Methods for administering and administering manipulated islet cells

Abstract

This specification provides a method for administering engineered island cells containing functionally modified beta cells that include one or more modifications (such as genetic modifications). In some embodiments, the engineered island cells are low immunogenic cells. In some embodiments, the one or more modifications reduce or eliminate the expression of one or more MHC class I and / or MHC class II human leukocyte antigens and further increase the expression of one or more tolerance factors (such as CD47). In some embodiments, the subjects have a beta cell-related disorder such as diabetes (e.g., type 1 diabetes).

Description

[Technical Field]

[0001] Cross-reference of related applications This application is a U.S. Provisional Patent Application No. 63 / 463,885, filed on May 3, 2023, entitled "METHODS OF DOSING AND ADMINISTRATION OF ENGINEERED ISLET CELLS", U.S. Provisional Patent Application No. 63 / 468,217, filed on May 22, 2023, entitled "METHODS OF DOSING AND ADMINISTRATION OF ENGINEERED ISLET CELLS", U.S. Provisional Patent Application No. 63 / 580,934, filed on September 6, 2023, entitled "METHODS OF DOSING AND ADMINISTRATION OF ENGINEERED ISLET CELLS", and filed on October 27, 2023. This application claims priority to U.S. Provisional Patent Application No. 63 / 593,944 entitled “CELLS”, U.S. Provisional Patent Application No. 63 / 601,142 entitled “METHODS OF DOSING AND ADMINISTRATION OF ENGINEERED ISLET CELLS”, filed November 20, 2023, and U.S. Provisional Patent Application No. 63 / 551,010 entitled “METHODS OF DOSING AND ADMINISTRATION OF ENGINEERED ISLET CELLS”, filed February 7, 2024, with the contents of these U.S. Provisional Patent Applications incorporated in their entirety by reference.

[0002] Inclusion by referencing the sequence list This application is filed electronically along with the sequence listing. The sequence listing is provided as a file titled 186152009340SeqList.xml, created on May 2, 2024, with a size of 99,122 bytes. The electronic information of the sequence listing is incorporated entirely by reference.

[0003] field In certain embodiments, the disclosure relates to a method of administering engineered island cells containing functionally modified beta cells that include one or more modifications (such as genetic modifications). In some embodiments, the engineered islands are low immunogenic cells. In some embodiments, one or more modifications reduce or eliminate the expression of one or more MHC class I and / or MHC class II human leukocyte antigens and further increase the expression of one or more tolerance factors (such as CD47). In some embodiments, the subjects have beta cell-related disorders such as diabetes (e.g., type 1 diabetes). [Overview of the project]

[0004] overview In some embodiments, methods for treating or inhibiting beta-cell dysfunction in subjects requiring treatment or inhibition of beta-cell dysfunction are provided herein, the methods comprising administering a dose of manipulated low immunogenic islets to the subject, the dose being administered to the subject via intramuscular injection, the dose being approximately 1 × 10⁻¹⁶ 7 Individual cells ~ approx. 3×10 8 Individual cells, B) approx. 1.25×10 5 Individual cells / kg ~ approx. 1.2×10 7 The dosage is individual cells / kg, C) approximately 6,500 island equivalents (IEQ) to approximately 600,000 IEQ, or D) approximately 80 IEQ / kg to approximately 24,000 IEQ / kg.

[0005] In some embodiments, methods for reducing exogenous insulin dependence in subjects having or at risk of having beta cell dysfunction are provided herein, the methods comprising administering a dose of manipulated low immunogenic islands to the subject, the dose being administered via intramuscular injection, and the dose being approximately 1 × 10⁻¹⁶ 7 Individual cells ~ approx. 3×10 8 Individual cells, B) approx. 1.25×10 5 Individual cells / kg ~ approx. 1.2×10 7a dose of from about 1 cell / kg, C) from about 6,500 islet equivalents (IEQ) to about 600,000 IEQ, or D) from about 80 IEQ / kg to about 24,000 IEQ / kg, and the amount of exogenous insulin required is less than the amount of exogenous insulin required for a subject treated with non-low-immunogenic islets or less than the amount of exogenous insulin required for an untreated subject with beta cell damage.

[0006] In some embodiments, provided herein is a method of promoting insulin independence in a subject having or at risk of having beta cell damage, the method comprising administering to the subject a dose of engineered low-immunogenic islets, the dose being administered via intramuscular injection, the dose being A) from about 1×10 7 cells to about 3×10 8 cells, B) from about 1.25×10 5 cells / kg to about 1.2×10 7 cells / kg, C) from about 6,500 islet equivalents (IEQ) to about 600,000 IEQ, or D) from about 80 IEQ / kg to about 24,000 IEQ / kg.

[0007] In some embodiments, provided herein is a method of improving graft function in a subject having or at risk of having beta cell damage, the method comprising administering to the subject a dose of engineered low-immunogenic islets, the dose being administered via intramuscular injection, the dose being A) from about 1×10 7 cells to about 3×10 8 cells, B) from about 1.25×10 5 cells / kg to about 1.2×10 7 cells / kg, C) from about 6,500 islet equivalents (IEQ) to about 600,000 IEQ, or D) from about 80 IEQ / kg to about 24,000 IEQ / kg.

[0008] In some embodiments, provided herein is a method of enhancing engraftment in a subject having or at risk of having beta cell damage, the method comprising administering to the subject a dose of engineered low-immunogenic islets, the dose being administered via intramuscular injection, the dose being A) from about 1×10 7Individual cells ~ approx. 3×10 8 Individual cells, B) approx. 1.25×10 5 Individual cells / kg ~ approx. 1.2×10 7 The dosage is approximately 6,500 island equivalents (IEQ) to 600,000 island equivalents (IEQ) per cell / kg, or D) approximately 80 IEQ / kg to 24,000 IEQ / kg.

[0009] In some embodiments, methods for stabilizing glucose levels in subjects having or at risk of having beta cell dysfunction are provided herein, the methods comprising administering a dose of manipulated low immunogenic islets to the subject, the dose being administered via intramuscular injection, and the dose being approximately 1 × 10⁻¹⁶ 7 Individual cells ~ approx. 3×10 8 Individual cells, B) approx. 1.25×10 5 Individual cells / kg ~ approx. 1.2×10 7 The doses are individual cells / kg, C) approximately 6,500 island equivalents (IEQ) to approximately 600,000 IEQ, or D) approximately 80 IEQ / kg to approximately 24,000 IEQ / kg, and glucose levels are stabilized compared to subjects who received alternative island therapy or compared to untreated subjects.

[0010] In some embodiments, methods for stabilizing / increasing c-peptide levels in subjects having or at risk of having beta cell dysfunction are provided herein, the methods comprising administering a dose of manipulated low immunogenic islands to the subject, the dose being administered via intramuscular injection, and the dose being approximately 1 × 10⁷ cells to approximately 3 × 10⁷ cells. 8 Individual cells, B) approx. 1.25×10 5 Individual cells / kg ~ approx. 1.2×10 7 The doses were individual cells / kg, C) approximately 6,500 island equivalents (IEQ) to approximately 600,000 IEQ, or D) approximately 80 IEQ / kg to approximately 24,000 IEQ / kg, and c-peptide levels were stabilized or increased compared to subjects that received alternative island therapy or compared to untreated subjects.

[0011] In some embodiments, methods for reducing HbA1c levels in subjects having or at risk of having beta cell dysfunction are provided herein, the methods comprising administering a dose of manipulated low immunogenic islets to the subject, the dose being administered via intramuscular injection, and the dose being approximately 1 × 10⁻¹⁶ 7 Individual cells ~ approx. 3×10 8 Individual cells, B) approx. 1.25×10 5 Individual cells / kg ~ approx. 1.2×10 7 The doses were individual cells / kg, approximately 6,500 island equivalents (IEQ) to approximately 600,000 IEQ, or D) approximately 80 IEQ / kg to approximately 24,000 IEQ / kg, and HbA1c levels were reduced compared to subjects who received alternative island therapy or compared to untreated subjects.

[0012] In some embodiments, methods are provided herein for reducing adverse side effects associated with islet cell therapy in subjects having or at risk of beta cell dysfunction, the methods comprising: i) introducing a low immunogenic modification to a population of islet cells containing beta cells to generate engineered low immunogenic islets; and ii) administering a dose of the engineered low immunogenic islets to subjects having or at risk of beta cell dysfunction, the dose being administered via intramuscular injection, the dose being approximately 1 × 10⁻⁶ 7 Individual cells ~ approx. 3×10 8 Individual cells, B) approx. 1.25×10 5 Individual cells / kg ~ approx. 1.2×10 7 The dosage is individual cells / kg, C) approximately 6,500 island equivalents (IEQ) to approximately 600,000 IEQ, or D) approximately 80 IEQ / kg to approximately 24,000 IEQ / kg.

[0013] In some embodiments, methods for extending the time-in-range (TIR) ​​in subjects having or at risk of having beta-cell dysfunction are provided herein, the methods comprising administering a dose of manipulated low immunogenic islets to the subject, the dose being administered via intramuscular injection, and the dose being approximately 1 × 10⁻⁶ 7 Individual cells ~ approx. 3×10 8 Individual cells, B) approx. 1.25×10 5Individual cells / kg ~ approx. 1.2×10 7 The doses were approximately 6,500 island equivalents (IEQ) to 600,000 IEQ (individual cells / kg), or approximately 80 IEQ / kg to 24,000 IEQ / kg (individual cells / kg), and the time in response (TIR) ​​was prolonged compared to subjects who received alternative island therapy or compared to untreated subjects.

[0014] In some of the optional embodiments, the method results in a reduction in the amount of other drugs required to treat beta-cell dysfunction, and optionally, the diabetes drug is insulin. In some of the optional embodiments, the subject shows insulin-dependent reduction.

[0015] In some of the optional embodiments, the amount of exogenous insulin is reduced by more than 10% compared to the amount of exogenous insulin required for a subject treated with non-immunogenic islands for beta-cell damage, or the amount of exogenous insulin required for an untreated subject with beta-cell damage. In some of the optional embodiments, the amount of insulin is reduced by more than about 20%, more than about 30%, more than about 40%, more than about 50%, more than about 60%, more than about 70%, more than about 75%, more than about 80%, or more.

[0016] In some of the optional embodiments, the method is characterized in that the subject satisfies one or more of the following criteria: (i) fasting capillary glucose levels do not exceed 140 mg / dL (7.8 mmol / L) more than three times per week (based on measuring capillary glucose levels at least seven times over seven days); (ii) postprandial 2-hour capillary glucose levels do not exceed 180 mg / dL (10.0 mmol / L) more than three times per week (based on measuring capillary glucose levels at least 21 times over seven days); and (iii) evidence of endogenous insulin production, defined as fasting or stimulated C-peptide levels >0.5 ng / mL (0.16 pmol / L).

[0017] In some of the optional embodiments, the method enables the subject to exhibit insulin independence. In some of the optional embodiments, the subject exhibits insulin independence over a period of more than 1 month, more than 2 months, more than 3 months, more than 4 months, more than 5 months, more than 6 months, more than 7 months, more than 8 months, more than 9 months, more than 10 months, more than 11 months, or more than 12 months. In some of the optional embodiments, the subject exhibits insulin independence over a period of at least 1 year.

[0018] In some of the optional embodiments, subjects can be gradually reduced on insulin therapy for at least one week and meet one or more of the following criteria: (i) fasting capillary glucose levels do not exceed 140 mg / dL (7.8 mmol / L) more than three times per week (based on measuring capillary glucose levels at least seven times over seven days); (ii) postprandial 2-hour capillary glucose levels do not exceed 180 mg / dL (10.0 mmol / L) more than three times per week (based on measuring capillary glucose levels at least 21 times over seven days); and (iii) evidence of endogenous insulin production defined as fasting or stimulated C-peptide levels >0.5 ng / mL (0.16 pmol / L).

[0019] In some of the optional embodiments, subjects are able to gradually reduce insulin therapy over a period of time and meet one or more of the following criteria: (i) fasting capillary glucose levels do not exceed 140 mg / dL (7.8 mmol / L) more than three times per week (based on measuring capillary glucose levels at least seven times in seven days); (ii) 2-hour postprandial capillary glucose levels do not exceed 180 mg / dL (10.0 mmol / L) more than three times per week (based on measuring capillary glucose levels at least 21 times in seven days); and (iii) evidence of endogenous insulin production defined as fasting or stimulated C-peptide levels >0.5 ng / mL (0.16 pmol / L). In some of the optional embodiments, subjects are characterized by at least two of (i) to (iii). In some of the optional embodiments, subjects are characterized by each of (i) to (iii).

