes. In embodiments, the inactivated RNLS gene protects P-cells from autoimmune killing by inducing metabolic changes that suppress T cell proliferation and shift immune cell transcriptional profdes toward immune tolerance.

[0091] The terms “pluripotent stem cell” and “pluripotent cell” are used herein according to their plain and ordinary meaning and refer to a human cell capable of differentiating into cell types derived from all three primary germ layers: ectoderm, mesoderm, and endoderm. In embodiments, the pluripotent stem cell includes a human embryonic stem cell (hESC), a human pluripotent stem cell (hPSC), or a human induced pluripotent stem cell (iPSC). In embodiments, the pluripotent stem cell is derived from a non-pluripotent source through reprogramming. In embodiments, the pluripotent stem cell is capable of generating one or more differentiated cell types. In embodiments, the pluripotent stem cell is obtained from a publicly available or proprietary source. In embodiments, hPSC can be obtained from a variety of sources. For example, the International Stem Cell Registry provides a comprehensive, searchable database that includes current published and validated unpublished information on all known human pluripotent cell lines including human ESC and human iPSC lines. This includes cell lines approved by the National Institute of Health (N1H) for federal funding and those that were derived through other public or private funding sources such as non-profit institutions, academic centers, research enterprises, stem cell banks and industry based in the United States and abroad (for further information, see, e.g., www.umassmed.edu / iscr / index.aspx, which is incorporated herein in its entirety and for all purposes). Use of alternative or non-registered hPSC does not depart from the spirit and scope of the present disclosure. In embodiments, the pluripotent stem cell includes a cell line listed in the International Stem Cell Registry or a cell line not listed therein.

[0092] he term “hPSC-derived beta cell” is used herein according to its plain and ordinary meaning and refers to a pancreatic beta cell derived from a human pluripotent stem cell (hPSC). In embodiments, the hPSC-derived beta cell is generated from a human embryonic stem cell (hESC) or a human induced pluripotent stem cell (iPSC). In embodiments, the hPSC-derived beta cell isPATENTAttorney Docket No. 048440-851001 WO capable of producing insulin in response to glucose stimulation. In embodiments, methods for preparing hPSC-derived beta cells are described in Example 1 herein.

[0093] The terms “cell aggregate” and “cellular aggregate” are used herein according to their plain and ordinary meaning and refer to a group of cells. In embodiments, the cells within a cell aggregate are the same or different cell types. In embodiments, the cell aggregate includes one or more of a pluripotent stem cell, a beta cell, a mesenchymal stem cell, a native human islet cell, or a regulatory T cell. In embodiments, one or more of the cells within the cell aggregate include an inactivated gene. In embodiments, one or more of the cells within the cell aggregate include an exogenous gene. In embodiments, the cell aggregate has a three-dimensional structure. In embodiments, the cell aggregate is characterized by its largest diameter, which may be determined using a scanning electron microscope (SEM) or a light microscope. In embodiments, the cell aggregates described herein, including embodiments thereof, and / or the cells described herein (e.g., hPSCs, hPSC-derived beta cells, mesenchymal stem cells, regulatory T cells, native human islets), including embodiments thereof, can include a cell-surface modification. Cell-surface modifications includes those described in WO 2014 / 058359, the disclosure of which is incorporated by reference herein in its entirety and for all purposes.

[0094] The terms “islet cell” and “pancreatic islet cell” are used herein according to their plain and ordinary meaning and refer to a cell type normally found within the Islets of Langerhans in the pancreas. In embodiments, any cell normally found within the Islets of Langerhans is considered an “islet cell” or a “pancreatic islet cell”. In embodiments, the islet cell is a “beta cell” or a “beta islet cell.” In embodiments, the beta cell produces insulin. In embodiments, the islet cell is an “alpha cell” or an “alpha islet cell.” In embodiments, the alpha cell produces glucagon. In embodiments, the islet cell is a “delta cell” or a “delta islet cell.” In embodiments, the delta cell produces somatostatin. In embodiments, the islet cell is an “epsilon cell” or an “epsilon islet cells.” In embodiments, the epsilon cell produces ghrelin. In embodiments, the islet cell is a “gamma cell” or a “gamma islet cells,” (also known as “PP cells”). In embodiments, the gamma cell produces pancreatic polypeptide (PP). In embodiments, the islet cells used in the compositions and methods disclosed herein can be a mixture of one or more cell types (alpha, beta, gamma, delta and / or epsilon cells). In embodiments, the islet cells used in the methods of the present invention can be a pure or substantially pure population of alpha, beta, gamma, delta and / orPATENTAttorney Docket No. 048440-851001 WO epsilon cells. In embodiments, one or more islet cells include an inactivated gene. In embodiments, one or more islet cells include an exogenous gene.

[0095] The term “synthetic islet” is used herein according to its plain and ordinary meaning and refers to a cellular aggregate that includes one or more islet cells. In embodiments, the synthetic islet includes native human islet cells and / or human pluripotent stem cell (hPSC)-derived beta cells. In embodiments, the synthetic islet is a stem cell-derived synthetic islet (SC-SI). In embodiments, the synthetic islet is a cadaveric cell-derived synthetic islet (CC-SI). In embodiments, the synthetic islet includes a mixture of alpha, beta, gamma, delta, and / or epsilon cells. In embodiments, the synthetic islet has a three-dimensional structure and is characterized by its largest diameter.

[0096] The term “cadaveric cell” is used herein according to its plain and ordinary meaning and refers to a cell isolated from postmortem human tissue. In embodiments, the cadaveric cell is an islet cell obtained from a donor pancreas. In embodiments, the cadaveric cell is a beta cell, an alpha cell, a delta cell, an epsilon cell, or a gamma cell. In embodiments, the cadaveric cell is used to generate a cadaveric cell-derived synthetic islet (CC-SI). In embodiments, the cadaveric cell is included in a cell aggregate comprising one or more islet cell types. In embodiments, the cadaveric cell is modified to include an inactivated gene. In embodiments, the cadaveric cell is modified to include an exogenous gene.

[0097] The term “donor” is used herein according to its plain and ordinary meaning and refers to an individual organism that supplies living tissue for use in another body. In embodiments, the donor is a person who furnishes blood for transfusion or an organ for transplantation in a histocompatible recipient. In embodiments, the donor is a living human donor. In embodiments, the donor is a deceased human donor. In embodiments, the donor is a living human donor who does not have pre-diabetes, Type 1 diabetes, or Type 2 diabetes. In embodiments, the donor is a deceased human donor who did not have pre-diabetes, Type 1 diabetes, or Type 2 diabetes. In embodiments, the donor is a human donor.

[0098] The term “diabetes” is used herein according to its plain and ordinary meaning and refers to a group of metabolic disorders characterized by elevated blood glucose levels over a prolonged period of time. In embodiments, diabetes results from insufficient insulin production by the pancreas. In embodiments, diabetes results from impaired cellular responsiveness to insulin. InPATENTAttorney Docket No. 048440-851001 WO embodiments, diabetes includes Type 1 diabetes, Type 2 diabetes, and other forms of diabetes such as gestational diabetes or monogenic diabetes. In embodiments, diabetes is associated with dysregulated glucose metabolism and may lead to systemic complications if left untreated.

[0099] The term “Type 1 diabetes” or “T1D” is used herein according to its plain and ordinary meaning and refers to an autoimmune form of diabetes characterized by insufficient insulin production. In embodiments, the Type 1 diabetes is caused by immune-mediated destruction of pancreatic P-cells within the Islets of Langerhans. In embodiments, the Type 1 diabetes is caused by insufficient production of insulin by pancreatic P-cells within the Islets of Langerhans. In embodiments, Type 1 diabetes results in elevated blood glucose levels due to the absence or near absence of endogenous insulin. In embodiments, symptoms of Type 1 diabetes include frequent urination, increased thirst, increased hunger, weight loss, blurry vision, fatigue, and delayed wound healing. In embodiments, the symptoms of Type 1 diabetes develop over a short period of time. In embodiments, Type 1 diabetes is distinguished from other forms of diabetes by its autoimmune etiology and early onset.

[0100] The term “Type 2 diabetes” or “T2D” is used herein according to its plain and ordinary meaning and refers to a metabolic disease characterized by elevated blood glucose levels. In embodiments, the Type 2 diabetes is caused by insulin resistance and a relative deficiency in insulin production. In embodiments, Type 2 diabetes is interchangeably referred to as adult-onset diabetes. In embodiments, Type 2 diabetes arises when body cells respond inadequately to insulin, leading to impaired glucose uptake and persistent hyperglycemia. In embodiments, symptoms of Type 2 diabetes include increased thirst, frequent urination, unexplained weight loss, increased hunger, fatigue, and slow-healing sores. In embodiments, long-term complications associated with Type 2 diabetes include cardiovascular disease, stroke, and diabetic retinopathy.

[0101] The term “insulin-independent” is used herein according to its plain and ordinary meaning and refers to a diabetic subject who no longer requires exogenous insulin administration. In embodiments, the insulin-independent subject is insulin-free, meaning the individual does not require injectable insulin for glycemic control. In embodiments, insulin independence is achieved through restoration of endogenous insulin production. In embodiments, insulin independence is achieved through alternative therapeutic interventions that maintain blood glucose levels within a target range without the need for exogenous insulin.PATENTAttorney Docket No. 048440-851001 WO

[0102] The term “encapsulation” is used herein according to its plain and ordinary meaning and refers to a coating that encloses and physically isolates one or more cells (e.g., human pluripotent stem cells (hPSC), hPSC-derived beta-cells) from the surrounding environment. In embodiments, the encapsulation comprises a polymeric membrane that permits the diffusion of nutrients, oxygen, and metabolic waste products while preventing immune-mediated attack by the host in which the encapsulated cells or islets are transplanted. In embodiments, the encapsulation material includes one or more biocompatible polymers. In embodiments, the biocompatible polymer is agar, alginate, carrageenan, cellulose or its derivatives, chitosan, collagen, gelatin, epoxy resin, photo-crosslinkable resins, polyacrylamide, polyester, polystyrene, polyurethane, or polyethylene glycol (PEG). In embodiments, the cells are encapsulated by a cross-linked polymer comprising micelles of a maleimide-conjugated amphiphilic first polymer and a second polymer having multiple thiol groups, as described in WO 2014 / 058359, which is incorporated herein by reference in its entirety and for all purposes.

[0103] The term “polymeric microcapsule” is used herein according to its plain and ordinary meaning and refers to a microscopic structure comprising a polymer-based shell that encloses and physically isolates its contents from the surrounding environment. In embodiments, the polymeric microcapsule is a hydrogel microparticle. In embodiments, the hydrogel microparticle is within a biomimetic bead. In embodiments, the hydrogel includes one or more polymers that are capable of swelling in water, optionally including crosslinked polymers. In embodiments, the hydrogel microparticle includes polyethylene glycol. In embodiments, the polymeric microcapsule permits the diffusion of nutrients, oxygen, and metabolic waste products while providing a barrier to immune cell infiltration. In embodiments, the polymeric microcapsule is used to encapsulate cells such as human pluripotent stem cells (hPSC) or hPSC-derived beta cells for transplantation. In embodiments, the polymeric microcapsule is a hydrogel microparticle as described in WO 2022 / 120478, the disclosure of which is incorporated herein by reference in its entirety and for all purposes.

[0104] The terms “mesenchymal stem cell” and “MSC” are used herein according to their plain and ordinary meaning and refer to multipotent stromal cells capable of differentiating into multiple cell lineages. In embodiments, MSCs are capable of differentiating into osteoblasts, chondrocytes, adipocytes, and other mesodermal cell types. In embodiments, MSCs exhibit immunomodulatory properties and contribute to tissue repair and regeneration. In embodiments, mesenchymal stemPATENTAttorney Docket No. 048440-851001 WO cells are used in compositions or methods provided herein including embodiments thereof. In embodiments, the mesenchymal stem cells are used in compositions or methods described herein for treating diabetes or restoring glycemic control. MSCs are well known in the art and are described, for example, by Arzouni et al., Stem Cells Translational Medicine, 7:559-563 (2018), dx.doi.org / 10.1002 / sctm.18-0033, the disclosure of which is incorporated herein by reference in its entirety and for all purposes.

[0105] The term “extracellular vesicle” or “EV” is used herein according to its plain and ordinary meaning and refers to a membrane-bound particle that is naturally released by a cell and does not contain a functional nucleus. In embodiments, extracellular vesicles are composed of a lipid bilayer and contain proteins, peptides, lipids, and genetic material such as messenger RNA (mRNA), microRNA (miRNA), and DNA. In embodiments, extracellular vesicles mediate intercellular communication and influence physiological or pathological processes.

[0106] The terms “mesenchymal stem cell-derived extracellular vesicle,” “MSC-derived EV,” “MSC-EV,” and “extracellular vesicle derived from a mesenchymal stem cell” are used herein according to their plain and ordinary meaning and refer to extracellular vesicles released from mesenchymal stem cells. In embodiments, MSC-derived EVs display surface markers characteristic of mesenchymal stem cells (e.g., CD29, CD73, CD105) and classical markers of extracellular vesicles (e.g., CD63, CD9, CD81). In embodiments, MSC-derived EVs retain immunomodulatory and regenerative properties associated with their parent MSCs.

[0107] The term “native human islet cell” is used herein according to its plain and ordinary meaning and refers to a cell that is naturally present within the Islets of Langerhans in the human pancreas. In embodiments, the native human islet cell is isolated directly from human pancreatic tissue. In embodiments, the native human islet cell is an alpha cell, beta cell, delta cell, epsilon cell, or gamma cell (also known as a pancreatic polypeptide-producing cell). In embodiments, native human islet cells are capable of producing hormones such as insulin, glucagon, somatostatin, ghrelin, or pancreatic polypeptide. In embodiments, native human islet cells are used in compositions or methods provided herein including embodiments thereof. In embodiments, the native human islet cells are used in compositions or methods described herein for treating diabetes or restoring glycemic control.PATENTAttorney Docket No. 048440-851001 WO

[0108] The term “regulatory T cell” is used herein according to its plain and ordinary meaning and refers to a subset of T lymphocytes that function to suppress immune responses and maintain immune homeostasis. In embodiments, regulatory T cells inhibit the activation and proliferation of effector T cells and modulate antigen-presenting cell activity. In embodiments, regulatory T cells contribute to immune tolerance and prevent autoimmune responses. In embodiments, the regulatory T cell is a CD4+T cell expressing the transcription factor FOXP3. In embodiments, the regulatory T cell is a reprogrammed regulatory T cell as described in WO 2022 / 060854, the disclosure of which is incorporated herein by reference in its entirety and for all purposes. In embodiments, regulatory T cells are used in compositions or methods provided herein including embodiments thereof. In embodiments, the regulatory T cells are used in compositions or methods described herein for treating diabetes or restoring glycemic control.

[0109] The term “implantable device” is used herein according to its plain and ordinary meaning and refers to a device that is designed to be placed within the body of a subject. In embodiments, the human pluripotent stem cell, the beta cell, cell aggregate, and / or compositions provided herein including embodiments thereof are administered to a subject using an implantable device. In embodiments, the implantable device is configured to house and protect transplanted cells while permitting the exchange of nutrients, oxygen, and metabolic waste. In embodiments, the implantable device provides a physical barrier to immune cell infiltration and supports vascularization. Implantable devices are well known in the art and are described, for example, by Shapiro et al., Cell Reports Medicine, 2021, 2: 100466, the disclosure of which is incorporated herein by reference in its entirety and for all purposes.

[0110] The term “portal vein” is used herein according to its plain and ordinary meaning and refers to a large vein that carries blood from the gastrointestinal tract and spleen to the liver. In embodiments, the portal vein is used as a site for administration of the human pluripotent stem cell, the beta cell, the cell aggregate, and / or the composition provided herein including embodiments thereof.[OUl] The term “omental pouch” is used herein according to its plain and ordinary meaning and refers to a space within the omentum. In embodiments, the omental pouch is surgically created or naturally occurring. In embodiments, the omentum is a fold of peritoneal tissue that extends from the stomach and proximal duodenum to adjacent abdominal organs. In embodiments, thePATENTAttorney Docket No. 048440-851001 WO omental pouch is used as a site for implantation or administration of the human pluripotent stem cell, the beta cell, the cell aggregate, and / or the composition provided herein, including embodiments thereof.

[0112] The term “anterior rectus sheath” is used herein according to its plain and ordinary meaning and refers to a fibrous layer located on the anterior aspect of the rectus abdominis muscle. In embodiments, the anterior rectus sheath is formed by the aponeuroses of the abdominal wall muscles. In embodiments, the anterior rectus sheath is used as a site for implantation or administration of the human pluripotent stem cell, the beta cell, the cell aggregate, and / or the composition provided herein, including embodiments thereof. Methods for administering cell compositions (e.g., hPSCs and / or beta cells) to the anterior rectus sheath are well known in the art (See, e.g., Wang et al., “Transplantation of chemically induced pluripotent stem-cell -derived islets under abdominal anterior rectus sheath in a type 1 diabetes patient,” Cell, 2024;187(22):6152- 6164. el8; PMID: 39326417, which is incorporated herein in its entirety and for all purposes). The human pluripotent stem cells and beta cells provided herein including embodiments thereof may be administered to the anterior rectus sheath using any of the materials and methods disclosed in Wang et al., Cell, 2024; 187(22):6152-6164.el8.

[0113] The term “gastrin” is used herein according to its plain and ordinary meaning and refers to a peptide hormone that stimulates the secretion of gastric acid (hydrochloric acid). In embodiments, the gastrin promotes gastric motility. In embodiments, the gastric acid is secreted by parietal cells in the stomach In embodiments, gastrin is released by G cells located in the pyloric antrum of the stomach, the duodenum, and / or the pancreas. In embodiments, gastrin binds to cholecystokinin B receptors to stimulate histamine release from enterochromaffin-like cells and induces the insertion of potassium / hydrogen (K7H+) ATPase pumps into the apical membrane of parietal cells, thereby increasing hydrogen ion secretion into the gastric lumen. In embodiments, gastrin release is stimulated by the presence of peptides in the stomach lumen. In embodiments, the encoded polypeptide is preprogastrin, which undergoes post-translational processing to form progastrin and subsequently active gastrin isoforms, including gastrin-34, gastrin-17, and gastrin- 14.

[0114] “Nucleic acid” refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof; orPATENTAttorney Docket No. 048440-851001 WO nucleosides (e.g., deoxyribonucleosides or ribonucleosides). In embodiments, “nucleic acid” does not include nucleosides. The terms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term “nucleoside” refers, in the usual and customary sense, to a glycosylamine including a nucleobase and a five- carbon sugar (ribose or deoxyribose). Non limiting examples, of nucleosides include, cytidine, uridine, adenosine, guanosine, thymidine and inosine. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acid, e.g. polynucleotides, contemplated herein include any types of RNA, e.g. mRNA, siRNA, miRNA, and guide RNA and any types of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like. Nucleic acids can include nonspecific sequences. As used herein, the term “nonspecific sequence” refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to or are only partially complementary to any other nucleic acid sequence. By way of example, a nonspecific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism.

[0115] A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and / or modified nucleotides.PATENTAttorney Docket No. 048440-851001 WO

[0116] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0117] The term “complement,” as used herein, refers to a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides capable of base pairing with a complementary nucleotide or sequence of nucleotides. As described herein and commonly known in the art the complementary (matching) nucleotide of adenosine is thymidine and the complementary (matching) nucleotide of guanosine is cytosine. Thus, a complement may include a sequence of nucleotides that base pair with corresponding complementary nucleotides of a second nucleic acid sequence. The nucleotides of a complement may partially or completely match the nucleotides of the second nucleic acid sequence. Where the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, the complement forms base pairs with each nucleotide of the second nucleic acid sequence. Where the nucleotides of the complement partially match the nucleotides of the second nucleic acid sequence only some of the nucleotides of the complement form base pairs with nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding and a non-coding sequences, wherein the non-coding sequence contains complementary nucleotides to the coding sequence and thus forms the complement of the coding sequence. A further example of complementary sequences are sense and antisense sequences, wherein the sense sequence contains complementary nucleotides to the antisense sequence and thus forms the complement of the antisense sequence.