[0020] In some of the optional embodiments, the method is characterized in that the subject satisfies one or more of the following: a) peak c-peptide > 0.20 nmol / l (as assessed by a mixed food challenge test), b) non-fasting c-peptide > 0.10 nmol / l (as assessed by a mixed food challenge test), c) daily exogenous insulin requirement < 0.25 U / kg, d) daily exogenous insulin requirement = 0 U / kg, e) reduction in exogenous insulin requirement (per kg of body weight), f) reduction in HbA1c (per kg of body weight), g) reduction in glucose fluctuation (stabilization), h) reduction in the duration of hypoglycemia and / or hyperglycemia (improvement of euglycemic function), i) controlled HbA1c ≤ 6.5% (48 mmol / mol), and j) controlled HbA1c < 7.0% (53 mmol / mol). In some of the arbitrary embodiments, the method is characterized in that the subject satisfies two, three, four, five, six, seven, eight, nine, or ten of a) to j). In some of the arbitrary embodiments, the method is characterized in that the subject satisfies each of a) to j).

[0021] In some of the optional embodiments, the modified low immunogenic island includes modifications that inactivate or disrupt one or more alleles of (a)(i) one or more molecules that modulate the expression of one or more major histocompatibility complex (MHC) class I molecules, and / or (ii) one or more molecules that modulate the expression of one or more MHC class II molecules, and / or (b) increase the expression of one or more tolerance factors, the increase in expression being compared to a control or wild-type island without modifications.

[0022] In some of the optional embodiments, the engineered low immunogenic island comprises engineered beta islet cells. In some of the optional embodiments, the engineered low immunogenic island further comprises additional engineered islet cells, the additional engineered islet cells comprising alpha cells and / or delta cells. In some of the optional embodiments, the additional engineered islet cells comprises cells having the same modifications as those of the engineered beta islet cells.

[0023] In some of the embodiments, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the cells in the manipulated hypoimmunogenic islands include manipulated beta islet cells. In some of the embodiments, at least 60% of the cells in the manipulated hypoimmunogenic islands include manipulated beta islet cells. In some of the embodiments, the manipulated hypoimmunogenic islands are island clusters. In some of the embodiments, the manipulated hypoimmunogenic islands are manipulated from primary islets. In some of the embodiments, the primary islets are derived from the pancreas. In some of the embodiments, the primary islets are derived from a human subject. In some of the embodiments, the primary islets are derived from an animal subject. In some of the embodiments, the primary islets are from a pig, a cattle, or a sheep.

[0024] In some of the embodiments, the primary islands are derived from a donor subject not suspected of having beta-cell-related disorder. In some of the embodiments, the donor is cadaverous. In some of the embodiments, the engineered hypoimmunogenic islands are ABO blood type O. In some of the embodiments, the engineered hypoimmunogenic islands are Rh factor negative (Rh-).

[0025] In some of the optional embodiments, the manipulated low immunogenic islands are differentiated from stem cells. In some of the optional embodiments, the stem cells are selected from the group consisting of pluripotent stem cells (PSCs), induced pluripotent stem cells (iPSCs), embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells, germline stem cells, lung stem cells, umbilical cord blood stem cells, and multipotent stem cells. In some of the optional embodiments, the stem cells are induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), or embryonic stem cells (ESCs). In some of the optional embodiments, the stem cells are pluripotent stem cells (PSCs).

[0026] In some of the optional embodiments, beta-cell dysfunction is a metabolic disorder. In some of the optional embodiments, the metabolic disorder is selected from the group consisting of familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabbe disease, maple syrup urine disease, metachromatic leukodystrophy, mitochondrial encephalopathy, lactic acidosis, stroke-like episodes (MELAS), Niemann-Pick disease, phenylketonuria (PKU), porphyria, Tay-Sachs disease, Wilson's disease, type 1 diabetes mellitus, type 2 diabetes mellitus, obesity, hypertension, dyslipidemia, and carbohydrate intolerance. In some of the optional embodiments, beta-cell dysfunction is diabetes mellitus. In some of the optional embodiments, beta-cell dysfunction is type 1 diabetes mellitus.

[0027] In some of the optional embodiments, the subjects to be treated are characterized by one or more of the following: type 1 diabetes mellitus lasting more than 5 years, negative (or <0.01 nmol / l) C-peptide response to mixed meal challenge test (MMTT), positive antibody against either GAD or IA2, HbA1c ≥ 70 mmol / mol, and exogenous insulin requirement < 1 U / kg.

[0028] In some of the optional embodiments, the dose of the manipulated low immunogenic island comprises a pharmaceutically acceptable carrier. In some of the optional embodiments, the pharmaceutically acceptable carrier is a buffered aqueous solution. In some of the optional embodiments, the buffered aqueous solution is physiological saline. In some of the optional embodiments, the dose is administered intravenously to the subject. In some of the optional embodiments, if the dose is administered intravenously, the dose is administered intravenously via the portal vein. In some of the optional embodiments, the dose is administered to the subject via the renal capsule. In some of the optional embodiments, the dose is administered subcutaneously to the subject.

[0029] In some of the optional embodiments, the engineered low immunogenic islets are administered intramuscularly to the subject. In some of the optional embodiments, intramuscular administration is via the intramuscular lumen of the forearm. In some of the optional embodiments, the engineered low immunogenic islets are administered to the upper arm, hip, thigh, or buttocks.

[0030] In some of the optional embodiments, the dose is administered to the liver, kidney, spleen, muscle, subcutaneous tissue, or white adipose tissue of the subject. In some of the optional embodiments, the dose is administered to the liver, muscle, or white adipose tissue of the subject. In some of the optional embodiments, the white adipose tissue is a nettle.

[0031] In some of the optional embodiments, the dose includes the administration of one or more further doses of low immunogenic engineered cells.

[0032] In some of the optional embodiments, one or more additional doses of low immunogenic engineered cells are administered to the subject if (a) the subject does not show a reduction in the amount of other drugs required to treat beta-cell damage after the initial dose, and optionally the beta-cell damaging agent is insulin, and / or (b) the administered low immunogenic engineered cells are not detected by imaging. In some of the optional embodiments, the subject does not show an insulin-dependent reduction after the initial dose.

[0033] In some of the optional embodiments, one or more additional doses of low immunogenic engineered cells are administered to a subject if, after the initial dose, the subject does not meet one or more of the following criteria: (i) fasting capillary glucose levels not exceeding 140 mg / dL (7.8 mmol / L) more than three times per week (based on measuring capillary glucose levels at least seven times over seven days); (ii) postprandial 2-hour capillary glucose not exceeding 180 mg / dL (10.0 mmol / L) more than three times per week (based on measuring capillary glucose levels at least 21 times over seven days); and (iii) evidence of endogenous insulin production, defined as fasting or stimulated C-peptide levels >0.5 ng / mL (0.16 pmol / L).

[0034] In some of the optional embodiments, one or more additional doses of low immunogenic engineered cells are administered to the subject if (a) the subject does not achieve insulin independence within a period following the initial dose and / or (b) the subject does not show a reduction in the amount of other drugs required to treat beta-cell damage within a period of time, and optionally, the beta-cell damaging agent is insulin.

[0035] In some of the optional embodiments, the subject does not achieve insulin independence over a period of more than one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months, and optionally, the subject does not achieve insulin independence over a period of two weeks. In some of the optional embodiments, the subject does not achieve insulin independence over a period of at least one year.

[0036] In some of the optional embodiments, one or more additional doses of low immunogenic engineered cells are administered to a subject if, after the initial dose, the subject is unable to gradually reduce insulin therapy for at least one week and fails to meet one or more of the following criteria: (i) fasting capillary glucose levels not exceeding 140 mg / dL (7.8 mmol / L) more than three times per week (based on measuring capillary glucose levels at least seven times over seven days); (ii) postprandial 2-hour capillary glucose not exceeding 180 mg / dL (10.0 mmol / L) more than three times per week (based on measuring capillary glucose levels at least 21 times over seven days); and (iii) evidence of endogenous insulin production defined as fasting or stimulated C-peptide levels >0.5 ng / mL (0.16 pmol / L).

[0037] In some of the optional embodiments, one or more additional doses of low immunogenic engineered cells are administered to a subject if, after the initial dose, the subject is unable to gradually reduce insulin therapy over a period of time and fails to meet one or more of the following criteria: (i) fasting capillary glucose levels not exceeding 140 mg / dL (7.8 mmol / L) more than three times per week (based on measuring capillary glucose levels at least seven times over seven days); (ii) postprandial 2-hour capillary glucose not exceeding 180 mg / dL (10.0 mmol / L) more than three times per week (based on measuring capillary glucose levels at least 21 times over seven days); and (iii) evidence of endogenous insulin production defined as fasting or stimulated C-peptide levels >0.5 ng / mL (0.16 pmol / L). In some of the optional embodiments, the subject is characterized by failing to meet at least two of (i) to (iii) or each of (i) to (iii).

[0038] In some of the optional embodiments, one or more additional doses of low immunogenic engineered cells are administered to a subject if, after the initial dose, the subject does not meet one or more of the following criteria: a) peak c-peptide > 0.20 nmol / l (as assessed by mixed food challenge test), b) non-fasting c-peptide > 0.10 nmol / l (as assessed by mixed food challenge test), c) daily exogenous insulin requirement < 0.25 U / kg, d) daily exogenous insulin requirement = 0 U / kg, e) reduction in exogenous insulin requirement (per kg of body weight), f) reduction in HbA1c (per kg of body weight), g) reduction in glucose fluctuation (stabilization), h) reduction in the duration of hypoglycemia and / or hyperglycemia (improvement of euglycemic function), i) blood glucose control HbA1c ≤ 6.5% (48 mmol / mol), and j) blood glucose control HbA1c < 7.0% (53 mmol / mol). In some of the optional embodiments, one or more additional doses are administered to the subject if, after the initial dose, the subject does not meet two, three, four, five, six, seven, eight, nine, or ten of a) to j). In some of the optional embodiments, one or more additional doses are administered to the subject if, after the initial dose, the subject does not meet each of a) to j).

[0039] In some of the optional embodiments, the number of engineered hypoimmunogenic islands derived from the initial dose is eliminated or reduced in the subject before administering one or more further doses of the engineered hypoimmunogenic islands. In some of the optional embodiments, the number of engineered hypoimmunogenic islands is reduced in the subject after administration of an exogenously administered activator to induce targeted death of the engineered hypoimmunogenic islands. In some of the optional embodiments, the exogenously administered activator activates suicide genes or safety switches in the engineered cells or recognizes one or more tolerogenic factors on the surface of the engineered hypoimmunogenic islands.

[0040] In some of the optional embodiments, the subject undergoes an immunosuppressive regimen. In some of the optional embodiments, the immunosuppressive regimen comprises one or more of mycophenolate mofetil (MMF), an anti-CD25 antibody (e.g., basiliximab), and a calcineurin inhibitor (e.g., tacrolimus; FK-506). In some of the optional embodiments, the immunosuppressive regimen comprises administration of basiliximab (e.g., 2 × 20 mg iv), followed by administration of tacrolimus (starting dose 0.1 mg / kg / 24 hours; target concentration 10-12) and immunosuppression with MMF (500 mg 2x2, dose subsequently adjusted based on AUC). In some of the optional embodiments, the subject is further administered one or more of the following: CMV prophylaxis with valganciclovir (e.g., 450 mg twice daily), ulcer prophylaxis with omeprazole (e.g., 20 mg once daily), TNF-alpha inhibition with etanercept (e.g., 50 mg intravenously, followed by 25 mg sc on days 3, 7, and 10), and a standard antibiotic.

[0041] In some of the optional embodiments, the immunosuppressive regimen is administered only before the administration of the dose of the manipulated low immunogenic island. In some of the optional embodiments, the immunosuppressive regimen is administered only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 days prior to the administration of the dose of the manipulated low immunogenic island. In some of the optional embodiments, the immunosuppressive regimen is administered only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days prior to the administration of the dose of the manipulated low immunogenic island. In some of the optional embodiments, the immunosuppressive regimen is administered only 1, 2, 3, 4, or 5 weeks prior to the administration of the dose of the manipulated low immunogenic island. In some of the optional embodiments, the immunosuppressive regimen is administered only one, two, three, or four weeks prior to the administration of the dose of the manipulated low-immunogenic islets. In some of the optional embodiments, the immunosuppressive regimen is administered only after the administration of the dose of the manipulated low-immunogenic islets. In some of the optional embodiments, the immunosuppressive regimen is administered only to subjects 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 days after administration of the dose of the manipulated low immunogenic island.In some of the optional embodiments, the immunosuppressive regimen is administered only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days after administration of the dose of the manipulated low immunogenic island. In some of the optional embodiments, the immunosuppressive regimen is administered only 1, 2, 3, 4, or 5 weeks after administration of the dose of the manipulated low immunogenic island. In some of the optional embodiments, the immunosuppressive regimen is administered only one, two, three, or four weeks after the administration of the dose of the manipulated low-immunogenic island.

[0042] In some of the optional embodiments, the immunosuppressive regimen is administered intravenously to the subject. In some of the optional embodiments, the immunosuppressive regimen is administered to the subject via the renal capsule. In some of the optional embodiments, the immunosuppressive regimen is administered orally to the subject. In some of the optional embodiments, the immunosuppressive regimen is administered rectally to the subject. In some of the optional embodiments, the immunosuppressive regimen is administered subcutaneously to the subject. In some of the optional embodiments, the immunosuppressive regimen is administered intramuscularly to the subject. In some of the optional embodiments, the immunosuppressive regimen is administered in the forearm of the subject. In some of the optional embodiments, the immunosuppressive regimen is administered in the upper arm, hip, thigh, or buttocks. In some of the optional embodiments, the immunosuppressive regimen is administered at least once a day. In some of the optional embodiments, the immunosuppressive regimen is administered as a single regimen per day. In some of the optional embodiments, the immunosuppressive regimen is administered as a split regimen. In some of the arbitrary embodiments, the immunosuppressive regimen is divided into two regimens, three regimens, etc.

[0043] In some of the optional embodiments, the immunosuppressive regimen comprises one or more immunosuppressants. In some of the optional embodiments, one or more immunosuppressants are administered to the subject prior to the administration of a dose of the manipulated low immunogenic island. In some of the optional embodiments, one or more immunosuppressants are administered to the subject only prior to the first and / or second dose of the manipulated low immunogenic island. In some of the optional embodiments, one or more immunosuppressants are administered to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days prior to the administration of a dose of the manipulated low immunogenic island. In some of the optional embodiments, one or more immunosuppressants are administered to the subject at least one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, or more prior to the administration of a dose of the manipulated low immunogenic island. In some of the optional embodiments, one or more immunosuppressants are administered to the subject after the administration of a dose of the manipulated low immunogenic island. In some of the optional embodiments, one or more immunosuppressants are administered to the subject only after the first and / or second administration of a dose of the manipulated low immunogenic island. In some of the optional embodiments, one or more immunosuppressants are administered to the subject after the administration of a dose of the manipulated low immunogenic island. In some of the optional embodiments, one or more immunosuppressants are administered to the subject after the administration of a dose of the manipulated low immunogenic island. In some of the optional embodiments, one or more immunosuppressants are administered to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after administration of a dose of the manipulated low immunogenic island. In some of the optional embodiments, one or more immunosuppressants are administered to the subject at least 1 week, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or more after administration of a dose of the manipulated low immunogenic island.In some of the optional embodiments, one or more immunosuppressants are administered to the subject at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 16 weeks, or more, after administration of a dose of the manipulated low-immunogenic island. In some of the optional embodiments, one or more immunosuppressants are administered to the subject at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more, after administration of a dose of the manipulated low-immunogenic island. In some of the optional embodiments, one or more immunosuppressants are administered to the subject on the same day as the dose of the manipulated low-immunogenic island. In some of the optional embodiments, one or more immunosuppressants are administered to the subject concurrently with the dose of the manipulated low-immunogenic island. In some of the optional embodiments, one or more immunosuppressants are administered to the target only at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days prior to the first and / or second dose of the manipulated low immunogenic island dose. In some of the optional embodiments, one or more immunosuppressants are administered to the target only at least 1 week, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or more prior to the first and / or second dose of the manipulated low immunogenic island dose. In some of the optional embodiments, one or more immunosuppressants are administered to the subject only at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the first and / or second dose of the manipulated low immunogenic island dose. In some of the optional embodiments, one or more immunosuppressants are administered to the subject only at least 1 week, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after the first and / or second dose of the manipulated low immunogenic island dose. In some of the optional embodiments, one or more immunosuppressants are administered to the subject only at least 2 weeks, 4, 6, 8, 10, 12, or 16 weeks after the dose of the manipulated low immunogenic island dose.In some of the optional embodiments, one or more immunosuppressants are administered to the subject only at least one, two, three, four, five, or six months after administration of a dose of the manipulated low-immunogenic island.

[0044] In some of the optional embodiments, one or more immunosuppressants are administered to the subject before administration of a dose of the manipulated low immunogenic island and are administered continuously throughout the subject's lifetime. In some of the optional embodiments, one or more immunosuppressants are administered to the subject on the same day and / or concurrently with administration of a dose of the manipulated low immunogenic island and are administered continuously throughout the subject's lifetime. In some of the optional embodiments, one or more immunosuppressants are administered to the subject after administration of a dose of the manipulated low immunogenic island and are administered continuously throughout the subject's lifetime. In some of the optional embodiments, one or more immunosuppressants are administered to the subject before each administration of a dose of the manipulated low immunogenic island and are optionally administered continuously throughout the subject's lifetime. In some of the optional embodiments, one or more immunosuppressants are administered to the subject on the same day and / or concurrently with each administration of a dose of the manipulated low immunogenic island and are optionally administered continuously throughout the subject's lifetime. In some of the optional embodiments, one or more immunosuppressants are administered to the subject after each administration of a dose of the manipulated low immunogenic island and are optionally administered continuously throughout the subject's lifetime.

[0045] In some of the optional embodiments, one or more immunosuppressants are administered to a subject in a dose less than the dose of one or more immunosuppressants administered to the subject to reduce immune rejection of immunogenic cells, without modification of the dose of the engineered low immunogenic island. In some of the optional embodiments, one or more immunosuppressants comprise small molecules or biological products. In some of the optional embodiments, the biological product is a protein and / or antibody. In some of the optional embodiments, the small molecule is a chemical substance or nucleic acid. In some of the optional embodiments, one or more immunosuppressants comprise one or more immunomodulators. In some of the optional embodiments, one or more immunomodulators are small molecules or biological products. In some of the optional embodiments, the biological product is a protein or its peptide and / or antibody. In some of the optional embodiments, the small molecule is a chemical substance or nucleic acid. In some of the optional embodiments, one or more immunosuppressants are their pharmaceutically acceptable salts, their preliminary forms, and / or derivatives. In some of the optional embodiments, one or more immunomodulators are their pharmaceutically acceptable salts, their preliminary forms, and / or derivatives.

[0046] In some of the optional embodiments, one or more immunosuppressants are selected from the group consisting of calcineurin inhibitors, steroids, alkylating agents, antibiotics, analgesics, anti-inflammatory agents, antihistamines, antiviral agents, antifungal agents, anticoagulants, DNA synthesis inhibitors, anticoagulants, hemorheologic agents, inosine monophosphate dehydrogenase (IMDH) inhibitors, Janus kinase inhibitors, mTOR inhibitors, TNF inhibitors, and anti-CD25 inhibitors. In some of the optional embodiments, one or more immunosuppressants are antithymocyte globulin (ATG), corticosteroids, prednisone, cortisone, prednisolone methylprednisolone, dexamethasone, betamethasone, hydrocortisone, methotrexate, acetaminophen, diphenhydramine, sirolimus (rapamycin), tacrolimus (FK-506), mycophenolic acid (MPA), mycophenolate mofetil (MMF), mycophenol The group consists of sodium oxalate, cyclosporine, etanercept (TNFR-Fc), azathioprine, gold salt, sulfasalazine, antimalarial agents, brequinal, leflunomide, mizoribine, 15-deoxyspergualine, 6-mercaptopurine, cyclophosphamide, OKT3, antithymocyte globulin, thymopentin (thymosin-α), fludarabine, cyclophosphamide, and immunosuppressive antibodies.

[0047] In some of the optional embodiments, one or more immunosuppressants include anti-thymocyte globulin (ATG). In some of the optional embodiments, at least one regimen of ATG is administered to the subject before, concurrently with, and / or after, the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of ATG is administered to the subject before the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of ATG is administered to the subject before each administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of ATG is administered to the subject about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, or about 1 hour before the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of ATG is administered to the subject about two days and / or about one day before administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of ATG is administered to the subject on the same day and / or concurrently with administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of ATG is administered to the subject on the same day and / or concurrently with each administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, a first or second regimen of ATG is administered to the subject on the same day and / or concurrently with administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of ATG is administered to the subject after administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, a regimen of ATG is administered to the subject after each administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of ATG is administered to the subject approximately 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, or 48 hours after administration of a dose of the manipulated low immunogenic island to the subject.In some of the optional embodiments, at least one regimen of ATG is administered to the subject about 48 hours after administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of ATG is administered to the subject i) about 2 days prior, ii) about 1 day prior, iii) on the same day, iv) about 1 day after, and / or v) about 2 days after, the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one or at least two regimens of ATG include about 0.1 mg / kg to about 2.0 mg / kg of ATG administered to the subject. In some of the optional embodiments, the ATG regimen is administered at a lower dose. In some of the optional embodiments, the method includes a regimen in which i) at least one or at least two ATG regimens comprises a dose of approximately 0.5 mg / kg of ATG administered to the subject about two days prior to the administration of a dose of the manipulated hypoimmunogenic islets to the subject, ii) at least one or at least two ATG regimens comprises a dose of approximately 1.0 mg / kg of ATG administered to the subject about one day prior to the administration of a dose of the manipulated hypoimmunogenic islets to the subject, and / or iii) at least one or at least two ATG regimens comprises a regimen in which a dose of approximately 1.5 mg / kg of ATG administered to the subject on the same day as the administration of the dose of the manipulated hypoimmunogenic islets to the subject, about one day after the administration of the dose of the manipulated hypoimmunogenic islets to the subject, and about two days after the administration of the dose of the manipulated hypoimmunogenic islets to the subject. In some of the optional embodiments, the ATG regimen is carried out at a lower dose.

[0048] In some of the optional embodiments, one or more immunosuppressants include a corticosteroid. In some of the optional embodiments, one or more immunosuppressants include prednisone, cortisone, prednisolone-methylprednisolone, dexamethasone, betamethasone, or hydrocortisone. In some of the optional embodiments, one or more immunosuppressants include methylprednisolone. In some of the optional embodiments, at least one regimen of methylprednisolone is administered to the subject prior to administration of a dose of the manipulated low immunogenic island to the subject. In some of the optional embodiments, at least one regimen of methylprednisolone is administered to the subject about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, or about 1 hour before administration of a dose of the manipulated low immunogenic island to the subject. In some of the optional embodiments, at least one regimen of methylprednisolone is administered to the subject about two days before administration of a dose of the manipulated low immunogenic islands to the subject. In some of the optional embodiments, at least one regimen of methylprednisolone is administered to the subject before administration of the first regimen of ATG to the subject, and both the methylprednisolone regimen and the first regimen of ATG are administered to the subject before administration of a dose of the manipulated low immunogenic islands to the subject. In some of the optional embodiments, at least one regimen of methylprednisolone is administered to the subject about one hour before administration of the first regimen of ATG to the subject. In some of the optional embodiments, at least one regimen of methylprednisolone is administered to the subject approximately midway through the administration of the first regimen of ATG to the subject. In some of the optional embodiments, at least one regimen of methylprednisolone includes a dose of about 0.1 mg / kg to about 2.0 mg / kg. In some of the optional embodiments, the methylprednisolone regimen is carried out at a lower dose. In some of the optional embodiments, at least one regimen of methylprednisolone contains about 0.1 mg / kg of methylprednisolone. In some of the optional embodiments, the methylprednisolone regimen is carried out at a lower dose.In some of the optional embodiments, methylprednisolone is administered intravenously to the subject. In some of the optional embodiments, the method includes i) a regimen of methylprednisolone comprising about 1.0 mg / kg of methylprednisolone administered to the subject about 1 hour before the implementation of a first regimen of ATG to the subject, and / or ii) a regimen of methylprednisolone comprising about 1.0 mg / kg of methylprednisolone administered to the subject approximately midway through the implementation of a first regimen of ATG to the subject. In some of the optional embodiments, the methylprednisolone regimen and / or the ATG regimen are implemented at lower doses.

[0049] In some of the optional embodiments, one or more immunosuppressants include an analgesic. In some of the optional embodiments, the analgesic is acetaminophen, an opioid, or a nonsteroidal anti-inflammatory drug (NSAID). In some of the optional embodiments, at least one regimen of acetaminophen is administered to the subject prior to administration of a dose of the manipulated low immunogenic islands to the subject. In some of the optional embodiments, at least one regimen of acetaminophen is administered to the subject about 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour before administration of a dose of the manipulated low immunogenic islands to the subject. In some of the optional embodiments, at least one regimen of acetaminophen is administered to the subject about 2 days before administration of a dose of the manipulated low immunogenic islands to the subject. In some of the optional embodiments, at least one regimen of acetaminophen is administered to the subject prior to the administration of the first regimen of ATG to the subject, and both the acetaminophen regimen and the first regimen of ATG are administered to the subject prior to the administration of a dose of the manipulated low immunogenic island to the subject. In some of the optional embodiments, at least one regimen of acetaminophen is administered to the subject about 30 minutes before the administration of the first regimen of ATG to the subject. In some of the optional embodiments, at least one regimen of acetaminophen is administered to the subject approximately midway through the administration of the first regimen of ATG to the subject. In some of the optional embodiments, at least one regimen of acetaminophen is administered to the subject in doses ranging from about 100 mg to about 1,000 mg. In some of the optional embodiments, the acetaminophen regimen is administered in lower doses. In some of the optional embodiments, at least one regimen of acetaminophen is administered to the subject in doses of about 650 mg. In some of the optional embodiments, the acetaminophen regimen is carried out at lower doses. In some of the optional embodiments, acetaminophen is administered orally or rectally to the subject.In some of the optional embodiments, the method includes a regimen in which i) at least one regimen of acetaminophen at about 650 mg is administered to the subject about 30 minutes before the administration of a first regimen of ATG to the subject, and / or ii) at least one regimen of acetaminophen at about the middle of the administration of a first regimen of ATG to the subject. In some of the optional embodiments, the acetaminophen regimen and / or the ATG regimen are administered at lower doses.