[0118] The terms “isolate” or “isolated”, when applied to a nucleic acid, virus, or protein,PATENTAttorney Docket No. 048440-851001 WO denotes that the nucleic acid, virus, or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. An RNA that is the predominant species present in a preparation is substantially purified.

[0119] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0120] “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0121] The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (e.g., www.ncbi.nlm.nih.gov / BLAST / or the like). Such sequences are then the to be “substantially identical.” This definition also refers to, or may bePATENTAttorney Docket No. 048440-851001 WO applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0122] An amino acid or nucleotide base “position” is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence. The terms “numbered with reference to” or “corresponding to,” when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.

[0123] As used herein, the term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, “about” means within a standard deviation using measurements generally acceptable in the art. In embodiments, “about” means a range extending to + / - 10% of the specified value. In embodiments, “about” includes the specified value.

[0124] The singular terms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise.

[0125] The term “integrated nucleic acid sequence” as used herein refers to an exogenous nucleic acid sequence which has been inserted into the host chromosomal DNA.PATENTAttorney Docket No. 048440-851001 WO

[0126] The term “plasmid” or “expression vector” refers to a nucleic acid molecule that encodes for genes and / or regulatory elements necessary for the expression of genes. Expression of a gene from a plasmid can occur in cis or in trans. If a gene is expressed in cis, gene and regulatory elements are encoded by the same plasmid. Expression in trans refers to the instance where the gene and the regulatory elements are encoded by separate plasmids.

[0127] The term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid” which refers to a linear or circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. “Plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. Other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), serve equivalent functions. Additionally, some viral vectors are capable of targeting a particular cells type either specifically or non-specifically. Replication-incompetent viral vectors or replication-defective viral vectors refer to viral vectors that are capable of infecting their target cells and delivering their viral payload, but then fail to continue the typical lytic pathway that leads to cell lysis and death.

[0128] The term “lentiviral vector” is used in accordance with their plain ordinary meaning. A lentivirus is typically a type of retrovirus that can infect both dividing and non-dividing cells because their pre-integration complex (virus “shell”) can get through the intact membrane of the nucleus of the target cell. They have been used in the art as suitable tools for gene delivery in mammalian cells, with most of the viral genes removed. Lentiviral vectors can be used for introducing libraries of synthetic constructs, plasmids, complementary DNAs, short hairpin RNAs, and cis-regulatory elements into many targets, such as embryonic stem cells. In embodiments, lentivirus vectors have the ability to mediate potent transduction and stable expression intoPATENTAttorney Docket No. 048440-851001 WO dividing and non-dividing cells both in vitro and in vivo. Lentiviruses may have the ability integrate into host chromosomes, and to infect both dividing and non-dividing cells, for example, with sgRNA, gRNA, shRNA and Cas9 (e.g. dCas9, etc.) inserts.

[0129] The terms “transfection,” “transduction,” “transfecting” or “transducing” can be used interchangeably and are defined as a process of introducing a nucleic acid molecule and / or a protein to a cell. Nucleic acids may be introduced to a cell using non-viral or viral-based methods. The nucleic acid molecule can be a sequence encoding complete proteins or functional portions thereof. Typically, a nucleic acid vector, comprising the elements necessary for protein expression (e.g., a promoter, transcription start site, etc.). Non-viral methods of transfection include any appropriate method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell. Exemplary non-viral transfection methods include calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection through heat shock, magnetifection and electroporation. For viral-based methods, any useful viral vector can be used in the methods described herein. Examples of viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors. In aspects, the nucleic acid molecules are introduced into a cell using a retroviral vector following standard procedures well known in the art. The terms “transfection” or “transduction” also refer to introducing proteins into a cell from the external environment. Typically, transduction or transfection of a protein relies on attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell.

[0130] ‘ ‘Nuclease” and “endonuclease” are used interchangeably herein to mean an enzyme which possesses endonucleolytic catalytic activity for nucleic acid (e.g., polynucleotide) cleavage. The term includes site-specific endonucleases such as, designer zinc fingers, transcription activator-like effectors (TALEs), homing meganucleases, and site-specific endonucleases of clustered, regularly interspaced, short palindromic repeat (CRISPR) systems such as, e.g., Cas proteins.PATENTAttorney Docket No. 048440-851001 WO

[0131] The term “isolated”, when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

[0132] “Contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g., chemical compounds including biomolecules or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated; however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents that can be produced in the reaction mixture. The term “contacting” may include allowing two species to react, interact, or physically touch, wherein the two species may be a compound as described herein and a protein or enzyme. In embodiments contacting includes allowing a compound described herein to interact with a protein or enzyme that is involved in a signaling pathway.

[0133] The terms “activation,” “activate,” “activating,” “activator” and the like in reference to a protein-inhibitor interaction means positively affecting (e.g., increasing) the activity or function of the protein relative to the activity or function of the protein in the absence of the activator. In embodiments, activation means positively affecting (e.g., increasing) the concentration or levels of the protein relative to the concentration or level of the protein in the absence of the activator. The terms may reference activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein decreased in a disease. Thus, activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein associated with a disease (e.g., a protein which is decreased in a disease relative to a nondiseased control). Activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein.

[0134] The terms “agonist,” “activator,” “upregulator,” etc. refer to a substance capable of detectably increasing the expression or activity of a given gene or protein. The agonist can increase expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more inPATENTAttorney Docket No. 048440-851001 WO comparison to a control in the absence of the agonist. In embodiments, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or higher than the expression or activity in the absence of the agonist.

[0135] The terms “inhibition,” “inhibit,” “inhibiting,” and the like in reference to a proteininhibitor interaction means negatively affecting (e.g., decreasing) the activity or function of the protein relative to the activity or function of the protein in the absence of the inhibitor. In embodiments, inhibit means negatively affecting (e.g., decreasing) the concentration or level of the protein relative to the concentration or level of the protein in the absence of the inhibitor. In embodiments, inhibition refers to reduction of a disease or symptoms of disease. In embodiments, inhibition refers to a reduction in the activity of a particular protein target. Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein. In embodiments, inhibition refers to a reduction of activity of a target protein resulting from a direct interaction (e.g., an inhibitor binds to the target protein). In embodiments, inhibition refers to a reduction of activity of a target protein from an indirect interaction (e.g., an inhibitor binds to a protein that activates the target protein, thereby preventing target protein activation). In embodiments, inhibit refers to decreasing the expression or activity of a given gene or protein. The expression or activity of the protein can decrease by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control. In embodiments, expression or activity of the protein is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10- fold or lower than the expression or activity in the absence of the inhibitor.

[0136] The term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0137] ‘ ‘Patient” or “subject in need thereof’ refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In somePATENTAttorney Docket No. 048440-851001 WO embodiments, a patient is human.

[0138] “Allogeneic” is used in accordance with its plain and ordinary meaning and includes cells or tissues derived from different individuals of the same species. The cells or tissues may be sufficiently unlike genetically to interact antigenically. Thus, the term “allogeneic transplant” or “allogeneic transfusion” refers to the transfer of biological material to a recipient from a genetically non-identical donor of the same species.

[0139] “Autologous” is used in accordance with its plain and ordinary meaning and includes cells or tissues derived from the same individual. An autologous cell may be taken from an individual and genetically modified (e.g. nucleic acid integrated into cell genome) before being put back into the same individual.

[0140] A “cell” refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example, human cells. Cells may be useful when they are naturally nonadherent or have been treated not to adhere to surfaces, for example by trypsinization.CELL COMPOSITIONS

[0141] Provided herein are, inter alia, genetically engineered human pluripotent stem cells (hPSCs) useful for generating safe and functional insulin-producing cells for the treatment of diabetes. In embodiments, the hPSCs described herein include combinations of genetic modifications that enhance biosafety, reduce immunogenicity, and / or improve therapeutic function following differentiation into beta-like cells. In embodiments, the hPSCs include a suicide gene (e.g., mutant sr39 HSV-TK), inactivated immune recognition genes (e.g., B2M gene and / or CIITA gene), and / or exogenous expression of immune checkpoint and / or anti -phagocytic proteins (e.g., PD-L1 and / or CD47). In embodiments, the hPSCs may further include additional modifications such as inactivation of TPH1, RNLS, and / or expression of complement regulatory proteins (e.g., CD46, CD55 and / or CD59), transcription factors (e.g., MAFA), or non-classical HLA moleculesPATENTAttorney Docket No. 048440-851001 WO(e.g., HLA-E and / or HLA-G). The engineered hPSCs provided herein including embodiments thereof, are, inter alia, useful for generating stem cell-derived islets (SC-islets) capable of insulin secretion and resistant to immune-mediated destruction. Thus, in an aspect is provided a human pluripotent stem cell including: (i) a mutant sr39 HSV-TK protein-encoding gene; (ii) an inactivated B2M gene; (iii) an inactivated CIITA gene; (iv) an exogenous CD274 gene; and (v) an exogenous CD47 gene.

[0142] In another aspect is provided a human pluripotent stem cell including a mutant sr39 HSV-TK protein-encoding gene.

[0143] In another aspect is provided a human pluripotent stem cell including an inactivated TPH1 gene.

[0144] In another aspect is provided a human pluripotent stem cell including: (i) an inactivated B2M gene; and (ii) an inactivated CIITA gene.

[0145] In another aspect is provided a human pluripotent stem cell including: (i) an inactivated B2M gene; (ii) an HLA-E gene; and (ii) an inactivated CIITA gene.

[0146] In another aspect is provided a human pluripotent stem cell including a mutant sr39 HSV-TK protein-encoding gene. In embodiments, the human pluripotent stem cell further includes an inactivated B2M gene. In embodiments, the human pluripotent stem cell further includes an inactivated CIITA gene. In embodiments, the human pluripotent stem cell further includes an exogenous CD274 gene. In embodiments, the human pluripotent stem cell further includes an exogenous CD47 gene.

[0147] In embodiments, the human pluripotent stem cell further includes an inactivated TPH1 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene.

[0148] In embodiments, the human pluripotent stem cell further includes an HLA-E gene. In embodiments, the human pluripotent stem cell includes an HLA-E gene. In embodiments, the HLA-E gene is an exogenous HLA-E gene. In embodiments, the human pluripotent stem cell further includes an HLA-G gene. In embodiments, the human pluripotent stem cell includes an HLA-G gene. In embodiments, the HLA-G gene is an exogenous HLA-G gene. In embodiments, the human pluripotent stem cell includes an HLA-E gene and / or an HLA-G gene. In embodiments,PATENTAttorney Docket No. 048440-851001 WO the human pluripotent stem cell includes an HLA-E gene and an HLA-G gene. In embodiments, the human pluripotent stem cell includes an HLA-E gene or an HLA-G gene.

[0149] In embodiments, the human pluripotent stem cell further includes an exogenous CD59 gene. In embodiments, the human pluripotent stem cell includes an exogenous CD59 gene.

[0150] In embodiments, the human pluripotent stem cell further includes an exogenous transcription factor (e.g., MAFA) gene. In embodiments, the human pluripotent stem cell further includes an exogenous MAFA gene. In embodiments, the human pluripotent stem cell includes an exogenous MAFA gene. In embodiments, the exogenous MAFA gene is inserted into the reading frame of a PCSK1 gene. In embodiments, the exogenous MAFA gene is not inserted into the INS locus.

[0151] In embodiments, the human pluripotent stem cell further includes an exogenous CD46 gene, an exogenous CD55 gene, and / or an exogenous CD59 gene. In embodiments, the human pluripotent stem cell includes an exogenous CD46 gene, an exogenous CD 55 gene, and / or an exogenous CD59 gene. In embodiments, the human pluripotent stem cell further includes an exogenous CD46 gene, an exogenous CD55 gene, and an exogenous CD59 gene. In embodiments, the human pluripotent stem cell further includes an exogenous CD46 gene, an exogenous CD55 gene, or an exogenous CD59 gene. In embodiments, the human pluripotent stem cell further includes an exogenous CD46 gene. In embodiments, the human pluripotent stem cell further includes an exogenous CD55 gene.

[0152] In embodiments, the human pluripotent stem cell further includes a tissue factor pathway inhibitor (TFPI) protein-encoding gene. In embodiments, the human pluripotent stem cell includes a tissue factor pathway inhibitor (TFPI) protein-encoding gene. In embodiments, the TFPI proteinencoding gene is an exogenous TFPI protein-encoding gene.

[0153] In embodiments, the human pluripotent stem cell further includes an inactivated RNLS gene. In embodiments, the human pluripotent stem cell includes an inactivated RNLS gene.

[0154] In embodiments, the human pluripotent stem cell further includes an exogenous CD64 gene. In embodiments, the human pluripotent stem cell includes an exogenous CD64 gene.

[0155] In embodiments, the human pluripotent stem cell includes a TFPI protein-encoding gene, an inactivated RNLS gene, and / or an exogenous CD64 gene. In embodiments, the humanPATENTAttorney Docket No. 048440-851001 WO pluripotent stem cell includes a TFPI protein-encoding gene, an inactivated RNLS gene, and an exogenous CD64 gene. In embodiments, the human pluripotent stem cell includes a TFPI proteinencoding gene and an inactivated RNLS gene. In embodiments, the human pluripotent stem cell includes a TFPI protein-encoding gene and an exogenous CD64 gene. In embodiments, the human pluripotent stem cell includes an inactivated RNLS gene and an exogenous CD64 gene. In embodiments, the human pluripotent stem cell includes a TFPI protein-encoding gene, an inactivated RNLS gene, or an exogenous CD64 gene.

[0156] In another aspect is provided a composition including a plurality of the human pluripotent stem cells provided herein including embodiments thereof.

[0157] In embodiments, the human pluripotent stem cell provided herein including embodiments thereof is cultured on an extracellular matrix. In embodiments, the human pluripotent stem cell is expanded on an extracellular matrix. In embodiments, the extracellular matrix is a Matrigel extracellular matrix. In embodiments, the extracellular matrix is a laminin extracellular matrix. In embodiments, the extracellular matrix is a laminin 521 extracellular matrix. In embodiments, the extracellular matrix does not include a Matrigel extracellular matrix.

[0158] In embodiments, the human pluripotent stem cell provided herein including embodiments thereof may be differentiated into a beta cell. Materials and methods for differentiating human pluripotent stem cells into beta cells are well known in the art (See e.g., Wu etal., “Using gene editing to establish a safeguard system for pluripotent stem-cell-based therapies” iScience 2019;22:409-22; PMCID: PMC6909005, which is incorporated herein by reference in its entirety and for all purposes). The human pluripotent stem cell provided herein including embodiments thereof may be differentiated using any of the differentiation methods disclosed in Wu et al. iScience 2019; 22:409-22 to derive hematopoietic cells, neuronal cells, or beta cells.

[0159] In embodiments, the differentiation of the human pluripotent stem cell provided herein including embodiments thereof, includes seven stages. In embodiments, the differentiation of the hPSC includes culturing the hPSC in cell culture media including differentiation agents and / or factors. In embodiments, the cell culture media of stage 1 includes growth differentiation factor 8 (GDF8; e.g., myostatin) and / or a glycogen synthase kinase-3 (gsk-3) inhibitor (e g., CHIR99021). In embodiments, stage 1 includes decreasing the amount of a glycogen synthase kinase-3 (gsk-3)PATENTAttorney Docket No. 048440-851001 WO inhibitor (e.g., CHIR99021) in the cell culture media over consecutive days. In embodiments, stage 1 includes decreasing the amount of a glycogen synthase kinase-3 (gsk-3) inhibitor (e.g., CHIR99021)in the cell culture media over 3 days. In embodiments, the cell culture media of stage 1 on day 3 does not include a glycogen synthase kinase-3 (gsk-3) inhibitor (e.g., CHIR99021). In embodiments, the cell culture media of stage 2 includes fibroblast growth factor 7 (FGF7) and / or ascorbic acid. In embodiments, stage 2 lasts for 1-2 days. In embodiments, the cell culture media of stage 3 includes FGF7, ascorbic acid, a Smoothened antagonist (e.g., SANT-1), retinoic acid, LDN193189, an insulin-transferrin-selenium (ITS) supplement (e.g., ITS-X), a t, and / or combinations thereof. In embodiments, stage 3 lasts for 1-2 days. In embodiments, the cell culture media of stage 4 includes FGF7, ascorbic acid, a Smoothened antagonist (e.g., SANT-1), retinoic acid, LDN193189, an insulin-transferrin-selenium (ITS) supplement (e.g., ITS-X), a thyroid hormone (e.g., triiodothyronine or T3), a protein kinase C (PKC) activator (e.g., phorbol 12,13- diabutyrate (PDBu), betacellulin, and / or combinations thereof. In embodiments, the combination of a thyroid hormone (e.g., triiodothyronine or T3), a protein kinase C (PKC) activator (e.g., phorbol 12,13-diabutyrate (PDBu), and betacellulin is referred to herein as “TPB.” In embodiments, the cell culture media of stage 4 includes less FGF7, less retinoic acid, less TPB, and / or combinations thereof relative to the cell culture media of stage 3. In embodiments, the cell culture media of stage 4 includes more LDN193189 relative to the cell culture media of stage 3. In embodiments, stage 4 lasts for 1-3 days. In embodiments, the cell culture media of stage 5 includes a Smoothened antagonist (e g., SANT-1), retinoic acid, LDN193189, an insulin- transferrin-selenium (ITS) supplement (e g., ITS-X), a thyroid hormone (e g., triiodothyronine or T3), ALK5 inhibitor, zinc sulfate, heparin, and / or combinations thereof. In embodiments, stage 5 lasts for 1-3 days. In embodiments, the cell culture media of stage 6 includes LDN193189, an insulin-transferrin-selenium (ITS) supplement (e.g., ITS-X), a thyroid hormone (e.g., triiodothyronine or T3), ALK5 inhibitor, zinc sulfate, a G-protein signaling inhibitor (e.g., Gs inh XX), heparin, and / or combinations thereof. In embodiments, stage 6 lasts for 1-7 days. In embodiments, the cell culture media of stage 7 includes an insulin-transferrin-selenium (ITS) supplement (e.g., ITS-X), a thyroid hormone (e.g., triiodothyronine or T3), ALK5 inhibitor, zinc sulfate, N-acetyl cysteine, Trolox, an AXL kinase inhibitor (e.g., R428), heparin, and / or combinations thereof. In embodiments, stage 7 lasts for 1-7 days.PATENTAttorney Docket No. 048440-851001 WO

[0160] In another aspect is provided a beta cell including: (i) a mutant sr39 HSV-TK proteinencoding gene; (ii) an inactivated B2M gene; (iii) an inactivated CIITA gene; (iv) an exogenous CD274 gene; and (v) an exogenous CD47 gene.

[0161] In another aspect is provided a beta cell including a mutant sr39 HSV-TK proteinencoding gene. In embodiments, the beta cell further includes an inactivated B2M gene. In embodiments, the beta cell further includes an inactivated CIITA gene. In embodiments, the beta cell further includes an exogenous CD274 gene. In embodiments, the beta cell further includes an exogenous CD47 gene.

[0162] In embodiments, the beta cell further includes an inactivated TPH1 gene. In embodiments, the beta cell includes an inactivated TPH1 gene.