[0050] In some of the optional embodiments, one or more immunosuppressants include antihistamines. In some of the optional embodiments, the antihistamine is diphenhydramine. In some of the optional embodiments, at least one regimen of diphenhydramine is administered to the subject prior to administration of a dose of the manipulated low immunogenic islands to the subject. In some of the optional embodiments, at least one regimen of diphenhydramine is administered to the subject about 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour before administration of a dose of the manipulated low immunogenic islands to the subject. In some of the optional embodiments, at least one regimen of diphenhydramine is administered to the subject about 2 days before administration of a dose of the manipulated low immunogenic islands to the subject. In some of the optional embodiments, at least one diphenhydramine regimen is administered to the subject prior to the administration of the first ATG regimen to the subject, and both the diphenhydramine regimen and the first ATG regimen are administered to the subject prior to the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one diphenhydramine regimen is administered to the subject about 30 minutes before the administration of the first ATG regimen to the subject. In some of the optional embodiments, at least one diphenhydramine regimen is administered to the subject approximately midway through the administration of the first ATG regimen to the subject. In some of the optional embodiments, at least one diphenhydramine regimen contains about 10 mg to about 100 mg of diphenhydramine administered to the subject. In some of the optional embodiments, the diphenhydramine regimen is administered at a lower dose. In some of the optional embodiments, at least one regimen of about 50 mg of diphenhydramine is administered to the subject. In some of the optional embodiments, the diphenhydramine regimen is implemented at lower doses. In some of the optional embodiments, diphenhydramine is administered orally or rectally to the subject.In some of the optional embodiments, the method includes i) a diphenhydramine regimen comprising about 50 mg of diphenhydramine administered to the subject about 30 minutes before the administration of a first ATG regimen to the subject, and / or ii) a diphenhydramine regimen comprising about 50 mg of diphenhydramine administered to the subject about midway through the administration of a first ATG regimen to the subject. In some of the optional embodiments, the diphenhydramine regimen and / or the ATG regimen are administered at lower doses.

[0051] In some of the optional embodiments, one or more immunosuppressants include an anti-inflammatory agent. In some of the optional embodiments, the anti-inflammatory agent is a TNF inhibitor. In some of the optional embodiments, the TNF inhibitor is selected from the group consisting of infliximab, adalimumab, etanercept, golimumab, and certolizumab. In some of the optional embodiments, the TNF inhibitor is etanercept (TNFR-Fc). In some of the optional embodiments, at least one regimen of etanercept is administered to the subject before, concurrently with, and / or after, the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of etanercept is administered to the subject before the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of etanercept is administered to the subject approximately 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour before administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of etanercept is administered to the subject on the same day and / or concurrently with the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, a first regimen of etanercept is administered to the subject on the same day and / or concurrently with the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of etanercept is administered to the subject after the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of etanercept is administered to the subject approximately 1 hour, 5 hours, 10 hours, 24 hours, 2 days, 3 days, 5 days, 7 days, or 10 days after administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of etanercept is administered to the subject approximately 3 days, 7 days, and / or 10 days after administration of a dose of the manipulated low immunogenic islets to the subject.In some of the optional embodiments, at least one regimen of etanercept is administered to the subject i) on the same day as the administration of a dose of the manipulated low immunogenic islets to the subject, ii) about 3 days thereafter, iii) about 7 days thereafter, and / or iv) about 10 days thereafter. In some of the optional embodiments, at least one regimen of etanercept contains about 10 mg to about 100 mg of etanercept. In some of the optional embodiments, the etanercept regimen is administered at a lower dose. In some of the optional embodiments, at least one regimen of etanercept contains about 50 mg of etanercept. In some of the optional embodiments, the etanercept regimen is administered at a lower dose. In some of the optional embodiments, at least one regimen of etanercept contains about 25 mg of etanercept. In some of the optional embodiments, the etanercept regimen is administered at a lower dose. In some of the optional embodiments, etanercept is administered intravenously and / or subcutaneously to the subject. In some of the optional embodiments, the method includes i) administering at least one regimen of etanercept at about 50 mg to the subject on the same day as the administration of a dose of the manipulated low immunogenic islets to the subject, and / or ii) administering at least one regimen of etanercept at about 3 days, about 7 days, and / or about 10 days after the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, the etanercept regimen is administered at a lower dose. In some of the optional embodiments, the subject is administered at least one regimen of etanercept and at least one regimen of ATG. In some of the optional embodiments, the subject is administered at least one regimen of ATG prior to at least one regimen of etanercept.In some of the optional embodiments, i) at least one regimen of ATG comprises approximately 40 mg / kg of ATG mg administered daily to the subject for four consecutive days; ii) at least one regimen of etanercept comprises approximately 25 mg of etanercept administered twice weekly to the subject for two consecutive weeks following i); and iii) at least one regimen of etanercept comprises approximately 25 mg of etanercept administered monthly to the subject for approximately four months following ii). In some of the optional embodiments, at least one etanercept regimen and / or at least one ATG regimen are implemented at lower doses. In some of the optional embodiments, the subject undergoes at least one regimen of etanercept and at least one regimen of an IL-1 receptor antagonist.

[0052] In some of the optional embodiments, one or more immunosuppressants include an mTOR inhibitor. In some of the optional embodiments, the mTOR inhibitor is sirolimus (rapamycin). In some of the optional embodiments, at least one regimen of sirolimus is administered to the subject before, concurrently with, and / or after, the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of sirolimus is administered to the subject before the administration of a dose of the manipulated low immunogenic islets to the subject.

[0053] In some of the optional embodiments, at least one regimen of sirolimus is administered to the subject approximately 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour prior to the administration of a dose of the manipulated low immunogenic island to the subject.

[0054] In some of the optional embodiments, at least one regimen of sirolimus is administered to the subject on the same day and / or concurrently with the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of sirolimus is administered to the subject after the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of sirolimus is administered to the subject approximately 1 hour, 5 hours, 10 hours, or 24 hours, approximately 3 months, 6 months, 12 months, 24 months, 36 months, 48 ​​months, 60 months, or more after the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, the total daily dose of sirolimus administered to the subject gives a trough blood level of approximately 1 ng / mL to approximately 30 ng / mL, approximately 2 ng / mL to approximately 25 ng / mL, approximately 5 ng / mL to approximately 20 ng / mL, or approximately 10 ng / mL to approximately 15 ng / mL (including both extreme values). In some of the optional embodiments, a sirolimus regimen of approximately 0.1 mg / kg to approximately 2.0 mg / kg is administered to the subject. In some of the optional embodiments, the sirolimus regimen is administered at a lower dose. In some of the optional embodiments, sirolimus is administered orally to the subject.

[0055] In some of the optional embodiments, i) a regimen of approximately 0.2 mg / kg of sirolimus is administered to the subject on the same day as the administration of a dose of the manipulated low immunogenic islets to the subject, and ii) a regimen of approximately 0 / 1 mg / kg of sirolimus is administered to the subject daily until approximately 3 months after the administration of a dose of the manipulated low immunogenic islets to the subject, with the total daily dose of sirolimus administered to the subject giving a blood trough level of approximately 12 ng / mL to approximately 15 ng / mL for approximately 3 months after administration of the composition, and thereafter giving a blood trough level of approximately 7 ng / mL to approximately 10 ng / mL. In some of the optional embodiments, the sirolimus regimen is administered at a lower dose.

[0056] In some of the optional embodiments, one or more immunosuppressants include calcineurin inhibitors. In some of the optional embodiments, the calcineurin inhibitor is tacrolimus (FK-506). In some of the optional embodiments, at least one regimen of tacrolimus is administered to the subject before, concurrently with, and / or after, the administration of a dose of the manipulated low immunogenic island to the subject.

[0057] In some of the optional embodiments, at least one regimen of tacrolimus is administered to the subject prior to the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of tacrolimus is administered to the subject approximately 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour before the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of tacrolimus is administered to the subject on the same day and / or concurrently with the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, a first regimen of tacrolimus is administered to the subject on the same day and / or concurrently with the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of tacrolimus is administered to the subject after administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of tacrolimus is administered to the subject approximately 1 hour, 5 hours, 10 hours, or 24 hours, 3 months, 6 months, 12 months, 24 months, 36 months, 48 ​​months, 60 months, or more after administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, the total daily dose of tacrolimus administered to the subject gives blood trough levels of approximately 1 ng / mL to 30 ng / mL, 2 ng / mL to 25 ng / mL, 5 ng / mL to 20 ng / mL, or 10 ng / mL to 15 ng / mL (including both extreme values). In some of the optional embodiments, the total daily dose of tacrolimus administered to the subject gives a blood trough level of approximately 5 ng / mL to approximately 10 ng / mL (including both extreme values). In some of the optional embodiments, the total daily dose of tacrolimus administered to the subject gives a blood trough level of approximately 10 ng / mL to approximately 15 ng / mL (including both extreme values).

[0058] In some of the optional embodiments, a regimen of tacrolimus ranging from about 0.1 mg to about 5 mg is administered to the subject. In some of the optional embodiments, the tacrolimus regimen is administered at a lower dose. In some of the optional embodiments, one or more immunosuppressants include an inosine-''-monophosphate dehydrogenase (IMPDH) inhibitor. In some of the optional embodiments, the IMPDH inhibitor is MPA, MMF, or MS. In some of the optional embodiments, the IMPDH inhibitor is mycophenolic acid (MPA). In some of the optional embodiments, at least one regimen of MPA is administered to the subject before, concurrently with, and / or after, the administration of a dose of the manipulated low immunogenic island to the subject. In some of the optional embodiments, at least one regimen of MPA is administered to the subject before the administration of a dose of the manipulated low immunogenic island to the subject. In some of the optional embodiments, at least one regimen of MPA is administered to the subject approximately 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour before administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of MPA is administered to the subject on the same day and / or concurrently with the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, a first regimen of MPA is administered to the subject on the same day and / or concurrently with the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of MPA is administered to the subject after the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of MPA is administered to the subject approximately 1 hour, 5 hours, 10 hours, or 24 hours after administration of a dose of the manipulated low immunogenic island to the subject, approximately 3 months, 6 months, 12 months, 24 months, 36 months, 48 ​​months, 60 months, or more.

[0059] In some of the optional embodiments, MPA is mycophenolate mofetil (MMF). In some of the optional embodiments, the total daily dose of MMF is approximately 10 mg to approximately 3000 mg, approximately 500 mg to approximately 3000 mg, approximately 1000 mg to approximately 2500 mg, or approximately 1500 mg to approximately 2000 mg (including both extreme values). In some of the optional embodiments, the total daily dose of MMF is approximately 100 mg, 500 mg, 1000 mg, approximately 1500 mg, approximately 2000 mg, or approximately 2500 mg. In some of the optional embodiments, the total daily dose of MMF is less.

[0060] In some of the optional embodiments, MPA is sodium mycophenolate (MS). In some of the optional embodiments, the total daily dose of MS is approximately 10 mg to approximately 2700 mg, approximately 360 mg to approximately 2700 mg, approximately 720 mg to approximately 2160 mg, or approximately 720 mg to approximately 1620 mg (including both extreme values). In some of the optional embodiments, the total daily dose of MS is approximately 100 mg, approximately 360 mg, approximately 720 mg, approximately 1080 mg, or approximately 1440 mg. In some of the optional embodiments, the total daily dose of MS is less.

[0061] In some of the optional embodiments, the subject undergoes at least one regimen of tacrolimus and at least one regimen of MPA.

[0062] In some of the optional embodiments, one or more immunosuppressants include cyclosporine. In some of the optional embodiments, at least one regimen of cyclosporine is administered to the subject before, concurrently with, and / or after, the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of cyclosporine is administered to the subject if the subject is intolerant to the tacrolimus regimen. In some of the optional embodiments, at least one regimen of cyclosporine is administered to the subject on the same day and / or concurrently with the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, a first regimen of cyclosporine is administered to the subject on the same day and / or concurrently with the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of cyclosporine is administered to the subject after the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of cyclosporine is administered to the subject approximately 1 hour, 5 hours, 10 hours, or 24 hours, 3 months, 6 months, 12 months, 24 months, 36 months, 48 ​​months, 60 months, or more after administration of a dose of the manipulated low immunogenic island to the subject. In some of the optional embodiments, the total daily dose of cyclosporine administered to the subject gives blood trough levels of approximately 50 ng / mL to 300 ng / mL, 100 ng / mL to 250 ng / mL, 200 ng / mL to 300 ng / mL, or 150 ng / mL to 200 ng / mL (including both extreme values).

[0063] In some of the optional embodiments, a cyclosporine regimen of approximately 2 mg / kg to approximately 10 mg / kg is administered daily. In some of the optional embodiments, the cyclosporine regimen is administered at a lower dose. In some of the optional embodiments, a cyclosporine regimen of approximately 6 mg / kg is administered daily. In some of the optional embodiments, the cyclosporine regimen is administered at a lower dose.

[0064] In some of the optional embodiments, the subject is administered at least one regimen of cyclosporine and at least one regimen of MPA. In some of the optional embodiments, the subject is administered at least one regimen of cyclosporine and at least one regimen of ATG. In some of the optional embodiments, the subject is administered at least one regimen of ATG before at least one regimen of cyclosporine. In some of the optional embodiments, i) a regimen of approximately 40 mg / kg of ATG mg is administered to the subject daily for 4 consecutive days, and ii) a regimen of approximately 10 mg / kg to approximately 12 mg / kg of cyclosporine is administered to the subject daily for 6 months after i) the previous administration. In some of the optional embodiments, the cyclosporine regimen and / or ATG regimen are administered at lower doses.

[0065] In some of the optional embodiments, one or more immunosuppressants include antibodies for binding to MHC, CD2, CD3, CD4, CD7, CD28, B7, CD25, CD40, CD45, CD95, IFN-gamma, TNF-alpha, IL-2R-alpha, IL-4, IL-5, IL-6R, IL-6, IGF, IGFR1, IL-7, IL-8, IL-10, CD11-alpha, or CD58, and antibodies for binding to any of their ligands. In some of the optional embodiments, one or more immunosuppressants include soluble IL-15R, IL-10, B7 molecules, e.g., B7-1, B7-2, their variants and fragments, ICOS, and OX40. In some of the optional embodiments, one or more immunosuppressants include inhibitors of negative T cell regulators, e.g., antibodies against CTLA-4 or similar agents. In some of the optional embodiments, one or more immunosuppressants include anti-CD25 antibodies or anti-IL-2R-alpha antibodies. In some of the optional embodiments, the anti-CD25 antibody or anti-IL-2R alpha antibody is selected from the group consisting of basiliximab, daclizumab, and alemtuzumab.

[0066] In some of the optional embodiments, one or more immunosuppressants include basiliximab. In some of the optional embodiments, at least one regimen of basiliximab is administered to the subject on the same day, concurrently with, and / or after, the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of basiliximab is administered to the subject approximately 1 hour, approximately 5 hours, approximately 10 hours, or approximately 24 hours, approximately 3 months, approximately 6 months, approximately 12 months, approximately 24 months, approximately 36 months, approximately 48 months, approximately 60 months, or more after the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of basiliximab is administered to the subject after the administration of at least one regimen of ATG to the subject. In some of the optional embodiments, at least one dose of basiliximab is administered to the subject after administration of at least one regimen of ATG to the subject and after administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of basiliximab is administered to the subject about 4 days after administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, a regimen of basiliximab ranging from about 10 mg to about 30 mg is administered to the subject. In some of the optional embodiments, a regimen of basiliximab ranging from about 20 mg is administered to the subject. In some of the optional embodiments, the basiliximab regimen is administered at a lower dose. In some of the optional embodiments, i) a regimen of approximately 20 mg of basiliximab is administered to the subject on the same day as the administration of a dose of the manipulated low immunogenic islets to the subject, and / or ii) a regimen of approximately 20 mg of basiliximab is administered to the subject approximately 4 days after the administration of a dose of the manipulated low immunogenic islets to the subject.

[0067] In some of the optional embodiments, one or more immunosuppressants include daclizumab. In some of the optional embodiments, at least one regimen of daclizumab is administered to the subject on the same day, concurrently with, and / or after, the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of daclizumab is administered to the subject approximately 1 hour, 5 hours, 10 hours, or 24 hours after the administration of a dose of the manipulated low immunogenic islets to the subject, approximately 3 months, 6 months, 12 months, 24 months, 36 months, 48 ​​months, 60 months, or more after. In some of the optional embodiments, at least one regimen of daclizumab is administered to the subject approximately every 14 days starting from the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, a regimen of daclizumab at approximately 0.5 mg / kg to approximately 2 mg / kg is administered to the subject. In some of the optional embodiments, the daclizuma regimen is administered at a lower dose. In some of the optional embodiments, a regimen of approximately 1 mg / kg of daclizumab is administered. In some of the optional embodiments, the daclizuma regimen is administered at a lower dose.

[0068] In some of the optional embodiments, the subject undergoes at least one regimen of tacrolimus and at least one regimen of sirolimus. In some of the optional embodiments, the subject undergoes at least one regimen of tacrolimus and at least one regimen of daclizumab. In some of the optional embodiments, the subject undergoes at least one regimen of sirolimus and at least one regimen of daclizumab. In some of the optional embodiments, the subject undergoes at least one regimen of tacrolimus, at least one regimen of sirolimus, and at least one regimen of daclizumab.

[0069] In some of the optional embodiments, i) a regimen of approximately 0.2 mg / kg of sirolimus is administered to the subject on the same day as the administration of a dose of the manipulated low immunogenic islets to the subject, and ii) a regimen of approximately 0.1 mg / kg of sirolimus is administered to the subject daily after the administration of a dose of the manipulated low immunogenic islets to the subject, and the total daily dose of sirolimus administered to the subject gives a blood trough level of approximately 12 ng / mL to approximately 15 ng / mL (including both extremes) over the first three months after administration of the composition to the subject, and thereafter the first three months, the total daily dose of sirolimus administered to the subject is approximately 7 ng / mL to approximately 10 ng / mL (each iii) administering a regimen of approximately 1 mg of tacrolimus to the subject on the same day as the administration of the manipulated low immunogenic islet dose to the subject, and iv) administering a regimen of approximately 1 mg of tacrolimus twice daily to the subject starting approximately 12 hours after the administration of the manipulated low immunogenic islet dose to the subject, with a total daily dose of tacrolimus administered to the subject giving a trough blood level of approximately 3 ng / mL to approximately 6 ng / mL (including both ends), and / or v) administering a regimen of approximately 1 mg / kg of daclizumab to the subject approximately every 14 days after the administration of the manipulated low immunogenic islet dose to the subject.

[0070] In some of the optional embodiments, the sirolimus regimen, the tacrolimus regimen, and / or the daclizuma regimen are administered at lower doses. In some of the optional embodiments, the subject is not administered glucocorticoids. In some of the optional embodiments, one or more immunosuppressants include alemtuzumab. In some of the optional embodiments, at least one regimen of alemtuzumab is administered to the subject prior to, on the same day as, concurrently with, and / or after, the administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of alemtuzumab is administered to the subject before at least one regimen of tacrolimus and / or MPA is administered to the subject. In some of the optional embodiments, at least one regimen of alemtuzumab and at least one regimen of tacrolimus and / or MPA are administered to the subject after the administration of a dose of the manipulated low immunogenic islets to the subject.

[0071] In some of the embodiments, one or more immunosuppressants comprise an anti-CD3 antibody. In some of the embodiments, the anti-CD3 antibody is an anti-CD3ε antibody. In some of the embodiments, the anti-CD3 antibody is OKT3. In some of the embodiments, one or more immunosuppressants comprise an anti-IL-33 antibody. In some of the embodiments, one or more immunosuppressants comprise an anti-CD95 antibody. In some of the embodiments, one or more immunosuppressants comprise fingolimod hydrochloride. In some of the embodiments, one or more immunosuppressants comprise clodronate-encapsulated liposomes. In some of the embodiments, one or more immunosuppressants comprise CTLA4-Ig. In some of the embodiments, one or more immunosuppressants comprise aryl hydrocarbon receptor (AhR) ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylate methyl ester (ITE). In some of the embodiments, one or more immunosuppressants comprise the T1D autoantigen proinsulin. In some of the embodiments, one or more immunosuppressants include TGF-β1. In some of the embodiments, one or more immunosuppressants include dexamethasone. In some of the embodiments, one or more immunosuppressants include methotrexate. In some of the embodiments, one or more immunosuppressants include gold salts. In some of the embodiments, one or more immunosuppressants include sulfasalazine. In some of the embodiments, one or more immunosuppressants include one or more antimalarial agents. In some of the embodiments, one or more immunosuppressants include brachynal. In some of the embodiments, one or more immunosuppressants include leflunomide. In some of the embodiments, one or more immunosuppressants include mizoribine. In some of the embodiments, one or more immunosuppressants include 15-deoxysperguarine. In some of the embodiments, one or more immunosuppressants include 6-mercaptopurine. In some of the embodiments, one or more immunosuppressants include cyclophosphamide. In some of the optional embodiments, one or more immunosuppressants include antithymocyte globulin. In some of the optional embodiments, one or more immunosuppressants include antibiotic formulations.

[0072] In some of the optional embodiments, the antibiotic formulation is selected from the group consisting of trimethoprim / sulfamethoxaxole, penicillin, amoxicillin, cephalexin, erythromycin (E-Mycin), clarithromycin (Biaxin), azithromycin (Zithromax), ciprofolxacin (Cipro), levofloxacin (Levaquin), ofloxacin (Floxin), cotrimoxazole (Bactrim), and trimethoprim (Proloprim), tetracycline (Sumycin, Panmycin), and doxycycline (Vibramycin), gentamicin (Garamycin), and tobramycin (Tobrex). In some of the optional embodiments, the antibiotic formulation is trimethoprim / sulfamethoxaxole. In some of the optional embodiments, at least one regimen of trimethoprim / sulfamethoxaxol is administered to the subject after administration of a dose of the manipulated low immunogenic island to the subject.

[0073] In some of the optional embodiments, at least one regimen of trimethoprim / sulfamethoxaxol is administered to the subject approximately 1 hour, 5 hours, 10 hours, or 24 hours, approximately 3 months, approximately 6 months, approximately 12 months, approximately 24 months, approximately 36 months, approximately 48 months, approximately 60 months, or more, following administration of a dose of the manipulated low-immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of trimethoprim / sulfamethoxaxol is administered to the subject daily for approximately 6 months following administration of a dose of the manipulated low-immunogenic islets to the subject. In some of the optional embodiments, a regimen of trimethoprim / sulfamethoxaxol ranging from approximately 50 mg to approximately 500 mg is administered to the subject. In some of the optional embodiments, the trimethoprim / sulfamethoxaxol regimen is administered at a lower dose. In some of the optional embodiments, a trimethoprim / sulfamethoxaxol regimen of approximately 80 mg to approximately 400 mg is administered to the subject. In some of the optional embodiments, the trimethoprim / sulfamethoxaxol regimen is administered at a lower dose. In some of the optional embodiments, one or more immunosuppressants include an antifungal agent. In some of the optional embodiments, the antifungal agent is selected from the group consisting of clotrimazole, miconazole, ketoconazole, itraconazole, and fluconazole. In some of the optional embodiments, the antifungal agent is clotrimazole. In some of the optional embodiments, at least one regimen of clotrimazole is administered to the subject before, on the same day as, concurrently with, and / or after, the administration of a dose of the manipulated low immunogenic island to the subject. In some of the optional embodiments, the clotrimazole regimen is administered to the subject approximately four times daily. In some of the optional embodiments, at least one regimen of clotrimazole is administered to the subject daily for up to approximately 3 months after administration of a dose of the manipulated, low immunogenic islets to the subject.

[0074] In some of the optional embodiments, one or more immunosuppressants include an antiviral agent. In some of the optional embodiments, the antiviral agent is selected from the group consisting of darunavir, atazanavir, ritonavir, acyclovir, valacyclovir, valganciclovir, tenofovir, and raltegravir. In some of the optional embodiments, the antiviral agent is an anticytomegalovirus agent. In some of the optional embodiments, the antiviral agent is valganciclovir. In some of the optional embodiments, at least one regimen of valganciclovir is administered to the subject after administration of a dose of the manipulated low immunogenic island to the subject. In some of the optional embodiments, at least one regimen of valganciclovir is administered to the subject about 1 hour, about 5 hours, about 10 hours, or about 24 hours, about 3 months, about 6 months, about 12 months, about 24 months, about 36 months, about 48 months, about 60 months, or more after administration of a dose of the manipulated low immunogenic island to the subject. In some of the optional embodiments, a regimen of valganciclovir ranging from approximately 300 mg to approximately 1,000 mg is administered to the subject. In some of the optional embodiments, the valganciclovir regimen is administered at a lower dose. In some of the optional embodiments, a regimen of approximately 450 mg of valganciclovir is administered to the subject daily after administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, the valganciclovir regimen is administered at a lower dose. In some of the optional embodiments, a regimen of approximately 900 mg of valganciclovir is administered to the subject daily starting approximately 12 days after administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, the valganciclovir regimen is administered at a lower dose. In some of the optional embodiments, a regimen of 900 mg of valganciclovir is administered to the subject throughout the period from administration of a dose of the manipulated low immunogenic islets to approximately 14 weeks.

[0075] In some of the optional embodiments, one or more immunosuppressants include a blood rheological modifier. In some of the optional embodiments, the blood rheological modifier is pentoxifylline. In some of the optional embodiments, at least one regimen of pentoxifylline is administered to the subject prior to, on the same day as, and / or after, the administration of a dose of the manipulated low immunogenic islands to the subject. In some of the optional embodiments, at least one regimen of pentoxifylline is administered to the subject prior to the administration of a dose of the manipulated low immunogenic islands to the subject.