[0163] In embodiments, the beta cell further includes an HLA-E gene. In embodiments, the beta cell includes an HLA-E gene. In embodiments, the HLA-E gene is an exogenous HLA-E gene. In embodiments, the beta cell further includes an HLA-G gene. In embodiments, the beta cell includes an HLA-G gene. In embodiments, the HLA-G gene is an exogenous HLA-G gene. In embodiments, the beta cell includes an HLA-E gene and / or an HLA-G gene. In embodiments, the beta cell includes an HLA-E gene and an HLA-G gene. In embodiments, the beta cell includes an HLA-E gene or an HLA-G gene.

[0164] In embodiments, the beta cell further includes an exogenous CD59 gene.

[0165] In embodiments, the beta cell further includes an exogenous transcription factor (e.g., MAFA) gene. In embodiments, the beta cell further includes an exogenous MAFA gene. In embodiments, the exogenous MAFA gene is inserted into the reading frame of a PCSK1 gene. In embodiments, the exogenous MAFA gene is not inserted into the INS locus.

[0166] In embodiments, the beta cell further includes an exogenous CD46 gene, an exogenous CD55 gene, and / or an exogenous CD59 gene. In embodiments, the beta cell includes an exogenous CD46 gene, an exogenous CD55 gene, and / or an exogenous CD59 gene. In embodiments, the beta cell includes an exogenous CD46 gene, an exogenous CD55 gene, and an exogenous CD59 gene. In embodiments, the beta cell includes an exogenous CD46 gene and an exogenous CD55 gene. In embodiments, the beta cell includes an exogenous CD55 gene and an exogenous CD59 gene. In embodiments, the beta cell includes an exogenous CD46 gene and anPATENTAttorney Docket No. 048440-851001 WO exogenous CD59 gene. In embodiments, the beta cell includes an exogenous CD46 gene, an exogenous CD55 gene, or an exogenous CD59 gene. In embodiments, the beta cell includes an exogenous CD46 gene. In embodiments, the beta cell includes an exogenous CD55 gene. In embodiments, the beta cell includes an exogenous CD59 gene.

[0167] In embodiments, the beta cell further includes a tissue factor pathway inhibitor (TFPI) protein-encoding gene. In embodiments, the beta cell further includes an inactivated RNLS gene. In embodiments, the beta cell further includes an exogenous CD64 gene.

[0168] In embodiments, the beta cell includes a TFPI protein-encoding gene, an inactivated RNLS gene, and / or an exogenous CD64 gene. In embodiments, the beta cell includes a TFPI protein-encoding gene, an inactivated RNLS gene, and an exogenous CD64 gene. In embodiments, the beta cell includes a TFPI protein-encoding gene and an inactivated RNLS gene. In embodiments, the beta cell includes a TFPI protein-encoding gene and an exogenous CD64 gene. In embodiments, the beta cell includes an inactivated RNLS gene and an exogenous CD64 gene. In embodiments, the beta cell includes a TFPI protein-encoding gene, an inactivated RNLS gene, or an exogenous CD64 gene. In embodiments, the beta cell includes a TFPI proteinencoding gene. In embodiments, the beta cell includes an inactivated RNLS gene. In embodiments, the beta cell includes an exogenous CD64 gene.

[0169] In another aspect is provided a composition including a plurality of the beta cells provided herein including embodiments thereof.

[0170] In embodiments, the beta cell provided herein including embodiments thereof is cultured on an extracellular matrix. In embodiments, the beta cell is expanded on an extracellular matrix. In embodiments, the extracellular matrix is a Matrigel extracellular matrix. In embodiments, the extracellular matrix is a laminin extracellular matrix. In embodiments, the extracellular matrix is a laminin 521 extracellular matrix. In embodiments, the extracellular matrix does not include a Matrigel extracellular matrix. Materials and methods for generating beta cells from human pluripotent stem cells are well known in the art (See e.g., Wu et al., “Using gene editing to establish a safeguard system for pluripotent stem-cell-based therapies” iScience 2019;22:409-22; PMCID: PMC6909005, which is incorporated herein by reference in its entirety and for all purposes). The beta cell provided herein including embodiments thereof may be generated by any of the differentiation methods disclosed in Wu et al. iScience 2019; 22:409-22.PATENTAttorney Docket No. 048440-851001 WO

[0171] In embodiments, the composition further includes mesenchymal stem cells. In embodiments, the composition further includes native human islets. In embodiments, the composition further includes regulatory T cells. In embodiments, the composition further includes mesenchymal stem cells and native human islets. In embodiments, the composition further includes mesenchymal stem cells and regulatory T cells. In embodiments, the composition further includes native human islets and regulatory T cells. In embodiments, the composition further includes mesenchymal stem cells, native human islets, and regulatory T cells.

[0172] In embodiments, the mesenchymal stem cells further include an exogenous CD274 gene. In embodiments, the mesenchymal stem cells further include an exogenous CD47 gene. In embodiments, the mesenchymal cell includes an exogenous CD274 gene and an exogenous CD47 gene. Materials and methods for genetically modifying mesenchymal cells to express exogenous CD274 and / or CD47 are well known in the art (See e.g., Wang el al., “Engineered immunomodulatory accessory cells improve experimental allogeneic islet transplantation without immunosuppression,” Sci Adv, 2022;8(29):eabn0071; PMCID: 9307254, which is incorporated herein by reference in its entirety and for all purposes). The mesenchymal cells provided herein including embodiments thereof may be genetically modified using any of the exogenous genes and / or methods disclosed in Wang et al. Sci Adv, 2022; 8(29) :eabn0071.

[0173] In embodiments, the native human islets further include an exogenous CD274 gene. In embodiments, the native human islets further include an exogenous CD47 gene. In embodiments, the native human islets includes an exogenous CD274 gene and an exogenous CD47 gene. The native human islets provided herein including embodiments thereof may be genetically modified using any of the exogenous genes and / or methods disclosed in Wang et al. Sci Adv, 2022; 8(29) :eabn0071.

[0174] In embodiments, the composition further includes a mesenchymal stem cell-derived extracellular vesicle.

[0175] In another aspect is provided a cell aggregate including a plurality of the human pluripotent stem cells provided herein including embodiments thereof.

[0176] In another aspect is provided a cell aggregate including a plurality of the beta cells provided herein including embodiments thereof.PATENTAttorney Docket No. 048440-851001 WO

[0177] In embodiments, the cell aggregate further includes mesenchymal stem cells. In embodiments, the cell aggregate further includes native human islets. In embodiments, the cell aggregate further includes regulatory T cells. In embodiments, the cell aggregate further includes mesenchymal stem cells and native human islets. In embodiments, the cell aggregate further includes mesenchymal stem cells and regulatory T cells. In embodiments, the cell aggregate further includes native human islets and regulatory T cells. In embodiments, the cell aggregate further includes mesenchymal stem cells, native human islets, and regulatory T cells.

[0178] In embodiments, the mesenchymal stem cells further include an exogenous CD274 gene. In embodiments, the mesenchymal stem cells further include an exogenous CD47 gene. In embodiments, the mesenchymal stem cells include an exogenous CD274 gene and an exogenous CD47 gene.

[0179] In embodiments, the native human islets further include an exogenous CD274 gene. In embodiments, the native human islets further include an exogenous CD47 gene. In embodiments, the native human islets include an exogenous CD274 gene and an exogenous CD47 gene.

[0180] In embodiments, the cell aggregate further includes a mesenchymal stem cell-derived extracellular vesicle.

[0181] In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 250 microns. In embodiments, the longest diameter of the cell aggregate is from about 110 microns to about 250 microns. In embodiments, the longest diameter of the cell aggregate is from about 120 microns to about 250 microns. In embodiments, the longest diameter of the cell aggregate is from about 130 microns to about 250 microns. In embodiments, the longest diameter of the cell aggregate is from about 140 microns to about 250 microns. In embodiments, the longest diameter of the cell aggregate is from about 150 microns to about 250 microns. In embodiments, the longest diameter of the cell aggregate is from about 160 microns to about 250 microns. In embodiments, the longest diameter of the cell aggregate is from about 170 microns to about 250 microns. In embodiments, the longest diameter of the cell aggregate is from about 180 microns to about 250 microns. In embodiments, the longest diameter of the cell aggregate is from about 190 microns to about 250 microns. In embodiments, the longest diameter of the cell aggregate is from about 200 microns to about 250 microns. In embodiments, the longest diameter of the cell aggregate is from about 210 microns to about 250 microns. In embodiments, the longest diameterPATENTAttorney Docket No. 048440-851001 WO of the cell aggregate is from about 220 microns to about 250 microns. In embodiments, the longest diameter of the cell aggregate is from about 230 microns to about 250 microns. In embodiments, the longest diameter of the cell aggregate is from about 240 microns to about 250 microns.

[0182] In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 240 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 230 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 220 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 210 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 200 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 190 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 180 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 170 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 160 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 150 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 140 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 130 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 120 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 110 microns.

[0183] In embodiments, the longest diameter of the cell aggregate is about 100 microns. In embodiments, the longest diameter of the cell aggregate is about 110 microns. In embodiments, the longest diameter of the cell aggregate is about 120 microns. In embodiments, the longest diameter of the cell aggregate is about 130 microns. In embodiments, the longest diameter of the cell aggregate is about 140 microns. In embodiments, the longest diameter of the cell aggregate is about 150 microns. In embodiments, the longest diameter of the cell aggregate is about 160 microns. In embodiments, the longest diameter of the cell aggregate is about 170 microns. In embodiments, the longest diameter of the cell aggregate is about 180 microns. In embodiments, the longest diameter of the cell aggregate is about 190 microns. In embodiments, the longest diameter of the cell aggregate is about 200 microns. In embodiments, the longest diameter of the cell aggregate is about 210 microns. In embodiments, the longest diameter of the cell aggregate isPATENTAttorney Docket No. 048440-851001 WO about 220 microns. In embodiments, the longest diameter of the cell aggregate is about 100 microns. In embodiments, the longest diameter of the cell aggregate is about 230 microns. In embodiments, the longest diameter of the cell aggregate is about 240 microns. In embodiments, the longest diameter of the cell aggregate is about 250 microns.

[0184] In another aspect is provided a polymeric microcapsule including: (i) the human pluripotent stem cell provided herein including embodiments thereof; (ii) the beta cell provided herein including embodiments thereof; (iii) the composition provided herein including embodiments thereof; or (iv) the cell aggregate provided herein including embodiments thereof.

[0185] In embodiments, the polymeric microcapsule includes the human pluripotent stem cell provided herein including embodiments thereof. In embodiments, the polymeric microcapsule includes the beta cell provided herein including embodiments thereof. In embodiments, the polymeric microcapsule includes the composition provided herein including embodiments thereof. In embodiments, the polymeric microcapsule includes the cell aggregate provided herein including embodiments thereof.

[0186] In another aspect is provided an implantable device including: (i) the human pluripotent stem cell provided herein including embodiments thereof; (ii) the beta cell provided herein including embodiments thereof; (iii) the composition provided herein including embodiments thereof; (iv) the cell aggregate provided herein including embodiments thereof; or (v) the polymeric microcapsule provided herein including embodiments thereof.

[0187] In embodiments, the implantable device includes the human pluripotent stem cell provided herein including embodiments thereof. In embodiments, the implantable device includes the beta cell provided herein including embodiments thereof. In embodiments, the implantable device includes the composition provided herein including embodiments thereof. In embodiments, the implantable device includes the cell aggregate provided herein including embodiments thereof. In embodiments, the implantable device includes the polymeric capsule provided herein including embodiments thereof.

[0188] Provided herein is a human pluripotent stem cell including a mutant sr39 HSV-TK protein-encoding gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene and an inactivated B2M gene. In embodiments, the humanPATENTAttorney Docket No. 048440-851001 WO pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene and an inactivated CIITA gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an inactivated B2M gene, and an inactivated CIITA gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene and an HLA-E gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an HLA-E gene, and an inactivated B2M gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an HLA-E gene, and an inactivated CIITA gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an HLA-E gene, an inactivated B2M gene, and an inactivated CIITA gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an inactivated TPH1 gene, an HLA-E gene, an inactivated B2M gene, and an inactivated CIITA gene. Provided herein is a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a beta cell derived from any of the hPSC described herein, including embodiments thereof (i.e., “hPSC-derived beta cell”). Provided herein is a composition including a plurality of the hPSC-derived beta cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof and a plurality of the hPSC-derived beta cells described herein, including embodiments thereof.

[0189] In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an inactivated B2M gene, and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an inactivated CIITA gene, and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK proteinencoding gene, an inactivated B2M gene, an inactivated CIITA gene, and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK proteinencoding gene, an HLA-E gene, and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an HLA-E gene, an inactivated B2M gene, and an exogenous CD47 gene. In embodiments, the human pluripotentPATENTAttorney Docket No. 048440-851001 WO stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an HLA-E gene, an inactivated CUT A gene, and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an HLA-E gene, an inactivated B2M gene, an inactivated CIITA gene, and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an inactivated TPH1 gene, an HLA-E gene, an inactivated B2M gene, an inactivated CIITA gene, and an exogenous CD47 gene. Provided herein is a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a beta cell derived from any of the hPSC described herein, including embodiments thereof (i.e., “hPSC-derived beta cell”). Provided herein is a composition including a plurality of the hPSC- derived beta cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof and a plurality of the hPSC-derived beta cells described herein, including embodiments thereof.

[0190] In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an inactivated B2M gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an inactivated CIITA gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK proteinencoding gene, an inactivated B2M gene, an inactivated CIITA gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK proteinencoding gene, an HLA-E gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an HLA-E gene, an inactivated B2M gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an HLA-E gene, an inactivated CIITA gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an HLA-E gene, an inactivated B2M gene, an inactivated CIITA gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an inactivated TPH1PATENTAttorney Docket No. 048440-851001 WO gene, an HLA-E gene, an inactivated B2M gene, an inactivated CIITA gene, and an exogenous CD274 gene. Provided herein is a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a beta cell derived from any of the hPSC described herein, including embodiments thereof (i.e., “hPSC-derived beta cell”). Provided herein is a composition including a plurality of the hPSC- derived beta cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof and a plurality of the hPSC-derived beta cells described herein, including embodiments thereof.

[0191] In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an exogenous CD47 gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an inactivated B2M gene, an exogenous CD47 gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an inactivated CIITA gene, an exogenous CD47 gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an inactivated B2M gene, an inactivated CIITA gene, an exogenous CD47 gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an HLA-E gene, an exogenous CD47 gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an HLA-E gene, an inactivated B2M gene, an exogenous CD47 gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an HLA-E gene, an inactivated CIITA gene, an exogenous CD47 gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an HLA-E gene, an inactivated B2M gene, an inactivated CIITA gene, an exogenous CD47 gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene, an inactivated TPH1 gene, an HLA-E gene, an inactivated B2M gene, an inactivated CIITA gene, an exogenous CD47 gene, and an exogenous CD274 gene. Provided herein is a plurality of the human pluripotent stem cells described herein, including embodiments thereof. ProvidedPATENTAttorney Docket No. 048440-851001 WO herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a beta cell derived from any of the hPSC described herein, including embodiments thereof (i.e., “hPSC-derived beta cell”). Provided herein is a composition including a plurality of the hPSC- derived beta cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof and a plurality of the hPSC-derived beta cells described herein, including embodiments thereof.

[0192] Provided herein is a human pluripotent stem cell including an inactivated TPH1 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene and a mutant sr39 HSV-TK protein-encoding gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene and an inactivated B2M gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene and an inactivated CIITA gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene an inactivated TPH1 gene, an inactivated B2M gene, and an inactivated CIITA gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene and an HLA-E gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an HLA-E gene, and an inactivated B2M gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an HLA-E gene, and an inactivated CIITA gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an HLA-E gene, an inactivated B2M gene, and an inactivated CIITA gene. Provided herein is a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a beta cell derived from any of the hPSC described herein, including embodiments thereof (i.e., “hPSC-derived beta cell”). Provided herein is a composition including a plurality of the hPSC-derived beta cells described herein, including embodiments thereof.Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof and a plurality of the hPSC-derived beta cells described herein, including embodiments thereof.PATENTAttorney Docket No. 048440-851001 WO

[0193] In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, a mutant sr39 HSV-TK protein-encoding gene, and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an inactivated B2M gene, and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an inactivated CIITA gene, and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK proteinencoding gene an inactivated TPH1 gene, an inactivated B2M gene, an inactivated CIITA gene, and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an HLA-E gene, and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an HLA-E gene, an inactivated B2M gene, and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an HLA-E gene, an inactivated CIITA gene, and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an HLA-E gene, an inactivated B2M gene, an inactivated CIITA gene, and an exogenous CD47 gene. Provided herein is a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a beta cell derived from any of the hPSC described herein, including embodiments thereof (i.e., “hPSC- derived beta cell”). Provided herein is a composition including a plurality of the hPSC-derived beta cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof and a plurality of the hPSC-derived beta cells described herein, including embodiments thereof.

[0194] In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, a mutant sr39 HSV-TK protein-encoding gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an inactivated B2M gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an inactivated CIITA gene, and an exogenousCD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TKPATENTAttorney Docket No. 048440-851001 WO protein-encoding gene an inactivated TPH1 gene, an inactivated B2M gene, an inactivated CIITA gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an HLA-E gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an HLA-E gene, an inactivated B2M gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an HLA-E gene, an inactivated CIITA gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an HLA-E gene, an inactivated B2M gene, an inactivated CIITA gene, and an exogenous CD274 gene. Provided herein is a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a beta cell derived from any of the hPSC described herein, including embodiments thereof (i.e., “hPSC-derived beta cell”). Provided herein is a composition including a plurality of the hPSC- derived beta cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof and a plurality of the hPSC-derived beta cells described herein, including embodiments thereof.

[0195] In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an exogenous CD274 gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, a mutant sr39 HSV-TK protein-encoding gene, an exogenous CD274 gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an inactivated B2M gene, an exogenous CD274 gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an inactivated CIITA gene, an exogenous CD274 gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes a mutant sr39 HSV-TK protein-encoding gene an inactivated TPH1 gene, an inactivated B2M gene, an inactivated CIITA gene, an exogenous CD274 gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an HLA-E gene, an exogenous CD274 gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an HLA-E gene, an inactivated B2M gene, an exogenous CD274 gene, and an exogenous CD274 gene. In embodiments, the humanPATENTAttorney Docket No. 048440-851001 WO pluripotent stem cell includes an inactivated TPH1 gene, an HLA-E gene, an inactivated CIITA gene, an exogenous CD274 gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated TPH1 gene, an HLA-E gene, an inactivated B2M gene, an inactivated CIITA gene, an exogenous CD274 gene, and an exogenous CD274 gene. Provided herein is a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a beta cell derived from any of the hPSC described herein, including embodiments thereof (i.e., “hPSC- derived beta cell”). Provided herein is a composition including a plurality of the hPSC-derived beta cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof and a plurality of the hPSC-derived beta cells described herein, including embodiments thereof.

[0196] Provided herein is a human pluripotent stem cell including an inactivated B2M gene and an inactivated CIITA gene. In embodiments, the human pluripotent stem cell includes an inactivated B2M gene. In embodiments, the human pluripotent stem cell includes an inactivated CIITA gene. In embodiments, the human pluripotent stem cell includes an inactivated B2M gene, an inactivated CIITA gene, and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes an inactivated B2M gene and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes an inactivated CIITA gene and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes an inactivated B2M gene, an inactivated CIITA gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated B2M gene and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated CIITA gene and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated B2M gene, an inactivated CIITA gene, an exogenous CD47 gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated B2M gene, an exogenous CD47 gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated CIITA gene, an exogenous CD47 gene, and an exogenous CD274 gene. Provided herein is a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cellsPATENTAttorney Docket No. 048440-851001 WO described herein, including embodiments thereof. Provided herein is a beta cell derived from any of the hPSC described herein, including embodiments thereof (i.e., “hPSC-derived beta cell”). Provided herein is a composition including a plurality of the hPSC-derived beta cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof and a plurality of the hPSC-derived beta cells described herein, including embodiments thereof.