[0076] In some of the optional embodiments, at least one regimen of pentoxifylline is administered to the subject approximately 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour before administration of a dose of the manipulated low immunogenic islands to the subject. In some of the optional embodiments, at least one regimen of pentoxifylline is administered to the subject approximately 2 days before administration of a dose of the manipulated low immunogenic islands to the subject.

[0077] In some of the optional embodiments, at least one regimen of pentoxifylline is administered to the subject after administration of a dose of manipulated low immunogenicity islands to the subject.

[0078] In some of the optional embodiments, at least one regimen of pentoxifylline is administered to the subject approximately 1 hour, 5 hours, 10 hours, or 24 hours, approximately 3 months, 6 months, 12 months, 24 months, 36 months, 48 ​​months, 60 months, or more after administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, at least one regimen of pentoxifylline is administered to the subject throughout the period from administration of a dose of the manipulated low immunogenic islets to approximately 7 days.

[0079] In some of the optional embodiments, a regimen of pentoxifylline ranging from approximately 300 mg to approximately 500 mg is used. In some of the optional embodiments, the pentoxifylline regimen is used at a lower dose.

[0080] In some of the optional embodiments, one or more immunosuppressants include one or more anticoagulants. In some of the optional embodiments, one or more anticoagulants are selected from the group consisting of aspirin, enoxaparin, and heparin. In some of the optional embodiments, one or more anticoagulants are aspirin. In some of the optional embodiments, at least one regimen of aspirin is administered to the subject after administration of a dose of manipulated low immunogenic islets to the subject. In some of the optional embodiments, one or more anticoagulants are enoxaparin. In some of the optional embodiments, at least one regimen of enoxaparin is administered to the subject after administration of a dose of manipulated low immunogenic islets to the subject. In some of the optional embodiments, one or more anticoagulants are heparin. In some of the optional embodiments, at least one regimen of heparin is administered to the subject after administration of at least one regimen of heparin to the subject.

[0081] In some of the optional embodiments, one or more immunosuppressants include DNA synthesis inhibitors. In some of the optional embodiments, the DNA synthesis inhibitor is fludarabine. In some of the optional embodiments, at least one regimen of fludarabine is administered to the subject before, concurrently with, and / or after, the administration of a dose of the manipulated low immunogenic islands to the subject. In some of the optional embodiments, at least one regimen of fludarabine is administered to the subject before the administration of a dose of the manipulated low immunogenic islands to the subject.

[0082] In some of the optional embodiments, at least one regimen of fludarabine is administered to the subject approximately 14 days, 10 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour before administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, a first regimen of fludarabine is administered to the subject approximately 2 to 14 days before administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, a regimen of fludarabine is administered to the subject daily for approximately 2, 3, or 4 days prior to administration of a dose of the manipulated low immunogenic islets to the subject. In some of the optional embodiments, the fludarabine regimen is administered to the subject on days 5, 4, and 3, counting back from the administration of doses of the manipulated low immunogenic islands to the subject.

[0083] In some of the optional embodiments, approximately 10 mg / m² 2 ~about 40mg / m 2 The fludarabine regimen is administered. In some of the optional embodiments, the fludarabine regimen is administered at a lower dose. In some of the optional embodiments, approximately 30 mg / m² 2 A fludarabine regimen is administered to the subject. In some of the optional embodiments, the fludarabine regimen is administered at a lower dose. In some of the optional embodiments, fludarabine is administered intravenously to the subject.

[0084] In some of the optional embodiments, one or more immunosuppressants include an alkylating agent. In some of the optional embodiments, the alkylating agent is cyclophosphamide. In some of the optional embodiments, at least one regimen of cyclophosphamide is administered to the subject before, concurrently with, and / or after, the administration of a dose of the manipulated low immunogenic islands to the subject. In some of the optional embodiments, at least one regimen of cyclophosphamide is administered to the subject before the administration of a dose of the manipulated low immunogenic islands to the subject. In some of the optional embodiments, at least one regimen of cyclophosphamide is administered to the subject about 14 days, about 10 days, 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, or about 1 hour before the administration of a dose of the manipulated low immunogenic islands to the subject. In some of the optional embodiments, the first regimen of cyclophosphamide is administered to the subject approximately 2 to 14 days before the administration of a dose of the manipulated low immunogenic islets. In some of the optional embodiments, the cyclophosphamide regimen is administered to the subject daily for approximately 2, 3, or 4 days prior to the administration of a dose of the manipulated low immunogenic islets. In some of the optional embodiments, the cyclophosphamide regimen is administered to the subject on the 5th, 4th, and 3rd day, counting backward from the administration of a dose of the manipulated low immunogenic islets.

[0085] In some of the optional embodiments, approximately 400 mg / m² 2 ~about 600mg / m 2 A cyclophosphamide regimen is administered. In some of the optional embodiments, the cyclophosphamide regimen is administered at a lower dose. In some of the optional embodiments, approximately 500 mg / m² 2 A cyclophosphamide regimen is administered to the subject. In some of the optional embodiments, the cyclophosphamide regimen is administered at a lower dose. In some of the optional embodiments, cyclophosphamide is administered intravenously to the subject.

[0086] In some of the optional embodiments, at least one regimen of fludarabine and at least one regimen of cyclophosphamide are administered to the subject. In some of the optional embodiments, at least one regimen of fludarabine is administered to the subject prior to the administration of at least one regimen of cyclophosphamide to the subject. In some of the optional embodiments, at least one regimen of fludarabine and at least one regimen of cyclophosphamide are administered to the subject prior to the administration of a dose of the manipulated low immunogenic island to the subject.

[0087] In some of the optional embodiments, i) approximately 30 mg / m² is administered to the subject about 2 to 7 days prior to the administration of a dose of the manipulated low immunogenicity island. 2 Fludarabine regimen and approximately 500 mg / m² 2 ii) The cyclophosphamide regimen is administered to the subject daily for three consecutive days, or approximately 30 mg / m² is administered to the subject approximately 2 to 14 days prior to the administration of the manipulated low immunogenicity islet dose. 2 Fludarabine regimen and approximately 500 mg / m² 2 The cyclophosphamide regimen is administered to the subject daily for two consecutive days, or iii) approximately 30 mg / m² is administered on the 5th, 4th, and 3rd days, calculated backward from the administration of the manipulated low immunogenicity island dose to the subject. 2 Fludarabine regimen and approximately 500 mg / m² 2 The cyclophosphamide regimen is administered to the target. In some of the optional embodiments, the fludarabine regimen and / or cyclophosphamide regimen are administered at lower doses.

[0088] In some of the optional embodiments, the procedure further includes tapering off the administration of one or more immunosuppressants. In some of the optional embodiments, tapering off includes gradually reducing the amount of one or more immunosuppressants administered to the subject. In some of the optional embodiments, tapering off is completed when at least one of the one or more immunosuppressants is no longer administered to the subject.

[0089] In some of the optional embodiments, the one or more molecules that regulate the cell surface protein expression of one or more MHC class I molecules is B2M. In some of the optional embodiments, the modification includes a modification that regulates the cell surface protein expression of one or more MHC class I molecules, and the modification inactivates or destroys one or more alleles of B2M. In some of the optional embodiments, the modification that inactivates or destroys one or more alleles of B2M reduces the mRNA expression of the B2M gene. In some of the optional embodiments, the modification that inactivates or destroys one or more alleles of B2M reduces the protein expression of B2M. In some of the optional embodiments, the modification that inactivates or destroys one or more alleles of B2M includes inactivation or destruction of one allele of the B2M gene, inactivation or destruction of both alleles of the B2M gene, or inactivation or destruction of all B2M coding alleles in the cell. In some of the optional embodiments, the inactivation or destruction includes an indel in the B2M gene. In some of the optional embodiments, inactivation or disruption involves frameshift mutations or deletions in a continuous section of the genomic DNA of the B2M gene.

[0090] In some of the optional embodiments, the modification is a modification that modulates the expression of one or more MHC class II molecules, and the modification inactivates or destroys one or more alleles of CIITA. In some of the optional embodiments, the modification that inactivates or destroys one or more alleles of CIITA reduces the protein expression of CIITA. In some of the optional embodiments, the modification that inactivates or destroys one or more alleles of CIITA includes inactivation or destruction of one allele of the CIITA gene, inactivation or destruction of both alleles of the CIITA gene, or inactivation or destruction of all CIITA coding alleles in a cell. In some of the optional embodiments, the inactivation or destruction includes an indel in the CIITA gene. In some of the optional embodiments, the inactivation or destruction is a frameshift mutation or deletion of a continuous section of genomic DNA of the CIITA gene.

[0091] In some of the optional embodiments, the expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR is reduced in the manipulated low-immunogenic islands.

[0092] In some of the arbitrary embodiments, one or more tolerogenic factors are selected from the group consisting of CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, C1 inhibitors, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc receptor, IL15-RF, H2-M3(HLA-G), A20 / TNFAIP3, CR1, HLA-F, and MANF.

[0093] In some of the various embodiments, at least one of the one or more tolerogenic factors is CD47. In some of the various embodiments, one or more tolerogenic factors are CD47. In some embodiments, CD47 is an engineered CD47 protein. In some embodiments, the engineered CD47 protein comprises (a) one or more extracellular domains and (b) one or more membrane tethers, the one or more extracellular domains comprising a signal-regulating protein alpha (SIRPα) interaction motif, and the engineered protein does not contain one or more full-length CD47 intracellular domains. In some embodiments, the SIRPα interaction motif is or comprises a CD47 extracellular domain or a portion thereof. In some embodiments, the SIRPα interaction motif is or comprises a SIRPα antibody or a portion thereof.

[0094] In some of the optional embodiments, a modification that increases the expression of one or more tolerancegenic factors comprises an exogenous polynucleotide encoding one or more tolerancegenic factors. In some of the optional embodiments, the exogenous polynucleotide encoding one or more tolerancegenic factors is incorporated into the genome of the engineered low immunogenic island. In some of the optional embodiments, the one or more tolerancegenic factors comprises CD47, and the engineered low immunogenic island expresses CD47 at a first level greater than 5 times or about 5 times above a second level expressed by control or wild-type island cells. In some of the optional embodiments, CD47 is expressed at a first level greater than 10 times or about 10 times, greater than 20 times or about 20 times, greater than 30 times or about 30 times, greater than 40 times or about 40 times, greater than 50 times or about 50 times, greater than 60 times or about 60 times, or greater than 70 times or about 70 times above a second level expressed by control or wild-type island cells.

[0095] In some of the arbitrary embodiments, one or more tolerance factors include CD47, which is expressed by engineered hypoimmunogenic islands in more than 20,000 molecules or approximately more than 20,000 molecules per cell. In some of the arbitrary embodiments, CD47 is expressed by engineered low immunogenic islands in quantities of more than 30,000 molecules or approximately 30,000 molecules per cell, more than 50,000 molecules or approximately 50,000 molecules per cell, more than 100,000 molecules or approximately 100,000 molecules per cell, more than 200,000 molecules or approximately 200,000 molecules per cell, more than 300,000 molecules or approximately 300,000 molecules per cell, more than 400,000 molecules or approximately 400,000 molecules per cell, more than 500,000 molecules or approximately 500,000 molecules per cell, or more than 600,000 molecules or approximately 600,000 molecules per cell.

[0096] In some of the optional embodiments, the manipulated hypoimmunogenic islands have the phenotype B2M indel / indel;CIITA indel / indel;CD47tg. In some of the optional embodiments, of the dose of manipulated hypoimmunogenic island cells, at least 85% of the cells have the modification. In some of the optional embodiments, at least 90%, at least 92%, at least 95%, or at least 98% of the cells have the modification. In some of the optional embodiments, of the dose of manipulated hypoimmunogenic island cells, at least 85% of the cells have the phenotype B2M indel / indel;CIITA indel / indel;CD47tg. In some of the optional embodiments, at least 90%, at least 92%, at least 95%, or at least 98% of the cells have the said phenotype.

[0097] In some of the optional embodiments, the engineered hypoimmunogenic islets exhibit one or more functions of wild-type or control beta islet cells, and optionally, one or more functions are selected from the group consisting of glucose-stimulated insulin secretion (GSIS) in vitro, glucose metabolism, maintenance of fasting blood glucose levels, insulin secretion in response to glucose injection in vivo, and glucose clearance after glucose injection in vivo. In some of the optional embodiments, the engineered hypoimmunogenic islets are capable of glucose-stimulated insulin secretion (GSIS), and optionally, insulin secretion is in a perfusion GSIS assay. In some of the optional embodiments, GSIS is dynamic GSIS, including dynamic insulin secretion in phases 1 and 2. In some of the optional embodiments, GSIS is static GSIS, and optionally, the static incubation index is greater than or about greater than 1, greater than or about greater than 2, greater than or about greater than 5, greater than or about greater than 10, or greater than or about greater than 20.

[0098] In some of the arbitrary embodiments, the level of insulin secretion from the manipulated low immunogenic islets is at least 20% of the level of insulin secretion observed in primary islets, and optionally in corpuscular islets. In some of the arbitrary embodiments, the level of insulin secretion from the manipulated low immunogenic islets is at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, and at least 70% of the level of insulin secretion observed in primary islets, and optionally in corpuscular islets.