[0197] Provided herein is a human pluripotent stem cell including an inactivated B2M gene; an HLA-E gene; and an inactivated CIITA gene. In embodiments, the human pluripotent stem cell includes an inactivated B2M gene, an HLA-E gene, an inactivated CIITA gene, and an exogenous CD47 gene. In embodiments, the human pluripotent stem cell includes an inactivated B2M gene, an HLA-E gene, an inactivated CIITA gene, and an exogenous CD274 gene. In embodiments, the human pluripotent stem cell includes an inactivated B2M gene, an HLA-E gene, an inactivated CIITA gene, an exogenous CD47 gene, and an exogenous CD274 gene. Provided herein is a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof. Provided herein is a beta cell derived from any of the hPSC described herein, including embodiments thereof (i.e., “hPSC-derived beta cell”). Provided herein is a composition including a plurality of the hPSC-derived beta cells described herein, including embodiments thereof. Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof and a plurality of the hPSC-derived beta cells described herein, including embodiments thereof.

[0198] Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof. In embodiments, the composition further includes mesenchymal stem cells. In embodiments, the composition further includes native human islets. In embodiments, the composition further includes regulatory T cells. In embodiments, the composition further includes MSC-derived EVs. In embodiments, the composition further includes mesenchymal stem cells and native human islets. In embodiments, the composition further includes mesenchymal stem cells and regulatory T cells. In embodiments, the composition further includes native human islets and regulatory T cells. In embodiments, the composition further includes mesenchymal stem cells, native human islets, and regulatory T cells. InPATENTAttorney Docket No. 048440-851001 WO embodiments, the composition further includes mesenchymal stem cells and MSC-derived EVs. In embodiments, the composition further includes MSC-derived EVs and regulatory T cells. In embodiments, the composition further includes native human islets and MSC-derived EVs. In embodiments, the composition further includes mesenchymal stem cells, MSC-derived EVs, and native human islets. In embodiments, the composition further includes mesenchymal stem cells, MSC-derived EVs, and regulatory T cells. In embodiments, the composition further includes native human islets, MSC-derived EVs, and regulatory T cells. In embodiments, the composition further includes mesenchymal stem cells, native human islets, MSC-derived EVs, and regulatory T cells.

[0199] Provided herein is a composition including a plurality of the hPSC-derived beta cells described herein, including embodiments thereof. In embodiments, the composition further includes mesenchymal stem cells. In embodiments, the composition further includes native human islets. In embodiments, the composition further includes regulatory T cells. In embodiments, the composition further includes MSC-derived EVs. In embodiments, the composition further includes mesenchymal stem cells and native human islets. In embodiments, the composition further includes mesenchymal stem cells and regulatory T cells. In embodiments, the composition further includes native human islets and regulatory T cells. In embodiments, the composition further includes mesenchymal stem cells, native human islets, and regulatory T cells. In embodiments, the composition further includes mesenchymal stem cells and MSC-derived EVs. In embodiments, the composition further includes MSC-derived EVs and regulatory T cells. In embodiments, the composition further includes native human islets and MSC-derived EVs. In embodiments, the composition further includes mesenchymal stem cells, MSC-derived EVs, and native human islets. In embodiments, the composition further includes mesenchymal stem cells, MSC-derived EVs, and regulatory T cells. In embodiments, the composition further includes native human islets, MSC-derived EVs, and regulatory T cells. In embodiments, the composition further includes mesenchymal stem cells, native human islets, MSC-derived EVs, and regulatory T cells.

[0200] Provided herein is a composition including a plurality of the human pluripotent stem cells described herein, including embodiments thereof and a plurality of the hPSC-derived beta cells described herein, including embodiments thereof. In embodiments, the composition furtherPATENTAttorney Docket No. 048440-851001 WO includes mesenchymal stem cells. In embodiments, the composition further includes native human islets. In embodiments, the composition further includes regulatory T cells. In embodiments, the composition further includes MSC-derived EVs. In embodiments, the composition further includes mesenchymal stem cells and native human islets. In embodiments, the composition further includes mesenchymal stem cells and regulatory T cells. In embodiments, the composition further includes native human islets and regulatory T cells. In embodiments, the composition further includes mesenchymal stem cells, native human islets, and regulatory T cells. In embodiments, the composition further includes mesenchymal stem cells and MSC-derived EVs. In embodiments, the composition further includes MSC-derived EVs and regulatory T cells. In embodiments, the composition further includes native human islets and MSC-derived EVs. In embodiments, the composition further includes mesenchymal stem cells, MSC-derived EVs, and native human islets. In embodiments, the composition further includes mesenchymal stem cells, MSC-derived EVs, and regulatory T cells. In embodiments, the composition further includes native human islets, MSC-derived EVs, and regulatory T cells. In embodiments, the composition further includes mesenchymal stem cells, native human islets, MSC-derived EVs, and regulatory T cells.

[0201] Provided herein is a cell aggregate including the human pluripotent stem cells described herein, including embodiments thereof. In embodiments, the cell aggregate further includes mesenchymal stem cells. In embodiments, the cell aggregate further includes native human islets. In embodiments, the cell aggregate further includes regulatory T cells. In embodiments, the cell aggregate further includes MSC-derived EVs. In embodiments, the cell aggregate further includes mesenchymal stem cells and native human islets. In embodiments, the cell aggregate further includes mesenchymal stem cells and regulatory T cells. In embodiments, the cell aggregate further includes native human islets and regulatory T cells. In embodiments, the cell aggregate further includes mesenchymal stem cells, native human islets, and regulatory T cells. In embodiments, the cell aggregate further includes mesenchymal stem cells and MSC-derived EVs. In embodiments, the cell aggregate further includes MSC-derived EVs and regulatory T cells. In embodiments, the cell aggregate further includes native human islets and MSC-derived EVs. In embodiments, the cell aggregate further includes mesenchymal stem cells, MSC-derived EVs, and native human islets. In embodiments, the cell aggregate further includes mesenchymal stem cells, MSC-derived EVs, and regulatory T cells. In embodiments, the cell aggregate further includesPATENTAttorney Docket No. 048440-851001 WO native human islets, MSC-derived EVs, and regulatory T cells. In embodiments, the cell aggregate further includes mesenchymal stem cells, native human islets, MSC-derived EVs, and regulatory T cells.

[0202] Provided herein is a cell aggregate including the hPSC-derived beta cells described herein, including embodiments, thereof. In embodiments, the cell aggregate further includes mesenchymal stem cells. In embodiments, the cell aggregate further includes native human islets. In embodiments, the cell aggregate further includes regulatory T cells. In embodiments, the cell aggregate further includes MSC-derived EVs. In embodiments, the cell aggregate further includes mesenchymal stem cells and native human islets. In embodiments, the cell aggregate further includes mesenchymal stem cells and regulatory T cells. In embodiments, the cell aggregate further includes native human islets and regulatory T cells. In embodiments, the cell aggregate further includes mesenchymal stem cells, native human islets, and regulatory T cells. In embodiments, the cell aggregate further includes mesenchymal stem cells and MSC-derived EVs. In embodiments, the cell aggregate further includes MSC-derived EVs and regulatory T cells. In embodiments, the cell aggregate further includes native human islets and MSC-derived EVs. In embodiments, the cell aggregate further includes mesenchymal stem cells, MSC-derived EVs, and native human islets. In embodiments, the cell aggregate further includes mesenchymal stem cells, MSC-derived EVs, and regulatory T cells. In embodiments, the cell aggregate further includes native human islets, MSC-derived EVs, and regulatory T cells. In embodiments, the cell aggregate further includes mesenchymal stem cells, native human islets, MSC-derived EVs, and regulatory T cells.

[0203] Provided herein is a cell aggregate including the human pluripotent stem cells described herein, including embodiments thereof, and the hPSC-derived beta cells described herein, including embodiments thereof. In embodiments, the cell aggregate further includes mesenchymal stem cells. In embodiments, the cell aggregate further includes native human islets. In embodiments, the cell aggregate further includes regulatory T cells. In embodiments, the cell aggregate further includes MSC-derived EVs. In embodiments, the cell aggregate further includes mesenchymal stem cells and native human islets. In embodiments, the cell aggregate further includes mesenchymal stem cells and regulatory T cells. In embodiments, the cell aggregate further includes native human islets and regulatory T cells. In embodiments, the cell aggregatePATENTAttorney Docket No. 048440-851001 WO further includes mesenchymal stem cells, native human islets, and regulatory T cells. In embodiments, the cell aggregate further includes mesenchymal stem cells and MSC-derived EVs. In embodiments, the cell aggregate further includes MSC-derived EVs and regulatory T cells. In embodiments, the cell aggregate further includes native human islets and MSC-derived EVs. In embodiments, the cell aggregate further includes mesenchymal stem cells, MSC-derived EVs, and native human islets. In embodiments, the cell aggregate further includes mesenchymal stem cells, MSC-derived EVs, and regulatory T cells. In embodiments, the cell aggregate further includes native human islets, MSC-derived EVs, and regulatory T cells. In embodiments, the cell aggregate further includes mesenchymal stem cells, native human islets, MSC-derived EVs, and regulatory T cells.

[0204] Provided herein is a cell aggregate including a composition described herein, including embodiments, thereof. In embodiments, the cell aggregate further includes mesenchymal stem cells. In embodiments, the cell aggregate further includes native human islets. In embodiments, the cell aggregate further includes regulatory T cells. In embodiments, the cell aggregate further includes MSC-derived EVs. In embodiments, the cell aggregate further includes mesenchymal stem cells and native human islets. In embodiments, the cell aggregate further includes mesenchymal stem cells and regulatory T cells. In embodiments, the cell aggregate further includes native human islets and regulatory T cells. In embodiments, the cell aggregate further includes mesenchymal stem cells, native human islets, and regulatory T cells. In embodiments, the cell aggregate further includes mesenchymal stem cells and MSC-derived EVs. In embodiments, the cell aggregate further includes MSC-derived EVs and regulatory T cells. In embodiments, the cell aggregate further includes native human islets and MSC-derived EVs. In embodiments, the cell aggregate further includes mesenchymal stem cells, MSC-derived EVs, and native human islets. In embodiments, the cell aggregate further includes mesenchymal stem cells, MSC-derived EVs, and regulatory T cells. In embodiments, the cell aggregate further includes native human islets, MSC-derived EVs, and regulatory T cells. In embodiments, the cell aggregate further includes mesenchymal stem cells, native human islets, MSC-derived EVs, and regulatory T cells.

[0205] In embodiments, the regulatory T cell can be a reprogrammed regulatory T cell as described in WO 2022 / 060854, the disclosure of which is incorporated by reference herein in its entirety and for all purposes.PATENTAttorney Docket No. 048440-851001 WO

[0206] In embodiments, the cell aggregates described herein, including embodiments thereof, and / or the cells described herein (e g., hPSCs, hPSC-derived beta cells, mesenchymal stem cells, regulatory T cells, native human islets), including embodiments thereof, can include a cell-surface modification. Cell-surface modifications includes those described in WO 2014 / 058359, the disclosure of which is incorporated by reference herein in its entirety and for all purposes.

[0207] In embodiments, the cell aggregate is a synthetic islet. In embodiments, the synthetic islet includes native islet cells. In embodiments, the synthetic islet includes hPSC-derived beta cells. In embodiments, the synthetic islet includes native islet cells and hPSC-derived beta cells. In embodiments, the synthetic islet further includes mesenchymal stem cells, MSC-derived EVs, regulatory T cells, or a combination of two or more thereof.

[0208] In embodiments, the longest diameter of the cell aggregate is about 550 microns or less. In embodiments, the longest diameter of the cell aggregate is about 500 microns or less. In embodiments, the longest diameter of the cell aggregate is about 450 microns or less. In embodiments, the longest diameter of the cell aggregate is about 400 microns or less. In embodiments, the longest diameter of the cell aggregate is about 350 microns or less. In embodiments, the longest diameter of the cell aggregate is about 300 microns or less. In embodiments, the longest diameter of the cell aggregate is about 250 microns or less. In embodiments, the longest diameter of the cell aggregate is about 200 microns or less.

[0209] In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 250 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 300 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 350 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 400 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 450 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 500 microns. In embodiments, the longest diameter of the cell aggregate is from about 100 microns to about 550 microns.

[0210] Provided herein is a polymeric microcapsule including the human pluripotent stem cells described herein, including embodiments, thereof. Provided herein is a polymeric microcapsule including a hPSC-derived beta cells described herein, including embodiments, thereof. Provided herein is a polymeric microcapsule including a composition described herein, includingPATENTAttorney Docket No. 048440-851001 WO embodiments, thereof. Provided herein is a polymeric microcapsule including a synthetic islet described herein, including embodiments thereof. Provided herein is a polymeric microcapsule including a cell aggregate described herein, including embodiments, thereof. In embodiments, the polymeric microcapsule is a hydrogel microparticle. In embodiments, the polymeric microcapsule is a hydrogel microparticle described in WO 2022 / 120478, the disclosure of which is incorporated by reference herein in its entirety and for all purposes. In embodiments, the hydrogel microparticle is within a biomimetic bead as described in WO 2022 / 120478, the disclosure of which is incorporated by reference herein in its entirety and for all purposes. The term “hydrogel” refers to polymers (optionally crosslinked) that can swell in water. In embodiments, the hydrogel microparticle includes polyethylene glycol.METHODS OF TREATMENT

[0211] Provided herein are, inter alia, methods for treating diabetes using genetically engineered stem cell-derived islets (SC-islets). The methods described herein include administering to a subject in need thereof a therapeutically effective amount of SC-islets derived from genetically modified hPSCs, including embodiments thereof. In embodiments, the SC-islets are derived from hPSCs including a suicide gene (e.g., mutant sr39 HSV-TK), inactivated immune recognition genes (e g., B2M gene and / or CIITA gene), and / or exogenous expression of immune checkpoint and / or anti-phagocytic proteins (e.g., PD-L1 and / or CD47). In embodiments, the hPSCs may further include additional modifications such as inactivation of TPH1, RNLS, and / or expression of complement regulatory proteins (e.g., CD46, CD55, and / or CD59), transcription factors (e.g., MAFA), or non-classical HLA molecules (e.g., HLA-E and / or HLA-G). These modifications enable the SC-islets to secrete insulin in response to glucose stimulation, while reducing the risk of immune rejection and minimizing the potential for malignancy. In embodiments, the SC-islets restore glycemic control in diabetic subjects following transplantation. Thus, in an aspect is provided a method of treating diabetes in a subject in need thereof, the method including administering to the subject a therapeutically effective amount of SC-islets derived from a human pluripotent stem cell including: (i) a mutant sr39 HSV-TK protein-encoding gene; (ii) an inactivated B2M gene; (iii) an inactivated CIITA gene; (iv) an exogenous CD274 gene; and (v) an exogenous CD47 gene, thereby treating diabetes.PATENTAttorney Docket No. 048440-851001 WO

[0212] In another aspect is provided a method of treating diabetes in a patient in need thereof, the method including administering to the patient an effective amount of: (i) the human pluripotent stem cell provided herein including embodiments thereof; (ii) the beta cell provided herein including embodiments thereof; (iii) the composition provided herein including embodiments thereof; (iv) the cell aggregate provided herein including embodiments thereof; or (v) the polymeric microcapsule provided herein including embodiments thereof.

[0213] In embodiments, the method includes administering to the patient an effective amount of the human pluripotent stem cell provided herein including embodiments thereof. In embodiments, the method includes administering to the patient an effective amount of the beta cell provided herein including embodiments thereof. In embodiments, the method includes administering to the patient an effective amount of the composition provided herein including embodiments thereof. In embodiments, the method includes administering to the patient an effective amount of the cell aggregate provided herein including embodiments thereof. In embodiments, the method includes administering to the patient an effective amount of the polymeric microcapsule provided herein including embodiments thereof.

[0214] In another aspect is provided a method of treating diabetes in a patient in need thereof, the method including administering to the patient an effective amount of the human pluripotent stem cell provided herein including embodiments thereof.

[0215] In another aspect is provided a method of treating diabetes in a patient in need thereof, the method including administering to the patient an effective amount of the beta cell provided herein including embodiments thereof.

[0216] In another aspect is provided a method of treating diabetes in a patient in need thereof, the method including administering to the patient an effective amount of the composition provided herein including embodiments thereof.

[0217] In another aspect is provided a method of treating diabetes in a patient in need thereof, the method including administering to the patient an effective amount of the cell aggregate provided herein including embodiments thereof.PATENTAttorney Docket No. 048440-851001 WO

[0218] In another aspect is provided a method of treating diabetes in a patient in need thereof, the method including administering to the patient an effective amount of the polymeric microcapsule provided herein including embodiments thereof.

[0219] In embodiments, the administering is administering via an implanted device; wherein the device includes (i), (ii), (iii), (iv), or (v). In embodiments, the administering is administering via an implanted device; wherein the device includes (i). In embodiments, the administering is administering via an implanted device; wherein the device includes (ii). In embodiments, the administering is administering via an implanted device; wherein the device includes (iii). In embodiments, the administering is administering via an implanted device; wherein the device includes (iv). In embodiments, the administering is administering via an implanted device; wherein the device includes (v).

[0220] In embodiments, the administering is administering via an omental pouch; wherein the omental pouch includes (i), (ii), (iii), (iv), or (v). In embodiments, the administering is administering via an omental pouch; wherein the omental pouch includes (i). In embodiments, the administering is administering via an omental pouch; wherein the omental pouch includes (ii). In embodiments, the administering is administering via an omental pouch; wherein the omental pouch includes (iii). In embodiments, the administering is administering via an omental pouch; wherein the omental pouch includes (iv). In embodiments, the administering is administering via an omental pouch; wherein the omental pouch includes (v).

[0221] In embodiments, the administering is administering via an anterior rectus sheath, wherein the anterior rectus sheath includes (i), (ii), (iii), (iv), or (v). In embodiments, the administering is administering via an anterior rectus sheath, wherein the anterior rectus sheath includes (i) In embodiments, the administering is administering via an anterior rectus sheath, wherein the anterior rectus sheath includes (ii). In embodiments, the administering is administering via an anterior rectus sheath, wherein the anterior rectus sheath includes (iii). In embodiments, the administering is administering via an anterior rectus sheath, wherein the anterior rectus sheath (iv). In embodiments, the administering is administering via an anterior rectus sheath, wherein the anterior rectus sheath includes (v).

[0222] In embodiments, the diabetes is type 1 diabetes. In embodiments, the diabetes is type 2 diabetes.PATENTAttorney Docket No. 048440-851001 WO

[0223] In embodiments, the method further includes administering to the patient an effective amount of ganciclovir. In embodiments, the effective amount of ganciclovir is a low dose.

[0224] In embodiments, the method further includes monitoring the patient for serotonin production.

[0225] In embodiments, the patient had previously rejected a tissue graft or a cell graft. In embodiments, the patient had previously rejected a tissue graft. In embodiments, the patient had previously rejected a cell graft.

[0226] In embodiments, the method further includes administering to the patient an effective amount of gastrin.

[0227] In embodiments, the method includes administering to the patient an effective amount of a stem cell-derived synthetic islet (SC-SI). In embodiments, the method includes administering to the patient an effective amount of a cadaveric cell -derived synthetic islet (CC-SI).

[0228] In embodiments, the effective amount of ganciclovir is a low dose. In embodiments, the effective amount of ganciclovir is about 10 mg / kg / day or less. In embodiments, the effective amount of ganciclovir is about 9 mg / kg / day or less. In embodiments, the effective amount of ganciclovir is about 8 mg / kg / day or less. In embodiments, the effective amount of ganciclovir is about 7 mg / kg / day or less. In embodiments, the effective amount of ganciclovir is about 6 mg / kg / day or less. In embodiments, the effective amount of ganciclovir is about 5 mg / kg / day or less. In embodiments, the effective amount of ganciclovir is about 4 mg / kg / day or less. In embodiments, the effective amount of ganciclovir is about 3 mg / kg / day or less. In embodiments, the effective amount of ganciclovir is about 2 mg / kg / day or less. In embodiments, the effective amount of ganciclovir is about 1 mg / kg / day or less. In embodiments, the effective amount of ganciclovir is about 0.9 mg / kg / day or less. In embodiments, the effective amount of ganciclovir is about 0.8 mg / kg / day or less. In embodiments, the effective amount of ganciclovir is about 0.7 mg / kg / day or less. In embodiments, the effective amount of ganciclovir is about 0.6 mg / kg / day or less. In embodiments, the effective amount of ganciclovir is about 0.5 mg / kg / day or less. In embodiments, the effective amount of ganciclovir is about 0.4 mg / kg / day or less. In embodiments, the effective amount of ganciclovir is about 0.3 mg / kg / day or less. In embodiments, the effectivePATENTAttorney Docket No. 048440-851001 WO amount of ganciclovir is about 0.2 mg / kg / day or less. In embodiments, the effective amount of ganciclovir is about 0.1 mg / kg / day or less.

[0229] In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / day to about 10 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.2 mg / kg / day to about 10 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.3 mg / kg / day to about 10 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.4 mg / kg / day to about 10 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.5 mg / kg / day to about 10 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.6 mg / kg / day to about 10 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.7 mg / kg / day to about 10 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.8 mg / kg / day to about 10 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.9 mg / kg / day to about 10 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 1 mg / kg / day to about 10 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 2 mg / kg / day to about 10 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 3 mg / kg / day to about 10 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 4 mg / kg / day to about 10 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 5 mg / kg / day to about 10 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 6 mg / kg / day to about 10 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 7 mg / kg / day to about 10 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 8 mg / kg / day to about 10 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 9 mg / kg / day to about 10 mg / kg / day.

[0230] In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / day to about 9 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / day to about 8 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / day to about 7 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / day to about 6 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / day to about 5 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / day to about 4 mg / kg / day. In embodiments, thePATENTAttorney Docket No. 048440-851001 WO effective amount of ganciclovir is from about 0.1 mg / kg / day to about 3 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / day to about 2 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / day to about 1 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / day to about 0.9 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / day to about 0.8 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / day to about 0.7 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / day to about 0.6 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / day to about 0.5 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / day to about 0.4 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / day to about 0.3 mg / kg / day. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / day to about 0.2 mg / kg / day.

[0231] In embodiments, the effective amount of ganciclovir is about 10 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 9 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 8 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 7 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 6 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 5 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 4 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 3 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 2 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 1 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 0.9 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 0.8 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 0.7 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 0.6 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 0.5 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 0.4 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 0.3 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 0.2 mg / kg / week or less. In embodiments, the effective amount of ganciclovir is about 0.1 mg / kg / week or less.PATENTAttorney Docket No. 048440-851001 WO

[0232] In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / week to about 10 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.2 mg / kg / week to about 10 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.3 mg / kg / week to about 10 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.4 mg / kg / week to about 10 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.5 mg / kg / week to about 10 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.6 mg / kg / week to about 10 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.7 mg / kg / week to about 10 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.8 mg / kg / week to about 10 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.9 mg / kg / week to about 10 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 1 mg / kg / week to about 10 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 2 mg / kg / week to about 10 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 3 mg / kg / week to about 10 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 4 mg / kg / week to about 10 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 5 mg / kg / week to about 10 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 6 mg / kg / week to about 10 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 7 mg / kg / week to about 10 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 8 mg / kg / week to about 10 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 9 mg / kg / week to about 10 mg / kg / week.

[0233] In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / week to about 9 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / week to about 8 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / week to about 7 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / week to about 6 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / week to about 5 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / week to about 4 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / week to about 3 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / week to about 2 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.1PATENTAttorney Docket No. 048440-851001 WO mg / kg / week to about 1 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.01 mg / kg / week to about 1 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / week to about 0.9 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / week to about 0.8 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / week to about 0.7 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / week to about 0.6 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / week to about 0.5 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / week to about 0.4 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / week to about 0.3 mg / kg / week. In embodiments, the effective amount of ganciclovir is from about 0.1 mg / kg / week to about 0.2 mg / kg / week.

[0234] The method of treating diabetes can further include administering a gastrin compound to the subject. In embodiments, administering a gastrin compound can be pre-admini strati on of the islet cells, post-administration of the islet cells, or both. In embodiments, the gastrin compound can be administered to the subject daily, twice daily, or more. In embodiments, the gastrin compound can be administered for at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, or more days. In embodiments, the gastrin compound is administered daily for about 1 day to about 60 days. In embodiments, the gastrin compound is administered daily for about 7 days to about 60 days. In embodiments, the gastrin compound is administered daily for about 14 days to about 60 days. In embodiments, the gastrin compound is administered daily for about 21 days to about 60 days. In embodiments, the gastrin compound is administered daily for about 28 days to about 60 days. In embodiments, the gastrin compound is administered daily for about 35 days to about 60 days. In embodiments, the gastrin compound is administered daily for about 42 days to about 60 days. In embodiments, the gastrin compound is administered daily for about 49 days to about 60 days. In embodiments, the gastrin compound is administered daily for about 56 days to about 60 days.

[0235] In embodiments, the gastrin compound is administered daily for about 1 day to about 56 days. In embodiments, the gastrin compound is administered daily for about 1 day to about 49 days. In embodiments, the gastrin compound is administered daily for about 1 day to about 42 days. In embodiments, the gastrin compound is administered daily for about 1 day to about 35PATENTAttorney Docket No. 048440-851001 WO days. In embodiments, the gastrin compound is administered daily for about 1 day to about 28 days. In embodiments, the gastrin compound is administered daily for about 1 day to about 21 days. In embodiments, the gastrin compound is administered daily for about 1 day to about 14 days. In embodiments, the gastrin compound is administered daily for about 1 day to about 7 days.

[0236] In embodiments, the gastrin compound is administered daily for about 14 days to about 42 days. In embodiments, the gastrin compound is administered daily for about 1 day. In embodiments, the gastrin compound is administered daily for about 7 days. In embodiments, the gastrin compound is administered daily for about 14 days. In embodiments, the gastrin compound is administered daily for about 21 days. In embodiments, the gastrin compound is administered daily for about 28 days. In embodiments, the gastrin compound is administered daily for about 30 days. In embodiments, the gastrin compound is administered daily for about 35 days. In embodiments, the gastrin compound is administered daily for about 42 days. In embodiments, the gastrin compound is administered daily for about 49 days. In embodiments, the gastrin compound is administered daily for about 56 days. In embodiments, the gastrin compound is administered daily for about 60 days.

[0237] In embodiments, the gastrin compound is administered twice daily for about 1 day to about 60 days. In embodiments, the gastrin compound is administered twice daily for about 7 days to about 60 days. In embodiments, the gastrin compound is administered twice daily for about 14 days to about 60 days. In embodiments, the gastrin compound is administered twice daily for about 21 days to about 60 days. In embodiments, the gastrin compound is administered twice daily for about 28 days to about 60 days. In embodiments, the gastrin compound is administered twice daily for about 35 days to about 60 days. In embodiments, the gastrin compound is administered twice daily for about 42 days to about 60 days. In embodiments, the gastrin compound is administered twice daily for about 49 days to about 60 days. In embodiments, the gastrin compound is administered twice daily for about 56 days to about 60 days.

[0238] In embodiments, the gastrin compound is administered twice daily for about 1 day to about 56 days. In embodiments, the gastrin compound is administered twice daily for about 1 day to about 49 days. In embodiments, the gastrin compound is administered twice daily for about 1 day to about 42 days. In embodiments, the gastrin compound is administered twice daily for about 1 day to about 35 days. In embodiments, the gastrin compound is administered twice daily forPATENTAttorney Docket No. 048440-851001 WO about 1 day to about 28 days. In embodiments, the gastrin compound is administered twice daily for about 1 day to about 21 days. In embodiments, the gastrin compound is administered twice daily for about 1 day to about 14 days. In embodiments, the gastrin compound is administered twice daily for about 1 day to about 7 days.

[0239] In embodiments, the gastrin compound is administered twice daily for about 14 days to about 42 days In embodiments, the gastrin compound is administered twice daily for about 1 day. In embodiments, the gastrin compound is administered twice daily for about 7 days. In embodiments, the gastrin compound is administered twice daily for about 14 days. In embodiments, the gastrin compound is administered twice daily for about 21 days. In embodiments, the gastrin compound is administered twice daily for about 28 days. In embodiments, the gastrin compound is administered twice daily for about 30 days. In embodiments, the gastrin compound is administered twice daily for about 35 days. In embodiments, the gastrin compound is administered twice daily for about 42 days. In embodiments, the gastrin compound is administered twice daily for about 49 days. In embodiments, the gastrin compound is administered twice daily for about 56 days. In embodiments, the gastrin compound is administered twice daily for about 60 days.

[0240] In embodiments, the gastrin compound is gastrin-34, gastrin-17, or gastrin-14. In embodiments, the gastrin compound is gastrin-34. In embodiments, the gastrin compound is gastrin- 17. In embodiments, the gastrin compound is gastrin- 14. In embodiments, the gastrin includes the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In embodiments, the gastrin includes the amino acid sequence of SEQ ID NO:3. In embodiments, the gastrin includes the amino acid sequence of SEQ ID NO:4. In embodiments, the gastrin includes the amino acid sequence of SEQ ID NO:5. In embodiments, the gastrin includes the amino acid sequence of SEQ ID NO:6. In embodiments, the gastrin includes the amino acid sequence of SEQ ID NO:7. In embodiments, the gastrin has the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In embodiments, the gastrin has the amino acid sequence of SEQ ID NO:3. In embodiments, the gastrin has the amino acid sequence of SEQ ID NO:4. In embodiments, the gastrin has the amino acid sequence of SEQ ID NO:5. In embodiments, the gastrin has the amino acid sequence of SEQ ID NO:6. In embodiments, the gastrin has the amino acid sequence of SEQ ID NO:7.PATENTAttorney Docket No. 048440-851001 WO

[0241] In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 0.005 micrograms to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 0.01 micrograms to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 0.05 micrograms to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 0.1 micrograms to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 0.5 micrograms to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 1 microgram to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 10 micrograms to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 50 micrograms to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 100 micrograms to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 200 micrograms to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 300 micrograms to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 400 micrograms to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 500 micrograms to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 600 micrograms to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 700 micrograms to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 800 micrograms to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 900 micrograms to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 1 gram to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 2 grams to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 3 grams to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 4 grams to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 5 grams to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 6 grams to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 7 grams to about 10 grams. In embodiments, the gastrinPATENTAttorney Docket No. 048440-851001 WO compound is administered in an amount from about 8 grams to about 10 grams. In embodiments, the gastrin compound is administered in an amount from about 9 grams to about 10 grams.

[0242] In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 9 grams. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 8 grams. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 7 grams. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 6 grams. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 5 grams. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 4 grams. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 3 grams. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 2 grams. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 1 gram. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 900 micrograms. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 800 micrograms. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 700 micrograms. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 600 micrograms. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 500 micrograms. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 400 micrograms. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 300 micrograms. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 200 micrograms. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 100 micrograms. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 50 micrograms. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 10 micrograms. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 1 microgram. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 0.5 micrograms. In embodiments, the gastrin compound is administered in an amount from aboutPATENTAttorney Docket No. 048440-851001 WO0.001 micrograms to about 0.1 micrograms. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 0.05 micrograms. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 0.01 micrograms. In embodiments, the gastrin compound is administered in an amount from about 0.001 micrograms to about 0.005 micrograms.

[0243] In embodiments, the gastrin compound is administered in an amount of about 0.001 micrograms. In embodiments, the gastrin compound is administered in an amount of about 0.005 micrograms. In embodiments, the gastrin compound is administered in an amount of about 0.01 micrograms. In embodiments, the gastrin compound is administered in an amount of about 0.05 micrograms. In embodiments, the gastrin compound is administered in an amount of about 0.1 micrograms. In embodiments, the gastrin compound is administered in an amount of about 0.5 micrograms. In embodiments, the gastrin compound is administered in an amount of about 1 microgram. In embodiments, the gastrin compound is administered in an amount of about 10 micrograms. In embodiments, the gastrin compound is administered in an amount of about 50 micrograms. In embodiments, the gastrin compound is administered in an amount of about 100 micrograms. In embodiments, the gastrin compound is administered in an amount of about 200 micrograms. In embodiments, the gastrin compound is administered in an amount of about 300 micrograms. In embodiments, the gastrin compound is administered in an amount of about 400 micrograms. In embodiments, the gastrin compound is administered in an amount of about 500 micrograms. In embodiments, the gastrin compound is administered in an amount of about 600 micrograms. In embodiments, the gastrin compound is administered in an amount of about 700 micrograms. In embodiments, the gastrin compound is administered in an amount of about 800 micrograms. In embodiments, the gastrin compound is administered in an amount of about 900 micrograms. In embodiments, the gastrin compound is administered in an amount of about 1 gram. In embodiments, the gastrin compound is administered in an amount of about 2 grams. In embodiments, the gastrin compound is administered in an amount of about 3 grams. In embodiments, the gastrin compound is administered in an amount of about 4 grams. In embodiments, the gastrin compound is administered in an amount of about 5 grams. In embodiments, the gastrin compound is administered in an amount of about 6 grams. In embodiments, the gastrin compound is administered in an amount of about 7 grams. In embodiments, the gastrin compound is administered in an amount of about 8 grams. InPATENTAttorney Docket No. 048440-851001 WO embodiments, the gastrin compound is administered in an amount of about 9 grams. In embodiments, the gastrin compound is administered in an amount of about 10 grams.

[0244] In embodiments, the sequence of big gastrin-34 and small gastrin-17 are shown herein. In embodiments, big gastrin-34 is essentially an extension form of small gastrin- 17 haying an additional amino acid sequence at the N-terminal end. In embodiments, big gastrin is cleaved in vivo to release gastrin- 17. In embodiments, the symbol “Glp” at the N-terminal end is a pyroglutamate residue, which is a naturally cyclized form of glutamate. In various embodiments, gastrins having an N-terminal pyroglutamate residues are modified to contain N-terminal cysteine or lysine residues by either replacing the pyroglutamate with a glutamate or glutamine, or deleting the pyroglutamate. Further, each of a gastrin 34 and gastrin-17 can be used in a modified form that has a methionine or a leucine at position 32 as shown herein in SEQ ID No: 3-4, respectively, or at position 15 as shown in SEQ ID No: 5-6, respectively. The symbol “Phe-NH2” is a phenylalaninamide residue.

[0245] (SEQ ID NO: 3) N-terminal Glp-Leu-Gly-Pro-Gln-Gly-Pro-Pro-His-Leu-Val-Ala-Asp- Pro-Ser-Lys-Lys-Gln-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe- NH2.

[0246] (SEQ ID NO: 4) N-terminal Glp-Leu-Gly-Pro-Gln-Gly-Pro-Pro-His-Leu-Val-Ala-Asp- Pro-Ser-Lys-Lys-Gln-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Leu-Asp-Phe- NH2.

[0247] (SEQ ID NO: 5) N-terminal Glp-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly- Trp-Met-Asp-Phe-NH2.

[0248] (SEQ ID NO: 6) N-terminal Glp-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly- T rp-Leu- Asp-Phe-NH2.

[0249] “Treating” or “treatment” as used herein and as well-understood in the art includes any approach for obtaining beneficial clinical results for a patient. Beneficial clinical results includes, but is not limited to, alleviation or amelioration of one or more symptoms of a disease, diminishment of the extent of the disease, stabilizing the disease, delaying or slowing progression of the disease, amelioration or palliation of the disease, and remission, whether partial or total and whether detectable or undetectable. Treatment may relieve the disease’s symptoms, fully orPATENTAttorney Docket No. 048440-851001 WO partially remove the disease’s underlying cause, shorten a disease’s duration, or do a combination of these things. Treatment methods include administering to a subject a therapeutically effective amount of the therapeutic agents described herein. The administering step may include a single administration or a series of administrations. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, or a combination thereof. It will also be appreciated that the effective amount used for the treatment may increase or decrease over the course of a particular treatment regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. Treating does not include preventing.

[0250] “Patient” or “subject in need thereof’ refers to a living organism suffering from or prone to diabetes that can be treated by administration of a therapeutic agent as provided herein. Nonlimiting examples include humans and other mammals, such as dogs and cats. In embodiments, a patient is human.

[0251] An “effective amount” or “therapeutically effective amount” is an amount sufficient for a therapeutic agent to accomplish a stated purpose relative to the absence of the therapeutic agent (e.g., achieve the effect for which it is administered, treat a diabetes). An example of an “effective amount” or “therapeutically effective amount” is an amount sufficient to contribute to the treatment or reduction of a symptom or symptoms of diabetes. A reduction of a symptom or symptoms means decreasing of the severity or frequency of the symptom, or elimination of the symptom. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. For any therapeutic agent described herein, the therapeutically effective amount can be initially determined from cell culture assays. Target concentrations will be those concentrations of therapeutic agents that are capable of achieving the methods described herein. As is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan. Dosages may be varied depending upon the requirements of the patient. The dose administered to a patient should be sufficient to effect a beneficial therapeuticPATENTAttorney Docket No. 048440-851001 WO response in the patient over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Dosage amounts and intervals can be adjusted individually to provide levels of the administered therapeutic agent effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.

[0252] The term “administering” is used in accordance with its plain and ordinary meaning and includes parenteral administration to a patient and / or implantation into a subject. Parenteral administration includes, e.g., intravenous, subcutaneous, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. In embodiments, administration is intravenously into the portal vein. In embodiments, administration is intravenously (via infusion or bolus injection) into the liver. In embodiments, administration is via the omentum. The omentum is a large apron-like fold of membrane inside the abdomen that drapes over the intestines. In embodiments, administration is via an omental pouch. In embodiments, administration is through an implantable device. Implantable devices are known in the art. An exemplary implantable device is described, e.g., by Shapiro et al, Cell Reports Medicine 2 (doi . org / 10.1016 / j .xcrm .2021.100466) .