[0099] In some of the arbitrary embodiments, the total insulin content of the manipulated low immunogenic islands is greater than or about 500 μIU of insulin per 5000 cells, greater than or about 1000 μIU of insulin per 5000 cells, greater than or about 2000 μIU of insulin per 5000 cells, greater than or about 3000 μIU of insulin per 5000 cells, or greater than or about 4000 μIU of insulin per 5000 cells. In some of the arbitrary embodiments, the ratio of proinsulin to insulin in modified SC-beta cells is 0.02–0.1 or about 0.02–about 0.1, and optionally, any value between 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or about 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 and any of the aforementioned.

[0100] In some of the available embodiments, the manipulated hypoimmunogenic islands exhibit functionality for more than two weeks after transplantation into the subject. In some of the available embodiments, the manipulated hypoimmunogenic islands exhibit functionality for more than three weeks, more than four weeks, more than eight weeks, more than three months, more than six months, or more than twelve months after transplantation into the subject.

[0101] In some of the arbitrary embodiments, the functionality is selected from the group consisting of maintaining fasting blood glucose levels, insulin secretion in response to glucose injection in vivo, and glucose clearance after glucose injection in vivo.

[0102] In some of the arbitrary embodiments, the dose is approximately 1 × 10 7 Individual cells ~ approx. 3×10 8 It is individual cells. In some of the arbitrary embodiments, the dose is about 1.25 × 10⁻⁶. 5 Individual cells / kg ~ approx. 1.2×10 7 It is expressed as individual cells / kg.

[0103] In some of the optional embodiments, the dose is approximately 6,500 island equivalents (IEQ) to approximately 600,000 IEQ. In some of the optional embodiments, the dose is approximately 80 IEQ / kg to approximately 24,000 IEQ / kg. In some of the optional embodiments, the subjects do not undergo an immunosuppressive regimen. [Brief explanation of the drawing]

[0104] [Figure 1] Figures 1A and 1B show the results of an allogeneic transplantation study evaluating the immune response of NHP recipients to allogeneic non-human primate (NHP) primary islet cells. Quantification of luciferase expression BLI is provided for transplanted B2M- / -;CIITA- / -;CD47tg NHP primary islet cells (Figure 1A shows the quantification, Figure 1B shows the corresponding BLI image). [Figure 2] Figures 2A-2D provide the results of im-injected allogeneic transplantation studies in NHP to evaluate the immune response. Interferon-gamma (IFNg) levels are provided for NHP transplanted with B2M- / -;CIITA- / -;CD47tg NHP primary islet cells (Figure 2A). IgM levels (Figure 2B) and IgG levels (Figure 2C) of donor-specific antibodies (DSA) are provided for NHP transplanted with B2M- / -;CIITA- / -;CD47tg NHP primary islet cells. DSA IgG levels are also provided for NHP sensitized with B2M- / -;CIITA- / -;CD47tg NHP primary islet cell transplantation, where IgG levels were elevated before transplantation (Figure 2D). [Figure 3]This paper provides results of in vitro natural killer (NK) cell-mediated cell killing of primary NHP cells in B2M- / -;CIITA- / -;CD47tg NHP. [Figure 4A] Figures 4A-4D show phenotyping and allogeneic transplantation of primary rhesus macaque cells that have been edited to B2M- / -;CIITA- / -;CD47tg. Figure 4A shows immunofluorescence staining of somatostatin, insulin, and glucagon (upper panel) and CD47, MHC class I, and DAPI (lower panel) before and after B2M- / -;CIITA- / -;CD47tg editing. [Figure 4B] Figures 4A-4D show phenotyping and allogeneic transplantation of primary rhesus macaque insular cells edited with B2M- / -;CIITA- / -;CD47tg. Figure 4B shows the expression of MHC class I, MHC class II, and rhesus macaque CD47 in rhesus macaque insular cells before and after B2M- / -;CIITA- / -;CD47tg editing. [Figure 4C] Figures 4A-4D show phenotyping and allogeneic transplantation of primary rhesus macaque cells edited with B2M- / -;CIITA- / -;CD47tg. Figure 4C shows insulin release from rhesus macaque islets in vitro before and after B2M- / -;CIITA- / -;CD47tg editing. [Figure 4D] Figures 4A-4D show the phenotypic determination and allogeneic transplantation of primary rhesus macaque insular cells of B2M- / -;CIITA- / -;CD47tg. Figure 4D shows the composition of rhesus macaque insular cells before and after B2M- / -;CIITA- / -;CD47tg editing. [Figure 5] This report shows blood glucose measurements in diabetic non-human primates (NHPs) transplanted with allogeneic B2M- / -;CIITA- / -;CD47tg NHP primary islet cells. Blood samples were collected in the morning (morning glucose AM) and afternoon (afternoon glucose PM). Diabetes: >127 mg / dL; Abnormal fasting glucose: >80-127 mg / dL; Normal: <80 mg / dL; and Hypoglycemia: <30 mg / dL. [Figure 6]This shows blood glucose measurements extended to day 111 after STZ in diabetic non-human primates (NHPs) transplanted with allogeneic B2M- / -;CIITA- / -;CD47tg NHP primary islet cells. Blood was collected in the morning (morning glucose AM) and afternoon (afternoon glucose PM). Hyperglycemia (diabetes): >127 mg / dL; abnormal fasting glucose: >80-127 mg / dL; normal: <80 mg / dL; and hypoglycemia: <30 mg / dL. [Figure 7] This shows blood glucose measurements extended to day 226 after STZ in diabetic non-human primates (NHPs) transplanted with allogeneic B2M- / -;CIITA- / -;CD47tg NHP primary islet cells. Blood was collected in the morning (morning glucose AM) and afternoon (afternoon glucose PM). Hyperglycemia (diabetes): >127 mg / dL; abnormal fasting glucose: >80-127 mg / dL; normal: <80 mg / dL; and hypoglycemia: <30 mg / dL. [Figure 8A] This shows the administration of exogenous insulin (U / day) over time. [Figure 8B] This shows blood glucose levels (mg / dL) over time in the morning and evening. [Figure 8C] This shows serum c-peptide levels (ng / mL) over time. An asterisk indicates the time point at which the c-peptide was measured. [Figure 8D] This shows the weight (kg) over time. [Figure 9]This shows C-peptide measurements for diabetic non-human primates (NHP) transplanted with allogeneic B2M- / -;CIITA- / -;CD47tg NHP primary islet cells. Pre-STZ: C-peptide measurement before IV injection of streptozotocin (STZ); Post-STZ d50: C-peptide measurement 50 days after STZ injection (d50); d0 (Post-STZ d78): C-peptide measurement 78 days after STZ injection (d78) and 0 days after islet cell transplantation; d7 (Post-STZ d85): C-peptide measurement 85 days after STZ injection (d85) and 7 days after islet cell transplantation; d14 (Post-STZ d92): C-peptide measurement 92 days after STZ injection (d92) and islet cell transplantation C-peptide measurement on day 14 post-transplant (d14); d28 (d106 post-STZ): C-peptide measurement 106 days after STZ injection (d106) and 28 days after islet cell transplantation (d28); d42 (120 days post-STZ): C-peptide measurement 120 days after STZ injection (d120) and 42 days after islet cell transplantation (d42); d90 (d172 post-STZ): C-peptide measurement 172 days after STZ injection (d172) and 90 days after islet cell transplantation (d90). [Figure 10] This shows glucose tolerance measurements in diabetic non-human primates (NHPs) transplanted with allogeneic B2M- / -;CIITA- / -;CD47tg NHP primary islet cells. Pre-STZ: Glucose tolerance measurement before IV injection of streptozotocin (STZ); d50 (post-STZ): Glucose tolerance measurement 50 days after STZ injection (d50); d103 (d25 after cell transplantation): Glucose tolerance measurement 103 days after STZ injection (d103) and 25 days after islet cell transplantation (d25); merge: Pre-STZ, d50, and d103. [Figure 11-1]Figures 11A–11L show cell-mediated and antibody-mediated responses to primary insular cells of B2M- / -;CIITA- / -;CD47tg rhesus monkeys. Figure 11A shows the ELISpot assay using recipient monkey PBMCs collected at the scheduled time. Figures 11B–11E show killing assays using recipient cynomolgus monkey T cells (Figure 11B), PBMCs (Figure 11C), NK cells (Figure 11D), and macrophages (Figure 11E). The y-axis shows the percentage of target cell killing. Figures 11F–I show serum Ig levels, including total IgM (Figure 11F), IgG (Figure 11G), donor-specific antibody (DSA) IgM (Figure 11H), and DSA IgG (Figure 11I). Figures 11J–11L show antibody-dependent cell-mediated cytotoxicity (ADCC) assays using inactivated recipient cynomolgus monkey serum and NK cells (Figure 11J) or macrophages (Figure 11K), as well as CDC assays using complete recipient monkey serum (Figure 11L). The y-axis shows the percentage of target cell elimination. [Figure 11-2] See the explanation in Figure 11-1. [Figure 12] Figures 12A and 12B show the killing of rhesus macaque B2M- / -;CIITA- / -;CD47tg primary insular cells by cynomolgus macaque NK cells or macrophages in response to treatment with anti-CD47 antibody (maglorimab). [Figure 13] Figures 13A-C show immunohistochemical staining of the islets and primary muscle islets transplant sites. Figure 13A shows the pancreas derived from a healthy cynomolgus monkey. Figure 13B shows the pancreas of a recipient cynomolgus monkey. Figure 13C shows the muscle transplant site of a recipient cynomolgus monkey. [Modes for carrying out the invention]

[0105] Detailed explanation This specification provides a method involving the administration of a drug to an engineered island containing beta cells engineered to evade the immune system (also referred herein as modified immune-evading beta cells or hypoimmunogenic (HIP) beta cells). In some embodiments, the engineered island may be an engineered primary island. In some embodiments, the engineered island may be an engineered island cell differentiated from a pluripotent stem cell. In some embodiments, the engineered island cell containing engineered beta cells exhibits features that enable it to evade immune recognition. In some embodiments, the engineered island cell containing engineered beta cells is hypoimmunogenic (also referred to as hypoimmune or HIP). In some embodiments, the engineered island cell containing engineered beta cells is not subject to innate immune cell rejection. In some embodiments, the engineered island cell containing engineered beta cells provided herein exhibits reduced innate immune cell rejection and / or adaptive immune cell rejection (e.g., hypoimmunogenic cells). For example, in some embodiments, the engineered island cell containing engineered beta cells exhibits reduced sensitivity to NK cell-mediated lysis and / or macrophage phagocytosis. In some embodiments, the manipulated islands and cells are useful as a source of universally compatible cells or tissues (e.g., universal donor cells or tissues) to be transplanted into the recipient subject. Such low immunogenic cells retain cell-specific properties and characteristics upon administration to the subject (e.g., transplantation or engraftment). In some embodiments, the manipulated island cells, upon transplantation or engraftment in the subject, cluster into effective endocrine organoids (referred to as pseudo-islet grafts (p-islets)). Thus, in some embodiments, the manipulated islands are HIP pseudo-islets (HIP p-islets). In some embodiments, the effective endocrine organoids provide stable endocrine function through insulin production and secretion, thereby enabling insulin independence in the subject. In some embodiments, stable endocrine function and insulin independence occur in the absence of immunosuppression.In some embodiments, manipulated islet cells, including manipulated beta cells, can be used as a cell source for allogeneic therapy, regardless of the genetic makeup of the subject.

[0106] In some embodiments, the method provided is for the treatment of beta cell-related disorders (e.g., diabetes) in a subject (e.g., improving glucose tolerance in the subject). In certain embodiments, the method is for the treatment of type 1 diabetes in a subject (e.g., improving glucose tolerance in the subject). In other embodiments, the method improves the function of the provided islet cell grafts. In some embodiments, the method restores glucose metabolism in a subject.

[0107] Patients with type 1 diabetes mellitus (T1DM) or hypoglycemic altered consciousness (IAH) are at increased risk of severe hypoglycemic events due to a lack of basic hypoglycemia-inducing defense mechanisms (Hwang et al., J Clin Invest (2018) 128:1485-195; Lin et al., J Diabetes Investig (2020) 11:1388-1402). Current treatment for T1DM patients includes intensive insulin therapy. However, such treatments can cause severe hypoglycemia associated with altered mental status, seizures, arrhythmias, and even death (Bornstein et al., Nat Rev Endocrinol (2022) 18:389-390).

[0108] Islet transplantation has been shown to be superior to insulin therapy, resulting in improved patient survival and quality of life (Boughton et al., Diabetes Obes Metab (2021) 23:1389-1396). However, islet transplantation in T1DM patients is significantly hampered by the need for continuous immunosuppression, which is associated with significant pathological conditions, including chronic kidney injury, infections, and cancer, as well as a graft survival of only 4.4–5.9 years (Hering et al., Diabetes Care (2016) 39:1230-1240; Lemos et al., Diabetes Care (2021) 44:e67-e68; Marfil-Garza et al., Lancet Diabetes Endocrinol (2022) 10:519-532). Furthermore, despite being immunosuppressed, T1DM patients frequently experience sensitization with allogeneic grafts, leading to elevated panel-reactive antibodies and complicating any subsequent transplant. Therefore, improvements are needed in the methods for islet transplantation, including the treatment of diabetes.