[0253] In embodiments, the administering is via omental pouch. Methods of creating an omental pouch are known in the art. For example, an omental pouch can be created by lifting up edges of omentum; placing the hPSCs, hPSC-derived beta cells, compositions, cell aggregates or polymeric microcapsules described herein, into the omentum and optionally affixing them using a fibrin sealant hemostatic agent. After affixing with the hemostatic agent, the omental pouch is sealed (e.g., via suture). Stice et al, Cell Transplantation 27(10): 1561 -1568 (2018). In embodiments, an effective amount of the hPSCs described herein, including embodiments thereof, are administered to the patient via omental pouch. In embodiments, an effective amount of the hPSC-derived beta cells described herein, including embodiments thereof, are administered to the patient via omental pouch. In embodiments, an effective amount of the compositions described herein, including embodiments thereof, are administered to the patient via omental pouch. In embodiments, an effective amount of the cell aggregates described herein, including embodiments thereof, are administered to the patient via omental pouch. In embodiments, an effective amount of thePATENTAttorney Docket No. 048440-851001 WO polymeric microcapsules described herein, including embodiments thereof, are administered to the patient via omental pouch.EXAMPLESExample 1

[0254] The inventors adapted an improved protocol for differentiating human pluripotent stem cells (hPSCs) into beta-like cells in six distinct stages as monolayer (FIG. 1). This protocol generates beta-like cells with enhanced maturation status and restores normoglycemia in T1D mice at higher rate than other differentiation protocols (Hogrebe et al, Nat Biotechnol 38:460-470 (2020)). The beta-like cells derived from Hl cells were stained for Insulin, Glucagon and NKX6.1. The stage 6 (S6) beta-like cell clusters derived from the differentiation of human embryonic stem (hESC) line Hl contained cells expressing insulin or glucagon. These cells were transplanted into the kidney capsule of diabetic NODscid mice to determine whether they could restore normoglycemia. Fasting and non-fasting blood glucose levels in the transplanted mice showed a progressive improvement of hyperglycemia and was consistent with the findings that the process of in vivo cell maturation was required for hPSC-derived beta-like cells to become fully functional and respond to elevated blood glucose. Pagliuca et al, Cell 159:428-39 (2014); Rezania et al, Nat Biotechnol 32: 1121-33 (2014); Russ et al, EMBO J 34:1759-72 (2015).

[0255] The process of differentiating hPSCs into insulin-producing cells might leave some cells in a primordial state with tumor growth potential. To test whether undifferentiated hPSCs present in the beta-like cell clusters can be safely removed, the inventors employed the iC9 kill switch encoding a fusion protein of human Caspase 9 and FK506-binding protein (FKBP). Individual iC9 subunits do not induce cell apoptosis. iC9 dimerization by a clinically approved drug, AP1903, activates one of the last steps in the apoptotic cascade and triggers rapid cell apoptosis. Clackson et al, Proc Natl Acad Sci U S A 95: 10437-42 (1998). To restrict iC9 expression only in undifferentiated Hl cells, the inventors used CRISPR-Cas9 to insert the iC9 transgene in-frame into the stem cell-specific SOX2 locus (FIG. 2A). This locus remains active in undifferentiated hPSCs, and is rapidly silenced upon differentiation. API 903 induced apoptosis in undifferentiated Hl-iC9 cells without affecting the viability of parental Hl cells (FIG. 2B). Upon differentiation of Hl-iC9 cells into the beta-like cell clusters, API 903 treatment showed little effect on the viability of the differentiated cells (FIG. 2C). Since the efficiency of deriving the beta-like (INS+ / MAFA+)PATENTAttorney Docket No. 048440-851001 WO cells from Hl-iC9 cells was similar to that from the parental Hl cells (FIG. 2D), insertion of the iC9 gene did not seem to affect in vitro differentiation of the Hl cells. To determine whether undifferentiated Hl cells can be selectively removed, Hl-iC9-derived beta-like cells were cocultured with undifferentiated Hl-iC9 cells followed by an overnight API 903 treatment. Undifferentiated stem cells were identified by the expression of stem cell-specific marker OCT4. AP1903 treatment selectively eradicated undifferentiated Hl-iC9 cells without affecting the viability of differentiated beta-like cells (FIG. 2E). In addition, the in vivo killing of teratomas derived from Hl-iC9 cells in NSG mice was measured. Parental Hl cells were mixed with Hl-iC9 cells marked by GFP in equal ratio and were transplanted into the mice. API 903 administration effectively removed Hl-iC9 cells from teratomas, leaving only parental Hl-derived cells in the teratoma See Wu et al., “Using gene editing to establish a safeguard system for pluripotent stemcell-based therapies” iScience 22, 409-422 (2019) PMCID: PMC6909005).

[0256] This study showed the feasibility of installing a kill switch to eradicate residual undifferentiated hPSCs without affecting differentiated beta-like cells. (See, e. .,Wu et al, iScience 22:409-422 (2019)).Example 2

[0257] The strategy of the iC9 kill switch under the control of the endogenous SOX2 locus is used to destroy undifferentiated hPSCs which may be present in the beta-like cells before transplantation. However, this system will have limited capacity to eradicate malignant cells emerging after transplantation. The reasons could be due to the presence of a silent SOX2 locus in the malignant cells or over-expression of anti-apoptotic proteins such as BCL-2 in these cells that can bypass iC9-induced cell killing. The inventors therefore sought an alternative suicide system to remove all potential malignant cells emerging from grafted beta-like cell clusters. The gene encoding herpes simplex virus thymidine kinase (HSV-TK) is the most often used suicide gene in human cancer gene therapy. Shen et al, Cancer Gene Ther 13:975-92 (2006). Expression of this gene converts the anti-herpes drug, ganciclovir (GCV), to GCV triphosphate which inhibits DNA chain elongation and induces apoptosis of dividing cells. However, HSV-TK gene delivery via viral vectors frequently results in the emergence of escape mutants due to strong selection of HSV-TK gene mutation. Frank et al, Blood 104:3543-9 (2004). More recently, it has been shown that inserting the HSV-7V gene into the endogenous CDK1 locus avoids HSV-7V mutation inPATENTAttorney Docket No. 048440-851001 WO dividing cells. Liang et al, Nature 563:701-704 (2018). CDK1 mediates cell cycle transition from G2 to M and a kill switch modulated by this locus is predicted to express only in dividing cells, making these cells susceptible to GCV-induced toxicity. An HSV-TK mutant, SR39, containing five amino acid substitutions was used by the inventors. Black et al, Cancer Res 61 :3022-6 (2001). Compared with wild-type HSV-TK, SR39 requires substantially lower concentrations of GCV to kill dividing cells. Lowering the amount of GCV will avoid unwanted myelosuppression in patients. Qasim et al, Gene Ther 9:824-7 (2002). The SR39 gene was synthesized with optimized codon usage in human cells. A similar strategy as described in FIG. 2B was used to insert the SR39 gene in-frame into the CDK1 locus in Hl cells. GCV-resistant escapees emerged in mice when tumor lines with the mono-allelic HSV-7K gene insertion were used in transplantation. Liang et al, Nature 563:701-704 (2018). This was attributed to diploid loss of heterozygosity caused by mitotic recombination or chromosomal nondisjunction. However, no GCV escapee was detected with tumor lines containing homozygous HSV-' / 'V gene insertion at the CDK1 locus. Black et al, Cancer Res 61 :3022-6 (2001). Based on these observations, the inventors isolated Hl- CDK1-SR39 clones with homozygous HSV-5A39 gene insertion into the CDKI locus. Undifferentiated H1-CDK1-SR39 cells were sensitive to GCV treatment whereas the parental Hl cells were non-responsive (FIG. 3). GCV at a concentration of 10 nM was sufficient to eliminate all proliferating H1-CDK1-SR39 cells in culture after a 4-day treatment. By contrast, the viability of the parental Hl cells was not affected at this GCV concentration (FIG. 3).Example 3

[0258] Previous studies showed that the beta-like cell clusters contained a population of enterochromaffin (EC)-like cells could produce serotonin. Veres et al, Nature 569:368-373 (2019). The inventors determined whether the Hl-iC9-derived beta-like cells contained the EC-like cells. Stimulation of the beta-like cells with KC1 led to the secretion of serotonin into the culture media (FIG. 4). Excessive serotonin production could cause the life-threatening serotonin syndrome. Cote et al, Trends Mol Med 10:232-8 (2004). To inhibit serotonin production, the inventors employed CRISPR-Cas9 to knockout the tryptophan hydroxylase 1 (TPH1) gene in undifferentiated Hl-iC9 cells. This gene encodes the key TPH enzyme responsible for serotonin production in EC cells. After screening 72 clones, one clone with homozygous mutation in the gene was isolated. In Hl-iC9-ATPHl cells, the wild-type TTCT sequence in the second exon ofPATENTAttorney Docket No. 048440-851001 WO the TPH1 gene was converted to AGA, thus shifting the reading frame and generating a downstream stop codon nearby. The premature stop codon leads to the generation of the truncated TPH1 protein including the amino acids at positions 1-85 of the wildtype TPH1 (See SEQ ID NO: 12). Since a major portion of the TPH1 protein is predicted to be missing due to this mutation, we hypothesize that this mutation inactivates TPH1 expression and abolishes serotonin production from EC-like cells. As shown in FIG. 5A, basal-level secretion of serotonin from the Hl-iC9- ATPHl-derived beta-like cell clusters was reduced relative to that from the parental Hl-iC9- derived beta-like cell clusters, and KC1 treatment of these beta-like cells showed a significant reduction in serotonin secretion. This result indicates that the mutation effectively abolished serotonin production. The fact that the TPH1 gene knockout clone generated similar levels of insulin and glucagon4cells and exhibited similar GSIS activity (FIG. 5B) as the parental Hl-iC9 cells indicates that the TPH1 gene knockout did not affect normal hPSC differentiation into the beta-like cells.Example 4

[0259] To evaluate the feasibility of establishing hypoimmunogenic hPSCs, the inventors inactivated the Beta-2 Microglobulin (B2M) gene that encodes a common protein subunit required for surface expression of all polymorphic HLA class I heavy chains from Hl cells. However, the absence of HLA class 1 surface expression activates NK cells through the missing self response which can lyse the transplanted B2M'1' cells. Bix et al, Nature 349:329-31 (1991); Liao et al, Science 253: 199-202 (1991). This NK cell-dependent lysis is normally inhibited through interactions with class I molecules, including the minimally polymorphic HLA-E protein, which presents peptides derived from the signal sequences of other HLA class I molecules, and is a ligand for the inhibitory CD94 / NGK2A complex expressed on the majority of NK cells. Like other HLA class I molecules, HLA-E forms a heterodimer with a B2M subunit so it is not expressed on the surface of B2M~ / ~ cells. B2M~ ~ cells could be engineered to express HLA-E as a single-chain protein fused to B2M, thereby creating cells that express HLA-E as their only surface HLA class I molecule. Gornalusse et al, Nat Biotechnol 35:765-772 (2017). Using CRISPR-Cas9, the inventors inserted an HLA-E single-chain trimer construct into the B2M locus in Hl cells. This construct encodes a fusion protein of HLA-E covalently linked to B2M and the signal peptide from HLA-G normally presented by HLA-E that inhibits NK cell-dependent lysis through itsPATENTAttorney Docket No. 048440-851001 WO binding to CD94 / NGK2A. In-frame insertion of the construct not only inactivated B2M expression, it also placed the expression of the HLA-E single-chain trimer under the control of the B2M locus. Using CRISPR-Cas9, a Hl clone with insertion of the HLA-E fusion construct into the B2M locus (- / Etrimer) was isolated. Interferon-gamma stimulation of the undifferentiated - / Etrimer clone showed the down-regulation of HLA-A, HLA-B, HLA-C and the expression of HLA-E on cell surface (FIG. 6A). Differentiation of the - / Etrimer clone into S6 beta-like cells showed a similar pattern of cell surface expression of these HLA class 1 molecules (FIG. 6B). Loss of HLA-A, B, C cell surface expression in the beta-like cells will prevent the attack by the allogeneic host CD8+T cells upon transplantation whereas HLA-E single-chain trimer will suppress the missing self response mediated by the host NK cells.Example 5

[0260] Another strategy to avoid chronic immunosuppression will be generating hypoimmunogenic beta-like cells by using CRISPR-Cas9 to ablate the expression of HLAII through knocking out the CIITA gene in hPSCs. As the complete absence of HLAI activates NK cell-mediated cell killing, elevated CD47 expression can inhibit such NK activity. Thus, ectopic CD47 expression will be established in hPSCs with ablated HLA expression to protect transplanted beta-like cells from NK-mediated cytotoxicity. An alternative strategy to target immune surveillance is to over-express PD-L1 in hPSC-derived beta-like cells as inhibiting costimulation through the PD-1 / PD-L1 pathway is central to immune tolerance. We will therefore establish HLA-deficient hPSCs with ectopic expression of CD47 and PD-L1 to reduce the immunity of derived beta-like cells.Example 6

[0261] We plan to evaluate two GMP -grade human pluripotent stem cell (hPSC) lines for potential clinical application. ESI-017 is a human embryonic stem cell (ESC) line from Biotime. This cell line is NIH registered and backed with donor history and testing information in best compliance with current Good Tissue Practice (cGTP) and conform to Global Ethical Standards and Clinical Cell Regulations. TC-1133 is an induced pluripotent stem cell (iPSC) line from Lonza. This line was derived from human CD34+ umbilical cord blood cells. We will use the established protocol to determine the efficiency of beta cell differentiation of these two lines inPATENTAttorney Docket No. 048440-851001 WO vitro. The line that yields higher levels of beta-like cells will be subjected to further gene modifications.

[0262] SEQ ID NO lMASYPCHQHA SAFDQAARSR GHSNRRTALR PRRQQEATEV RLEQKMPTLLRVYI DGPHGM GKTTTTQLLV ALGSRDDIVY VPE PMTYWQV LGASE T IANIYTTQHRLDQG E I SAGDAAW MTSAQI TMGM PYAVTDAVLA PHI GGEAGSSHAPPPALTIF LDRHPIAFML CYPAARYLMG SMT PQAVLAF VALI PPTLPGTNIVLGALPE DRHIDRLAKR QRPGERLDLA MLAAIRRVYG LLANTVRYLQGGGSWREDWG QLSGTAVPPQ GAE PQSNAGP RPHI GDTLFT LFRAPELLAPNGDLYNVFAW ALDVLAKRLR PMHVFI LDYD QS PAGCRDAL LQLTSGMVQTHVTT PGS I PT I CDLARTFAR EMGEANExample 7

[0263] H1(AB2M) - This is an isolated clone with B2M gene knockout to eliminate HLA class I expression. To determine whether B2M knockout activates the NK cell activities, SC-islets were cocultured with NK cells. As shown in FIG. 31, live CFSE+SC-islet cells derived from H1(AB2M) differentiation were reduced significantly compared to those derived from parental Hl differentiation. Among CFSE+SC-islets, H1(AB2M) derived cells contained higher fractions of PI+cells compared to parental Hl derived cells (FIG. 31), suggesting that loss of HLA class I expression induced NK cell killing.

[0264] H1(SR39;AB2M) - This is an isolated clone with the herpes TK mutant SR39 transgene inserted at the CDK1 locus and B2M gene knockout to eliminate HLA class I expression. B2M gene knockout silences HLA class I expression (FIG. 33). The genetic modifications do not alter the ability of H1(SR39;AB2M) cells to differentiate into SC-islets as demonstrated by the production of INS+or NKX6.1 cells at a similar efficiency as the parental H1(SR39) cells.

[0265] H1(SR39;AB2M;PD-L1) - This is a pooled line containing the SR39 transgene, B2M gene knockout and ectopic PD-L1 expression. The PD-L1 transgene was delivered intoPATENTAttorney Docket No. 048440-851001 WOH1(SR39;AB2M) cells via lentiviral transduction and mCherry-expressing cells were pooled (FIG. 34)

[0266] H1(SR39;AB2M;PD-L1;CD47) - This is a pooled line containing the SR39 transgene, B2M gene knockout and ectopic PD-L1 and CD47 expression. The CD47 transgene gene was delivered into H1(SR39;AB2M;PD-L1) cells via lentiviral transduction and mCherry and GFP double-positive cells were pooled (FIG. 35).

[0267] H1(SR39;AB2M;ACIITA) - This is an isolated clone (clone 51) containing the SR39 transgene, and B2M and CIITA gene knockout to eliminate HLA class I and II expression. To isolate the clone, H1(SR39;AB2M) cells were treated with CRISPR-Cas9 to knockout CIITA gene expression. Two independent clones with mutations at the target site within exon 3 of the CIITA gene were isolated by capillary electrophoresis and fragment analysis. Verification of the mutation was confirmed by DNA sequencing. The two alleles in clone 51 have 4 and 14 bp deletion, respectively, resulting in significant shortening of the encoded CIITA protein. It is highly unlikely that the severely truncated CIITA can function normally.Example 8: Site-specific insertion of the SR39 gene to improve the biosafety of stem cell- derived SC-islets.

[0268] The iC9 kill switch has the limited capacity to eradicate malignant cells emerging after transplantation. The presence of a silent SOX2 locus or over-expression of anti-apoptotic proteins such as BCL-2 in such cells can bypass iC9-induced cell killing. For these reasons, we sought an alternative suicide system which can remove all proliferating cells if tumors emerge from grafted SC-islets. The gene encoding herpes simplex virus thymidine kinase (HSV-TK) is a suicide gene used most frequently in human cancer gene therapy. Cellular expression of the HSV-TK gene converts the anti-herpes drug, ganciclovir (GCV) to GCV triphosphate which inhibits DNA chain elongation and induces apoptosis of dividing cells. More recently, it was shown that inserting the HSV-TK gene into the endogenous CDK1 locus of human stem cells led to the killing of dividing cells (See Liang el al. “Linking a cell-division gene and a suicide gene to define and improve cell therapy safety”. Nature 563:701-704 (2018)). CDK1 mediates cell cycle transition from G2 to M. A kill switch modulated by this locus is predicted to express only in dividing cells, making these cells susceptible to GCV-induced toxicity. We used CRISPR-Cas9 for in-frame insertion of the HSV-TK gene or a mutant gene of HSV-TK, SR39, into the CDK1 locus in the Hl cells (FIG.PATENTAttorney Docket No. 048440-851001 WO27C) (See Black et al., Herpes simplex virus-1 thymidine kinase mutants created by semi-random sequence mutagenesis improve prodrug-mediated tumor cell killing. Cancer Res 61 :3022-3026 (2001)). SR39 has higher affinity for GCV than the wild-type HSV-TK, thus may avoid the unwanted myelosuppressive side effect with high GCV dose for a complete response. Treatment of undifferentiated Hl-TK and H1-SR39 cells with increasing concentration of GCV showed effective cell killing (FIG. 27A). As predicted, H1-SR39 cells were much more sensitive than Hl- TK cells to the GCV treatment. Differentiation of these cells into SC-islets rendered them insensitive to the drug treatment (FIG. 27B), consistent with the hypothesis that non-proliferating cells such as SC-islets failed to incorporate GCV.Example 9: Knocking out the serotonin production from SC-islets to improve the biosafety.

[0269] SC-islets often contain off-target enterochromaffin-like (SC-EC) cells, which express typical enterochromaffin cell (EC) markers such as TPH1 and LMX1A. Depending on the differentiation protocol used, SC-ECs constitute -15-25% of the SC-islet population. ECs typically are found in the gut but not in adult islets. Like ECs in the gut, SC-ECs secreted serotonin and survived in mice for up to six months after transplantation. The optimal dose of SC- islets to treat T1D patients is currently unknown. Assuming a similar dose as with human islets from deceased donors, on the order of 109 cells of SC-islets per patient may be required. Consistent with this assumption, Wang et al. injected SC-islets at a dose of -1.5 million islet equivalent (IEQ) (-1,000-2000 cells / IEQ) into the T1D patient in their clinical trial and restored glycemic control in the T1D patient (See Wang S, Du Y, Zhang B, Meng G, Liu Z, Liew SY, et al. “Transplantation of chemically induced pluripotent stem-cell-derived islets under abdominal anterior rectus sheath in a type 1 diabetes patient” Cell 187: 1-13 (2024)). If SC-ECs represent -15-25% of transplanted SC-islets, excessive serotonin production from the graft could represent a safety concern. There are two pools of serotonin in the body, one in the central nervous system and the other in the peripheral tissues. Since serotonin does not cross blood brain barrier, it is likely that serotonin generated from the SC-islet grafts in the liver or other sites will only affect its level in the peripheral pool. Peripheral serotonin plays a pivotal function in the regulation of glucose and lipid homeostasis. Serotonin has been found to promote insulin secretion from beta cells, promote gluconeogenesis in the liver, suppress hepatic glucose uptake, act on white adipocytes to promote lipolysis, suppress glucose uptake and insulin action. Chronic elevation ofPATENTAttorney Docket No. 048440-851001 WO the serotonin level caused by SC-islet graft therefore needs to be addressed, we employed gene editing to inactivate serotonin production from SC-islets. Serotonin synthesis involves the enzyme tryptophan hydroxylase (TPH), which converts L-tryptophan to 5-hydroxytryptophan, a precursor to serotonin. Two isoforms, TPH1 and TPH2, are encoded by separate genes: TPH1 is mainly expressed in peripheral tissues, whereas TPH2 is predominantly expressed in brain. In mice, TPHl-deficient animals exhibited a 90% reduction in pancreatic serotonin levels and impaired insulin secretion, suggesting that TPH1 might be involved in islet beta cell function. Mechanistic studies showed that covalent coupling of serotonin to the small GTPases Rab3a and Rab27a facilitated insulin secretion. This theory is somewhat in conflict with the data from human islet beta cells. Gene expression profiling of human islets reveals minimal TPH1 and TPH2 expression (See Veres et al. “Charting cellular identity during human in vitro P-cell differentiation” Nature 569:368-373 (2019)), casting doubt on a functional role of serotonin production in beta cells.

[0270] We used CRISPR-Cas9 to inactivate the TPH1 gene in Hl-iC9 cells containing the iC9 suicide system inserted at the SOX2 locus (see Fig. 1). A homozygous TPH1 knockout clone, Hl- iC9-ATPHl, was isolated. Sequence analysis showed a sequence alteration from TTCT to AGA in the 2nd exon of the coding region (See SEQ ID NO: 8 and 10). As a consequence, a frame shift mutation was created, leading to a severely truncated TPH1 protein (SEQ ID NO: 12).

[0271] Immunofluorescence (IF) staining of SC-islets derived from Hl-iC9 and Hl-iC9-ATPHl differentiation revealed no significant differences in the frequency of alpha and beta cells. IF staining of the TPH protein in SC-islets confirmed that the mutation severely diminished its expression in Hl-iC9-ATPHl cells.

[0272] Secretion of serotonin from SC-islets before and after KC1 stimulation was measured. As shown in FIG. 5A, serotonin secretion was significantly stimulated from SC-islets derived from Hl-iC9 parental cells whereas the fold of stimulation was diminished from SC-islets derived from Hl-iC9-ATPHl cells, consistent with the inactivation of TPH1 expression in Hl-iC9-ATPHl cells.

[0273] To determine whether TPH1 gene knockout affects SC-islet functions, we compared GSIS between SC-islets derived from Hl-iC9 and Hl-iC9-ATPHl cells (FIG. 5B). The data showed no significant difference in the GSIS activity between the two cell lines.PATENTAttorney Docket No. 048440-851001 WO

[0274] To measure the in vivo function of TPH1 knockout SC-islets, we transplanted SC-islets derived from Hl-iC9 and Hl-iC9-ATPHl cells into diabetic NODscid mice and measured their ability to restore glycemic control. Under nonfasting condition, normoglycemia was restored within approximately 7-8 weeks after SC-islet transplantation underneath the kidney capsule of diabetic animals (FIG. 5C). There is no significant difference between the SC-islets derived from the two cell lines. The timing of restoring glycemic control in diabetic mice is consistent with the previous reports that in vivo maturation of SC-islets is necessary for their activity in glycemic control. Together, these data suggest that TPH1 gene knockout does not seem to affect the ability of Hl-iC9 cells to differentiate into SC-islets and the SC-islets derived from Hl-iC9-ATPHl exhibit similar functions as those derived from Hl-iC9 parental cells. Utilization of SC-islets derived from Hl-iC9-ATPHl cells containing the iC9 suicide gene with impaired ability to produce serotonin would significantly improve the safety of applying SC-islets in clinics.Example 10: Genetic modification of Hl cells to generate safe and hypoimmunogenic SC- islets

[0275] Establish GM-H1 cell lines.

[0276] To establish Hl clones, when differentiated, generate SC-islets with improved biosafety and hypoimmunogenicity, we used CRISPR-Cas9 to first introduce the SR39 gene into the CDK1 locus in the Hl cells. An independent clone with biallelic insertion of the SR39 gene into the CDK1 locus was isolated (FIG. 32). This clone was then treated with CRISPR-Cas9 to knockout the B2M gene encoding the beta 2 microglobulin. One clone, H1(SR39;AB2M) with biallelic inactivation of the B2M gene was isolated. Sequence analysis of the two B2M alleles in this clone showed the destruction of the ATG initiation codon of the B2M gene and FACS analysis confirmed the loss of cells surface expression of the HLA class 1 expression (FIG. 33).

[0277] Using the H1(SR39; AB2M) clone, we further employed CRISPR-Cas9 to knockout the CIITA gene encoding a master transcription factor required for the expression of all HLA class 2. An independent clone, H1(SR39; AB2M; ACIITA), with biallelic mutation in the CIITA gene was isolated. Mutation in the two alleles cause the production of severely truncated CIITA protein (see e.g., SEQ ID NO: 17 and 18) and it was highly unlikely that the two truncated proteins could serve as functional transcription factors to modulate HLA class 2 expression.PATENTAttorney Docket No. 048440-851001 WO

[0278] For ectopic expression of PD-L1, the H1(SR39; AB2M; ACIITA) clone was transduced with a lentiviral vector carrying the PD-L1 cDNA and the mCherry gene (FIG. 34). Cells expressing high levels of mCherry were pooled and transduced with a second lentiviral vector carrying the CD47 cDNA and the GFP gene (FIG. 35). Cells coexpressing high levels of mCherry and GFP were GSIS assay with the pooled cells showed their responses upon glucose stimulation and secreted insulin (FIG. 37).

[0279] Coculture of SC-islets derived from either H1(SR39), H1(SR39; AB2M; ACIITA) or GM-H1 cells showed that HLA class 1 knockout in H1(SR39;AB2M; ACIITA) cells rendered these cells susceptible to NK cell-mediated killing (FIG. 38). By contrast, CD47 expression in GM-H1 cells avoided NK cell activation in the absence of HLA class 1 expression.

[0280] Transplantation of the GM-H1 cell pool into diabetic NODscid mice showed restoration of glycemic control in one mouse with a higher graft size (31mm) whereas the one with lower graft size (20 mm) failed to respond (FIG. 39, left panel). 1PGTT carried out on day 69 after transplantation was consistent with the graft size (FIG. 39, right panel).

[0281] To boost CD47 expression, we isolated an independent clone from the pooled GM-H1 cells. FACS analysis showed that CD47 expression from this clone, clone 11, was higher than the GM-H1 pool. (4.5 fold vs 2.5 fold above the parental control) (compare the MFI in FIG. 36 & 40). Clone 11 readily differentiated into SC-islets in vitro, generating NKX6.1+, INS+ and GCG+ cells efficiently. Transplantation of SC-islets derived from clone 11 showed rapid restoration of glycemic control in two out of three diabetic NODscid mice with large graft size whereas a mouse transplanted with smaller graft size failed to respond (Fig. 41). Together, these results show that a GM-H1 clone, clone 11, with five genetic modifications (a suicide gene, HLA class 1 and HLA class 2 knockout, PD-L1 and CD47 overexpression) was established. These modifications do not affect the ability of the clone to differentiate into SC-islets. The resulting SC-islets respond to glucose stimulation and secret insulin in vitro and have the ability to restore glycemic control in vivo.Example 11: Ectopic expression of transcription factors to enhance SC-islet functions.

[0282] Ectopic expression ofMAFA improved SC-islet functions.PATENTAttorney Docket No. 048440-851001 WO

[0283] MAFA is a beta cell-specific transcription factor (TF) implicated in beta cell maturation. Based on single cell RNA sequencing, its expression in stage 6 SC-beta cells is approximately 68 fold lower than that in mature beta cells (See Veres et al. “Charting cellular identity during human in vitro (3-cell differentiation” Nature 569:368-373 (2019)). To determine whether an elevation in MAFA expression in SC-beta cells can boost SC-islet functions, we used CRISPR-Cas9 to insert the MAFA cDNA into the PCSK1 locus in H1R cells with Hl cells carrying a tdTomato gene. This is an in-frame insertion of the MAFA coding region with the PCSK1 reading frame, therefore a disruption of normal PCSK1 expression is not expected. The level of PCSK1 in stage 6 SC-beta cells is much stronger than that of MAFA, insertion of the MAFA cDNA into the PCSK1 locus is therefore expected to boost overall MAFA expression. SC-islets derived from HIR-MafA cells showed a higher GSIS activity than H1R parental cells (FIG. 42), suggesting that an elevation in MAFA enhanced SC-islet’ s response to glucose stimulation for insulin secretion.

[0284] Establishment of an iPSC line and measure the effect of ectopic MAFA expression with this iPSC line.

[0285] To ensure that the effect of MAFA is not limited only to Hl cell line, we used Yamanaka’s reprogramming factors to establish another pluripotent stem cell line. Early passage of primary human mesenchymal stem cells (MSCs) from human umbilical cord were purchased from ATCC. They were transfected with the three episomal plasmids carrying the reprogramming factor genes (See Okita et al. “A more efficient method to generate integration-free human iPS cells” Nat Methods 8:409-412 (2011). An iPSC line was isolated. Immunofluorescence (IF) staining showed the expression of stem cell markers including TRA1-60 and TRA1-81. In vitro differentiation of this line showed its ability to generate cells from the three germ layers. Interestingly, SC-islets derived from this iPSC line failed to generate GSIS activity: high glucose stimulation did not result in increased insulin secretion (Fig. 43). However, insertion of the MAFA cDNA into the PCSK1 locus in this iPSC line (iPSC-MafA) led to stimulation of insulin secretion upon glucose stimulation. These results demonstrate that ectopic MAFA expression in SC-islets indeed boosts their response to glucose stimulation and secrets elevated levels of insulin. In contrast, insertion of the MAFA gene in the INS locus led to increased expression of MAFA, but also resulted in dysregulation of stem cell differentiation and / or maturation of the resulting SC-islets (FIG. 45A-45B). Our results also show that the PCSK1 locus can serve as an ideal site toPATENTAttorney Docket No. 048440-851001 WO introduce other TF genes for ectopic expression to enhance SC-islet functions as this locus is active only in more mature beta cells and its transcriptional activity is stronger than most of other loci in the beta cells. Beta cell-specific TF expression is important as premature expression of some TFs such as MAFA has been shown to block islet differentiation (See He et al.“Differentiation of pancreatic endocrine progenitors reversibly blocked by premature induction of MafA” Dev Biol 85: 2-12 (2014).Example 12: Replacement of Matrigel with Laminin for SC-islet differentiation.

[0286] Hl and all pluripotent stem cells we grow are expanded and differentiated in Matrigel- coated tissue culture dishes. Since Matrigel is purified from a mouse sarcoma cell line, it is highly unlikely that FDA will allow cells in contact with Matrigel to be used in clinical trials. We tested the feasibility of expanding and differentiating Hl cells in Laminin 521-coated dishes instead. As GMP Laminin 521 is available, stem cells cultured and differentiated in this extracellular matrix should address the concern of using Matrigel. We observed that SC-islets derived from either Laminin 521 or Matrigel exhibited very similar levels of INS- and GCG-positive cells. In fact, Laminin-coated dishes seem to generate slightly more SC-alpha and SC-beta cells than Matrigel- coated dishes. The data demonstrates that Laminin 521 can be substituted for Matrigel for stem cell expansion and differentiation into SC-islets in vitro.Example 13: Formation of MSC Reaggregates

[0287] Methods

[0288] Human Umbilical Cord derived MSCs (UCMSCs) at passage 3 (P3) were thawed and cultured in a T75 flask with 2.5xl06cells / flask in the alpha MEM media containing 25% human serum albumin, 5% human platelet lysate (hPL), 2.5 pM Rock inhibitor, and antibiotic- antimycotic solution until the confluency. The UCMSCs were harvested and plated in an ultra-low attachment 6-well plate (2, 4, or 6xl06cells / well) with CMRL 1066 supplemented CIT modified media (COH islet culture media) with 5% hPL and 10 pM Rock inhibitor, placed on a shaker (100 rpm) in a 37 °C incubator for 24 hours.PATENTAttorney Docket No. 048440-851001 WO

[0289] Results

[0290] MSCs reaggregated into uniform clusters within 24 hours. Except the large clusters (>400pm in diameter), the viability (assayed via FDA / PI staining) of a majority of the clusters were >95%.Example 14: Formation of restructured human islets

[0291] Methods

[0292] Isolated human islets were dissociated with TrypLE containing 10 pM Rock inhibitor and lOU / mL heparin for 10 minutes. The cells were then filtered through the 40 pm mesh and plated in a 24-well AggreWell 400 MicroCell Culture Plate at the seeding density 0.5 xlO6cells / well with COH islet culture media containing with or without 10 pM Rock inhibitor and lx cholesterol lipid concentrate (Gibco). The plate was centrifuged at 100g for 3 minutes, then cultured for 5 days. Medium change was performed every 2 days.

[0293] Results

[0294] The morphology of islets cultured with both Rock inhibitor (RI) or both RI and cholesterol lipid concentrate (CLC) showed better than control islets. The viability and dithizone (DTZ) staining of the restricted islets were all similar among the groups, resulting in over 90% viability and almost all the clusters were well stained with DTZ (100% purity). Most importantly, the restructured clusters responded well to high glucose (17 mM) (FIG. 44A) with the stimulation index >8, the highest by the group that contains both RI and CLC (FIG. 44B).PATENTAttorney Docket No. 048440-851001 WO

[0295] Table 1 : Baseline Characteristics of participants in the three islet transplant clinical trialsData reported as Mean (SD).All Participants had Undetectable C-peptide Prior to Transplant.P EMBODIMENTS

[0296] P Embodiment 1. A human pluripotent stem cell comprising a mutant sr39 HSV-TK protein-encoding gene.

[0297] P Embodiment 2. A human pluripotent stem cell comprising an inactivated TPH1 gene.

[0298] P Embodiment 3. The human pluripotent stem cell of P embodiment 2, further comprising a mutant sr39 HSV-TK protein-encoding gene.

[0299] P Embodiment 4. The human pluripotent stem cell of any one of P embodiments 1 to 3, further comprising an inactivated B2M gene.

[0300] P Embodiment 5. The human pluripotent stem cell of any one of P embodiments 1 to4, further comprising an HLA-E gene.

[0301] P Embodiment 6. The human pluripotent stem cell of any one of P embodiments 1 to5, further comprising an inactivated CIITA gene.PATENTAttorney Docket No. 048440-851001 WO

[0302] P Embodiment 7. The human pluripotent stem cell of any one of P embodiments 1 to 3, further comprising an inactivated B2M gene and an inactivated CIITA gene.

[0303] P Embodiment 8. The human pluripotent stem cell of any one of P embodiments 1 to3, further comprising an inactivated B2M gene, an HLA-E gene, and an inactivated CIITA gene.

[0304] P Embodiment 9. A human pluripotent stem cell comprising: (i) an inactivated B2M gene; and (ii) an inactivated CIITA gene.

[0305] P Embodiment 10. A human pluripotent stem cell comprising: (i) an inactivated B2M gene; (ii) an HLA-E gene; and (ii) an inactivated CIITA gene.

[0306] P Embodiment 11. The human pluripotent stem cell of any one of P embodiments 1 to 10, further comprising an exogenous CD47 gene.

[0307] P Embodiment 12. The human pluripotent stem cell of any one of P embodiments 1 to 10, further comprising an exogenous CD274 gene.

[0308] P Embodiment 13. The human pluripotent stem cell of any one of P embodiments 1 to 10, further comprising an exogenous CD47 gene and an exogenous CD274 gene.

[0309] P Embodiment 14. A beta cell derived from the human pluripotent stem cell of any one of P embodiments 1 to 13.

[0310] P Embodiment 15. A composition comprising a plurality of the human pluripotent stem cells of any one ofP embodiments 1 to 13.

[0311] P Embodiment 16. A composition comprising a plurality of the beta cells of P embodiment 14.

[0312] P Embodiment 17. The composition of P embodiment 15 or 16, further comprising mesenchymal stem cells.

[0313] P Embodiment 18. The composition of any one of P embodiments 15 to 17, further comprising native human islets.

[0314] P Embodiment 19. The composition of any one of P embodiments 15 to 18, further comprising regulatory T cells.PATENTAttorney Docket No. 048440-851001 WO

[0315] P Embodiment 20. The composition of P embodiment 15 or 16, further comprising mesenchymal stem cells and native human islets.

[0316] P Embodiment 21. The composition of P embodiment 15 or 16, further comprising mesenchymal stem cells and regulatory T cells.

[0317] P Embodiment 22. The composition of P embodiment 15 or 16, further comprising native human islets and regulatory T cells.

[0318] P Embodiment 23. The composition of P embodiment 15 or 16, further comprising mesenchymal stem cells, native human islets, and regulatory T cells.

[0319] P Embodiment 24. The composition of any one of P embodiments 15 to 23, further comprising a mesenchymal stem cell-derived extracellular vesicle.

[0320] P Embodiment 25. A cell aggregate comprising a plurality of the human pluripotent stem cells of any one of P embodiments 1 to 13.

[0321] P Embodiment 26. A cell aggregate comprising a plurality of the beta cells of P embodiment 14.

[0322] P Embodiment 27. The cell aggregate of P embodiment 25 or 26, further comprising mesenchymal stem cells.

[0323] P Embodiment 28. The cell aggregate of any one of P embodiments 25 to 27, further comprising native human islets.

[0324] P Embodiment 29. The cell aggregate of any one of P embodiments 25 to 28, further comprising regulatory T cells.

[0325] P Embodiment 30. The cell aggregate of P embodiment 25 or 26, further comprising mesenchymal stem cells and native human islets.

[0326] P Embodiment 31. The cell aggregate of P embodiment 25 or 26, further comprising mesenchymal stem cells and regulatory T cells.

[0327] P Embodiment 32. The cell aggregate of P embodiment 25 or 26, further comprising native human islets and regulatory T cells.PATENTAttorney Docket No. 048440-851001 WO

[0328] P Embodiment 33. The cell aggregate of P embodiment 25 or 26, further comprising mesenchymal stem cells, native human islets, and regulatory T cells.

[0329] P Embodiment 34. The cell aggregate of any one of P embodiments 25 to 33, further comprising a mesenchymal stem cell-derived extracellular vesicle.

[0330] P Embodiment 35. The cell aggregate of any one of P embodiments 25 to 34, wherein the longest diameter of the cell aggregate is from about 100 microns to about 250 microns.

[0331] P Embodiment 36. A polymeric microcapsule comprising: (i) the human pluripotent stem cell of any one of P embodiments 1 to 13; (ii) the beta cell of P embodiment 14; (iii) the composition of any one of P embodiments 15 to 24; or (iv) the cell aggregate of any one of P embodiments 25 to 35.

[0332] P Embodiment 37. An omental pouch comprising an effective amount of: (i) the human pluripotent stem cell of any one of P embodiments 1 to 13; (ii) the beta cell of P embodiment 14; (iii) the composition of any one of P embodiments 15 to 24; (iv) the cell aggregate of any one of P embodiments 25 to 35; or (v) the polymeric microcapsule of P embodiment 36.

[0333] P Embodiment 38. An implantable device comprising: (i) the human pluripotent stem cell of any one of P embodiments 1 to 13; (ii) the beta cell of P embodiment 14; (iii) the composition of any one of P embodiments 15 to 24; (iv) the cell aggregate of any one of P embodiments 25 to 35; or (v) the polymeric microcapsule of P embodiment 36.

[0334] P Embodiment 39. A method of treating diabetes in a patient in need thereof, the method comprising administering to the patient an effective amount of: (i) the human pluripotent stem cell of any one of P embodiments 1 to 13; (ii) the beta cell of P embodiment 14; (iii) the composition of any one of P embodiments 15 to 24; (iv) the cell aggregate of any one of P embodiments 25 to 35; or (v) the polymeric microcapsule of P embodiment 36.

[0335] P Embodiment 40. The method of P embodiment 39, wherein administering is administering via the portal vein.

[0336] P Embodiment 41. The method of P embodiment 40, wherein administering is intravenously administering via the portal vein.PATENTAttorney Docket No. 048440-851001 WO

[0337] P Embodiment 42. The method of P embodiment 39, wherein administering is administering via an implanted device; wherein the device comprises (i), (ii), (iii), (iv), or (v).

[0338] P Embodiment 43. The method of P embodiment 39, wherein administering is administering via an omental pouch; wherein the omental pouch comprises (i), (ii), (iii), (iv), or (v).

[0339] P Embodiment 44. The method of any one of P embodiments 39 to 43, wherein the diabetes is type 1 diabetes.

[0340] P Embodiment 45. The method of any one of P embodiments 39 to 43, wherein the diabetes is type 2 diabetes.

[0341] P Embodiment 46. The method of any one of P embodiments 39 to 45, further comprising administering to the patient an effective amount of ganciclovir.

[0342] P Embodiment 47. The method of P embodiment 46, wherein the effective amount of ganciclovir is a low dose.

[0343] P Embodiment 48. The method of any one of P embodiments 39 to 47, further comprising monitoring the patient for serotonin production.

[0344] P Embodiment 49. The method of any one of P embodiments 39 to 48, wherein the patient had previously rejected a tissue graft or a cell graft.

[0345] P Embodiment 50. The method of any one of P embodiments 39 to 49, further comprising administering to the patient an effective amount of gastrin.EMBODIMENTS

[0346] Embodiment 1. A human pluripotent stem cell comprising: (i) a mutant sr39 HSV- TK protein-encoding gene; (ii) an inactivated B2M gene; (iii) an inactivated CIITA gene; (iv) an exogenous CD274 gene; and (v) an exogenous CD47 gene.

[0347] Embodiment 2. The human pluripotent stem cell of embodiment 1, further comprising an inactivated TPH1 gene.PATENTAttorney Docket No. 048440-851001 WO

[0348] Embodiment 3. The human pluripotent stem cell of embodiment 1 or 2, further comprising an HLA-E gene.

[0349] Embodiment 4. The human pluripotent stem cell of any one of embodiments 1 to 3, further comprising an HLA-G gene.

[0350] Embodiment 5. The human pluripotent stem cell of any one of embodiments 1 to 4, further comprising an exogenous CD59 gene.

[0351] Embodiment 6. The human pluripotent stem cell of any one of embodiments 1 to 5, further comprising an exogenous MAFA gene.

[0352] Embodiment 7. The human pluripotent stem cell of embodiment 6, wherein the exogenous MAFA gene is inserted into the reading frame of a PCSK1 gene.

[0353] Embodiment 8. The human pluripotent stem cell of any one of embodiments 1 to 7, further comprising an exogenous CD46 gene, an exogenous CD55 gene, and / or an exogenous CD59 gene.

[0354] Embodiment 9. The human pluripotent stem cell of any one of embodiments 1 to 8, further comprising a tissue factor pathway inhibitor (TFPI) protein-encoding gene.

[0355] Embodiment 10. The human pluripotent stem cells of any one of embodiments 1 to 9, further comprising an inactivated RNLS gene.

[0356] Embodiment 11. The human pluripotent stem cell of any one of embodiments 1 to 10, further comprising an exogenous CD64 gene.

[0357] Embodiment 12. A composition comprising a plurality of the human pluripotent stem cells of any one of embodiments 1 to 11.

[0358] Embodiment 13. A beta cell comprising: (i) a mutant sr39 HSV-TK protein-encoding gene; (ii) an inactivated B2M gene; (iii) an inactivated CIITA gene; (iv) an exogenous CD274 gene; and (v) an exogenous CD47 gene.

[0359] Embodiment 14. The beta cell of embodiment 13, further comprising an inactivated TPH1 gene.PATENTAttorney Docket No. 048440-851001 WO

[0360] Embodiment 15. The beta cell of embodiment 13 or 14, further comprising an HLA-E gene.

[0361] Embodiment 16. The beta cell of any one of embodiments 13 to 15, further comprising an HLA-G gene.

[0362] Embodiment 17. The beta cell of any one of embodiments 13 to 16, further comprising an exogenous CD59 gene.

[0363] Embodiment 18. The beta cell of any one of embodiments 13 to 17, further comprising an exogenous MAFA gene.

[0364] Embodiment 19. The beta cell of embodiment 18, wherein the exogenous MAFA gene is inserted into the reading frame of a PC SKI gene.

[0365] Embodiment 20. The beta cell of any one of embodiments 13 to 19, further comprising an exogenous CD46 gene, an exogenous CD55 gene, and / or an exogenous CD59 gene.

[0366] Embodiment 21. The beta cell of any one of embodiments 13 to 20, further comprising a tissue factor pathway inhibitor (TFP1) protein-encoding gene.

[0367] Embodiment 22. The beta cells of any one of embodiments 13 to 21, further comprising an inactivated RNLS gene.

[0368] Embodiment 23. The beta cell of any one of embodiments 13 to 22, further comprising an exogenous CD64 gene.

[0369] Embodiment 24. A composition comprising a plurality of the beta cells of any one of embodiments 13 to 23.

[0370] Embodiment 25. The composition of embodiment 12 or 24, further comprising mesenchymal stem cells.

[0371] Embodiment 26. The composition of any one of embodiments 12, 24, or 25, further comprising native human islets.

[0372] Embodiment 27. The composition of any one of embodiments 12 or 24 to 26, further comprising regulatory T cells.PATENTAttorney Docket No. 048440-851001 WO

[0373] Embodiment 28. The composition of embodiment 12 or 24, further comprising mesenchymal stem cells and native human islets.

[0374] Embodiment 29. The composition of embodiment 12 or 24, further comprising mesenchymal stem cells and regulatory T cells.

[0375] Embodiment 30. The composition of embodiment 12 or 24, further comprising native human islets and regulatory T cells.

[0376] Embodiment 31. The composition of embodiment 12 or 24, further comprising mesenchymal stem cells, native human islets, and regulatory T cells.

[0377] Embodiment 32. The composition of any one of embodiments 25 to 31, wherein the mesenchymal stem cells further comprise an exogenous CD274 gene.

[0378] Embodiment 33. The composition of any one of embodiments 25 to 32, wherein the mesenchymal stem cells further comprise an exogenous CD47 gene.

[0379] Embodiment 34. The composition of any one of embodiments 26 to 33, wherein the native human islets further comprise an exogenous CD274 gene.

[0380] Embodiment 35. The composition of any one of embodiments 26 to 34, wherein the native human islets further comprise an exogenous CD47 gene.

[0381] Embodiment 36. The composition of any one of embodiments 12 or 24 to 35, further comprising a mesenchymal stem cell-derived extracellular vesicle.

[0382] Embodiment 37. A cell aggregate comprising a plurality of the human pluripotent stem cells of any one of embodiments 1 to 11.

[0383] Embodiment 38. A cell aggregate comprising a plurality of the beta cells of any one of embodiments 13 to 23.

[0384] Embodiment 39. The cell aggregate of embodiment 37 or 38, further comprising mesenchymal stem cells.

[0385] Embodiment 40. The cell aggregate of any one of embodiments 37 to 39, further comprising native human islets.PATENTAttorney Docket No. 048440-851001 WO

[0386] Embodiment 41. The cell aggregate of any one of embodiments 37 to 40, further comprising regulatory T cells.

[0387] Embodiment 42. The cell aggregate of embodiment 37 or 38, further comprising mesenchymal stem cells and native human islets.

[0388] Embodiment 43. The cell aggregate of embodiment 37 or 38, further comprising mesenchymal stem cells and regulatory T cells.

[0389] Embodiment 44. The cell aggregate of embodiment 37 or 38, further comprising native human islets and regulatory T cells.

[0390] Embodiment 45. The cell aggregate of embodiment 37 or 38, further comprising mesenchymal stem cells, native human islets, and regulatory T cells.

[0391] Embodiment 46. The cell aggregate of any one of embodiments 39 to 45, wherein the mesenchymal stem cells further comprise an exogenous CD274 gene.

[0392] Embodiment 47. The cell aggregate of any one of embodiments 39 to 46, wherein the mesenchymal stem cells further comprise an exogenous CD47 gene.

[0393] Embodiment 48. The cell aggregate of any one of embodiments 40 to 47, wherein the native human islets further comprise an exogenous CD274 gene.

[0394] Embodiment 49. The cell aggregate of embodiment 40 to 48, wherein the native human islets further comprise an exogenous CD47 gene.

[0395] Embodiment 50. The cell aggregate of any one of embodiments 37 to 49, further comprising a mesenchymal stem cell-derived extracellular vesicle.

[0396] Embodiment 51. The cell aggregate of any one of embodiments 37 to 50, wherein the longest diameter of the cell aggregate is from about 100 microns to about 250 microns.

[0397] Embodiment 52. A polymeric microcapsule comprising: (i) the human pluripotent stem cell of any one of embodiments 1 to 11; (ii) the beta cell of any one of embodiments 13 to 23; (iii) the composition of any one of embodiments 12 or 24 to 36; or (iv) the cell aggregate of any one of embodiments 37 to 51.PATENTAttorney Docket No. 048440-851001 WO

[0398] Embodiment 53. An implantable device comprising: (i) the human pluripotent stem cell of any one of embodiments 1 to 11; (ii) the beta cell of any one of embodiments 13 to 23; (iii) the composition of any one of embodiments 12 or 24 to 36; (iv) the cell aggregate of any one of embodiments 37 to 51; or (v) the polymeric microcapsule of embodiment 52.

[0399] Embodiment 54. A method of treating diabetes in a patient in need thereof, the method comprising administering to the patient an effective amount of: (i) the human pluripotent stem cell of any one of embodiments 1 to 11; (ii) the beta cell of any one of embodiments 13 to 23; (iii) the composition of any one of embodiments 12 or 24 to 36; (iv) the cell aggregate of any one of embodiments 37 to 51; or (v) the polymeric microcapsule of embodiment 52.

[0400] Embodiment 55. The method of embodiment 54, wherein administering is administering via the portal vein.

[0401] Embodiment 56. The method of embodiment 55, wherein administering is intravenously administering via the portal vein.

[0402] Embodiment 57. The method of embodiment 54, wherein administering is administering via an implanted device; wherein the device comprises (i), (ii), (iii), (iv), or (v).

[0403] Embodiment 58. The method of embodiment 54, wherein administering is administering via an omental pouch; wherein the omental pouch comprises (i), (ii), (iii), (iv), or (v).

[0404] Embodiment 59. The method of embodiment 54, wherein the administering is administering via an anterior rectus sheath, wherein the anterior rectus sheath comprises (i), (ii), (iii), (iv), or (v).

[0405] Embodiment 60. The method of any one of embodiments 54 to 59, wherein the diabetes is type 1 diabetes.

[0406] Embodiment 61. The method of any one of embodiments 54 to 59, wherein the diabetes is type 2 diabetes.

[0407] Embodiment 62. The method of any one of embodiments 54 to 61, further comprising administering to the patient an effective amount of ganciclovir.PATENT Attorney Docket No. 048440-851001 WO

[0408] Embodiment 63. The method of embodiment 62, wherein the effective amount of ganciclovir is a low dose.

[0409] Embodiment 64. The method of any one of embodiments 54 to 63, further comprising monitoring the patient for serotonin production.

[0410] Embodiment 65. The method of any one of embodiments 54 to 64, wherein the patient had previously rejected a tissue graft or a cell graft.

[0411] Embodiment 66. The method of any one of embodiments 54 to 65, further comprising administering to the patient an effective amount of gastrin.INFORMAL SEQUENCE LISTINGPATENTAttorney Docket No. 048440-851001 WOPATENTAttorney Docket No. 048440-851001 WOPATENTAttorney Docket No. 048440-851001 WOPATENTAttorney Docket No. 048440-851001 WOPATENTAttorney Docket No. 048440-851001 WO

Claims

PATENTAttorney Docket No. 048440-851001 WOCLAIMSWHAT IS CLAIMED IS:

1. A human pluripotent stem cell comprising:(i) a mutant sr39 HSV-TK protein-encoding gene;(ii) an inactivated B2M gene;(iii) an inactivated CIITA gene;(iv) an exogenous CD274 gene; and(v) an exogenous CD47 gene.

2. The human pluripotent stem cell of claim 1, further comprising an inactivated TPH1 gene.

3. The human pluripotent stem cell of claim 1, further comprising an HLA-E gene.

4. The human pluripotent stem cell of claim 1, further comprising an HLA-G gene.

5. The human pluripotent stem cell of claim 1, further comprising an exogenous CD59 gene.

6. The human pluripotent stem cell of claim 1, further comprising an exogenous MAFA gene.

7. The human pluripotent stem cell of claim 6, wherein the exogenous MAFA gene is inserted into the reading frame of a PCSK1 gene.

8. The human pluripotent stem cell of claim 1, further comprising an exogenous CD46 gene, an exogenous CD55 gene, and / or an exogenous CD59 gene.

9. The human pluripotent stem cell of claim 1, further comprising a tissue factor pathway inhibitor (TFPI) protein-encoding gene.

10. The human pluripotent stem cells of claim 1, further comprising an inactivated RNLS gene.

11. The human pluripotent stem cell of claim 1, further comprising an exogenous CD64 gene.

12. A composition comprising a plurality of the human pluripotent stem cells of claim 1.

13. A beta cell comprising:(i) a mutant sr39 HSV-TK protein-encoding gene;(ii) an inactivated B2M gene;(iii) an inactivated CIITA gene;(iv) an exogenous CD274 gene; and(v) an exogenous CD47 gene.

14. The beta cell of claim 13, further comprising an inactivated TPH1 gene.

15. The beta cell of claim 13, further comprising an HLA-E gene.

16. The beta cell of claim 13, further comprising an HLA-G gene.

17. The beta cell of claim 13, further comprising an exogenous CD59 gene.

18. The beta cell of claim 13, further comprising an exogenous MAFA gene.

19. The beta cell of claim 18, wherein the exogenous MAFA gene is inserted into the reading frame of a PCSK1 gene.

20. The beta cell of claim 13, further comprising an exogenous CD46 gene, an exogenous CD55 gene, and / or an exogenous CD59 gene.

21. The beta cell of claim 13, further comprising a tissue factor pathway inhibitor (TFPI) protein-encoding gene.

22. The beta cells of claim 13, further comprising an inactivated RNLS gene.PATENTAttorney Docket No. 048440-851001 WO23. The beta cell of claim 13, further comprising an exogenous CD64 gene.

24. A composition comprising a plurality of the beta cells of claim 13.

25. The composition of claim 12, further comprising mesenchymal stem cells.

26. The composition of claim 12, further comprising native human islets.

27. The composition of claim 12, further comprising regulatory T cells.

28. The composition of claim 12, further comprising mesenchymal stem cells and native human islets.

29. The composition of claim 12, further comprising mesenchymal stem cells and regulatory T cells.

30. The composition of claim 12, further comprising native human islets and regulatory T cells.

31. The composition of claim 12, further comprising mesenchymal stem cells, native human islets, and regulatory T cells.

32. The composition of claim 25, wherein the mesenchymal stem cells further comprise an exogenous CD274 gene.

33. The composition of claim 25, wherein the mesenchymal stem cells further comprise an exogenous CD47 gene.

34. The composition of claim 26, wherein the native human islets further comprise an exogenous CD274 gene.

35. The composition of claim 26, wherein the native human islets further comprise an exogenous CD47 gene.

36. The composition of claim 12, further comprising a mesenchymal stem cell- derived extracellular vesicle.PATENTAttorney Docket No. 048440-851001 WO37. A cell aggregate comprising a plurality of the human pluripotent stem cells of claim 1.

38. A cell aggregate comprising a plurality of the beta cells of claim 13.

39. The cell aggregate of claim 37, further comprising mesenchymal stem cells.

40. The cell aggregate of claim 37, further comprising native human islets.

41. The cell aggregate of claim 37, further comprising regulatory T cells.

42. The cell aggregate of claim 37, further comprising mesenchymal stem cells and native human islets.

43. The cell aggregate of claim 37, further comprising mesenchymal stem cells and regulatory T cells.

44. The cell aggregate of claim 37, further comprising native human islets and regulatory T cells.

45. The cell aggregate of claim 37, further comprising mesenchymal stem cells, native human islets, and regulatory T cells.

46. The cell aggregate of claim 39, wherein the mesenchymal stem cells further comprise an exogenous CD274 gene.

47. The cell aggregate of claim 39, wherein the mesenchymal stem cells further comprise an exogenous CD47 gene.

48. The cell aggregate of claim 40, wherein the native human islets further comprise an exogenous CD274 gene.

49. The cell aggregate of claim 40, wherein the native human islets further comprise an exogenous CD47 gene.

50. The cell aggregate of claim 37, further comprising a mesenchymal stem cell-derived extracellular vesicle.PATENTAttorney Docket No. 048440-851001 WO51. The cell aggregate of claim 37, wherein the longest diameter of the cell aggregate is from about 100 microns to about 250 microns.

52. A polymeric microcapsule comprising:(i) the human pluripotent stem cell of claim 1;(ii) the beta cell of claim 13;(iii) the composition of claim 12; or(iv) the cell aggregate of claim 37.

53. An implantable device comprising:(i) the human pluripotent stem cell of claim 1 ;(ii) the beta cell of claim 13;(iii) the composition of claim 12;(iv) the cell aggregate of claim 37; or(v) the polymeric microcapsule of claim 52.

54. A method of treating diabetes in a patient in need thereof, the method comprising administering to the patient an effective amount of:(i) the human pluripotent stem cell of claim 1;(ii) the beta cell of claim 13;(iii) the composition of claim 12;(iv) the cell aggregate of claim 37; or(v) the polymeric microcapsule of claim 52.

55. The method of claim 54, wherein administering is administering via the portal vein.

56. The method of claim 55, wherein administering is intravenously administering via the portal vein.

57. The method of claim 54, wherein administering is administering via an implanted device; wherein the device comprises (i), (ii), (iii), or (iv).PATENTAttorney Docket No. 048440-851001 WO58. The method of claim 54, wherein administering is administering via an omental pouch; wherein the omental pouch comprises (i), (ii), (iii), or (iv).

59. The method of claim 54, wherein the administering is administering via an anterior rectus sheath, wherein the anterior rectus sheath comprises (i), (ii), (iii), or (iv).

60. The method of claim 54, wherein the diabetes is type 1 diabetes.

61. The method of claim 54, wherein the diabetes is type 2 diabetes.

62. The method of claim 54, further comprising administering to the patient an effective amount of ganciclovir.

63. The method of claim 62, wherein the effective amount of ganciclovir is a low dose.

64. The method of claim 54, further comprising monitoring the patient for serotonin production.

65. The method of claim 54, wherein the patient had previously rejected a tissue graft or a cell graft.

66. The method of claim 54, further comprising administering to the patient an effective amount of gastrin.

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