[0109] The embodiments provided address these needs. The embodiments provided relate to primary islets that are engineered to be hypoimmune, thereby reducing or eliminating the need for immunosuppression. Specifically, allogeneic transplantation of hypoimmune engineered primary islet cells into fully immunocompetent non-human diabetic primate models has been shown by results herein to confer stable endocrine function and enable insulin independence without inducing any detectable immune response in the absence of immunosuppression. Thus, this disclosure demonstrates that hypoimmune primary islet cells provide a novel curative cell therapy for T1DM, enabling it with reduced or no need for immunosuppression.

[0110] In some embodiments, the engineered islands containing the engineered beta cells described herein are low immunogenic at the time of administration (e.g., at transplantation or graft transplantation) and, in some embodiments, avoid immune rejection. Non-limiting examples of modifications that result in avoidance of immune rejection include reduced expression of major histocompatibility complexes (MHC) (human leukocyte antigen (HLA) class I and HLA class II antigens) and increased expression of one or more tolerance factors (such as CD47). In some embodiments, the engineered islands containing the engineered beta cells are administered to MHC-incompatible allogeneic subjects.

[0111] In some embodiments, engineered island cells, including engineered beta cells, contain modifications that, compared to control or wild-type beta cells, (a) reduce the expression of one or more major histocompatibility complex (MHC) class I molecules and / or MHC class II molecules, and (b) increase the expression of one or more tolerance-generating factors in the engineered islands. In some embodiments, the modifications make the cells hypoimmune, which in some aspects allows the cells to avoid immune rejection compared to control or wild-type island cells (such as primary human island cell beta cells). For the purposes of this specification, the term engineered island may be used interchangeably with the term hypoimmune-inducing island.

[0112] The engineered islands include engineered cells (such as engineered beta cells) that utilize the expression of tolerancegenic factors and further regulate (e.g., reduce or eliminate) the expression (e.g., surface expression) of one or more MHC class I molecules and / or one or more MHC class II molecules. In some embodiments, the modification that reduces the expression of one or more MHC class I molecules is a modification that reduces the expression of β-2 microglobulin (B2M). In some embodiments, the modification that reduces the expression of one or more MHC class II molecules is a modification that reduces the expression of CIITA. In some embodiments, engineered cells containing the modifications described herein (including reduction or elimination of the expression of MHC class I or MHC class II molecules and increase the expression of CD47 or other tolerancegenic factors) survive, engraft, persist, and function after administration (e.g., transplantation or engraftment). In some embodiments, engineered island cells show enhanced survival and / or enhanced engraftment and / or function over the long term compared to control or wild-type islands (such as unmodified island cells that do not contain modifications that make the cells less immune).

[0113] In some embodiments, the manipulated island is administered via intramuscular injection (e.g., intramuscular injection into the forearm).

[0114] In some embodiments, genome editing techniques utilizing low-frequency endonucleases (e.g., CRISPR / Cas, TALEN, zinc finger nucleases, meganucleases, and homing endonuclease systems) are used to reduce or eliminate the expression of immunogenes described herein (such as genes involved in regulating the expression of MHC class I or MHC class II molecules) in island cells used to guide the engineered islands (e.g., by deleting the genomic DNA of critically important immunogenes). In certain embodiments, genome editing techniques or other gene regulation techniques are used to insert tolerance-inducing (tolerogenic) factors (e.g., CD47) into target genomic loci of island cells used to guide the engineered islands, thereby obtaining engineered islands that can evade immune recognition upon engraftment in the recipient. Therefore, the manipulated islands exhibit modulation (e.g., reduction or elimination) of the expression of one or more genes and factors that affect the expression of MHC class I and / or MHC class II molecules, and modulation (e.g., reduction or regulation (e.g., overexpression)) of the expression of tolerance-genic factors (such as CD47), resulting in reduced recognition by the immune system of the recipient. In some embodiments, the modified cells may also exhibit modulation (e.g., reduction) of the expression of CD142, and the expression of CD142 can also be reduced in some embodiments by genome editing techniques (e.g., CRISPR / Cas, TALEN, zinc finger nucleases, meganucleases, and homing endonuclease systems) to reduce or eliminate the expression of CD142 (e.g., by deleting genomic DNA of a very important immunogene). In some embodiments, the manipulated island may exhibit modulation (e.g., increased expression) of the expression of one or more complement inhibitors selected from CD46, CD59, CD55, and CD35, and in some embodiments, these complement inhibitors may also be increased by genome editing techniques to insert or incorporate exogenous polynucleotides encoding one or more complement inhibitors into genomic loci in the manipulated island.

[0115] In some embodiments, the beta-cell related disorder is a metabolic disorder. In some embodiments, the metabolic disorder is familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabbe disease, maple syrup urine disease, metachromatic leukodystrophy, mitochondrial encephalopathy, lactic acidosis, stroke-like episodes (MELAS), Niemann-Pick disease, phenylketonuria (PKU), porphyria, Tay-Sachs disease, Wilson's disease, type 1 diabetes mellitus, type 2 diabetes mellitus, obesity, hypertension, dyslipidemia, or carbohydrate intolerance. In some embodiments, the beta-cell related disorder is type 1 diabetes mellitus.

[0116] In carrying out specific embodiments, unless otherwise specified, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology within the scope of the skill in the art are used, many of which are described below for illustrative purposes. Such methods are fully described in the literature.For example, see Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998) Current Protocols in Immunology Q.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and research papers in scientific journals (such as Advances in Immunology).

[0117] All publications referenced in this application (including patent documents, scientific articles, and databases) are incorporated by reference in whole for any purpose to the same extent as if each individual publication were incorporated by reference individually. If any definition provided herein contradicts or otherwise conflicts with any definition provided herein in a patent, application, published application, or other publication incorporated herein by reference, the definition provided herein shall prevail over the definition incorporated herein by reference.

[0118] Section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein. Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this disclosure. The following descriptions are illustrative of this disclosure and should not be construed in any way as limiting the scope of the invention as described herein.

[0119] I. Methods and administration of beta cell therapy In some embodiments, methods for treating beta cell-related disorders in a subject are provided herein, the methods comprising administering engineered islands to the subject as described. Engineered islands administered to a subject according to the methods provided herein comprise cells that have been modified to avoid immune rejection. In some embodiments, the engineered islands are administered as island clusters. In certain embodiments, the engineered islands comprise engineered beta cells. In some embodiments, the engineered beta cells are present in a composition comprising additional island cells. In some embodiments, the islands (e.g., island clusters) further comprise alpha cells and / or delta cells. In some embodiments, the islands (e.g., island clusters) further comprise epsilon cells and / or PP cells. In some embodiments, the cells of the engineered islands comprise the same low immunomodulation. In certain embodiments, the cells of the engineered islands comprise beta cells modified using low immunomodulation. Section II describes exemplary features of engineered islands (including engineered or engineered islands) for use in the methods provided.

[0120] The manipulated cells provided herein can be administered to subjects for the treatment of beta cells and related diseases or disorders. In some embodiments, the subjects are mammals. In some embodiments, the subjects are humans.

[0121] In some embodiments, beta-cell-related disorders are metabolic disorders. Metabolic disorders can occur when abnormal chemical reactions in the subject's body disrupt metabolic processes (e.g., processes associated with the metabolism or breakdown of energy into sugars and acids or the storage of such energy). In some embodiments, metabolic disorders affect the breakdown of amino acids, carbohydrates, or lipids in the subject's body. In some embodiments, metabolic disorders affect the mitochondria of the subject (e.g., mitochondrial disease). In some embodiments, metabolic disorders manifest when the organ of the subject (e.g., the liver or pancreas) becomes diseased and / or does not function properly. Exemplary metabolic disorders as described herein may include, but are not limited to, any disease or disorder characterized by elevated blood pressure, hyperglycemia, excess body fat around the waist, and abnormal cholesterol or triglyceride levels. In some embodiments, the metabolic disorder is familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabbe disease, maple syrup urine disease, metachromatic leukodystrophy, mitochondrial encephalopathy, lactic acidosis, stroke-like episodes (MELAS), Niemann-Pick disease, phenylketonuria (PKU), porphyria, Tay-Sachs disease, Wilson's disease, type 1 diabetes mellitus, type 2 diabetes mellitus, obesity, hypertension, dyslipidemia, or carbohydrate intolerance. In some embodiments, the metabolic disorder is type 2 diabetes mellitus. In some embodiments, the metabolic disorder is type 1 diabetes mellitus. In some embodiments, the metabolic disorder is type 1 diabetes mellitus.

[0122] In some embodiments, the beta-cell disorder is a metabolic disorder. In some embodiments, the metabolic disorder is selected from the group consisting of familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabbe disease, maple syrup urine disease, metachromatic leukodystrophy, mitochondrial encephalopathy, lactic acidosis, stroke-like episodes (MELAS), Niemann-Pick disease, phenylketonuria (PKU), porphyria, Tay-Sachs disease, Wilson's disease, type 1 diabetes mellitus, type 2 diabetes mellitus, obesity, hypertension, dyslipidemia, and carbohydrate intolerance. In some embodiments, the disorder is diabetes mellitus. In some embodiments, the disorder is type 1 diabetes mellitus.

[0123] A. Island cells In some embodiments, engineered islands containing engineered beta cells have the ability to evade the immune system. In some embodiments, engineered islands containing engineered beta cells include modifications that (a) reduce the expression of one or more major histocompatibility complex (MHC) class I molecules and / or one or more MHC class II molecules in the engineered islands compared to control or wild-type island cells, and (b) increase the expression of one or more tolerance-generating factors in the engineered cells compared to control or wild-type island cells (e.g., compared to control or wild-type beta cells). In some embodiments, engineered islands containing engineered beta cells include modifications that reduce the expression of B2M in the engineered cells compared to control or wild-type island cells (e.g., control or wild-type beta cells). In some embodiments, engineered island cells include modifications that reduce the expression of CIITA in the modified island cells compared to control or wild-type island cells (e.g., compared to control or wild-type beta cells). In some embodiments, engineered island cells include modifications that increase the expression of CD47 in the engineered island cells compared to control or wild-type island cells (e.g., compared to control or wild-type beta cells). In some embodiments, the modified islet cells (such as modified beta cells) include modifications that (a) reduce B2M expression compared to control or wild-type islet cells, (b) reduce CIITA expression compared to control or wild-type islet cells, and (c) increase CD47 expression in the modified islet cells compared to control or wild-type islet cells.

[0124] In some embodiments, the islands are primary islands manipulated using the low-immunomodulation described. In some embodiments, the primary islands are human. In some embodiments, the island cells, including beta cells, are cells differentiated from stem cells and manipulated using the low-immunomodulation described. In some embodiments, the stem cells are selected from the group consisting of pluripotent stem cells (PSCs), induced pluripotent stem cells (iPSCs), embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells, germline stem cells, lung stem cells, umbilical cord blood stem cells, and plutopitous stem cells. In some embodiments, the stem cells are pluripotent stem cells (PSCs). In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), or embryonic stem cells (ESCs). In some embodiments, the stem cells are present in a suspension.

[0125] In some embodiments, the islet cells are primary islet cells (also referred to as pancreatic islet cells). In certain embodiments, the primary islet cells include primary beta islet cells (pancreatic beta islet cells). In some embodiments, the primary islets are isolated or obtained from one or more individual donor subjects (such as one or more individual healthy donors (e.g., subjects with no known or suspected disease or infection, e.g., those showing no clinical signs)). In some embodiments, the donor is a cadaver. As will be understood by those skilled in the art, methods for isolating or obtaining islets from an individual can be achieved using known techniques.

[0126] In some embodiments, islet cells are obtained from a subject or individual (e.g., collected, extracted, removed, or acquired). In some embodiments, primary islet cells are obtained from a pool of islet cells such that the islet cells originate from one or more subjects (e.g., one or more humans, including one or more healthy humans). In some embodiments, the pool of primary islet cells originates from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects. In some embodiments, the donor subjects are different from the patient (e.g., the recipient subject to whom the therapeutic cells are administered). In some embodiments, the pool of islet cells does not contain patient-derived cells. In some embodiments, one or more of the donor subjects from which the pool of islet cells is obtained are different from the patient.

[0127] Further descriptions of pancreatic islet cells, including those for use in this technology, can be found in WO2020 / 018615, which is incorporated herein by reference in its entirety.

[0128] In some embodiments, a population of engineered primary islet cells, including primary beta islet cells isolated from one or more individual donors (e.g., healthy donors), is maintained in culture and, in some cases, grown before administration. In certain embodiments, the population of engineered islet cells is cryopreserved before administration.

[0129] Exemplary islet cell types include, but are not limited to, islet progenitor cells, immature islet cells, mature islet cells, and similar types. In some embodiments, the pancreatic cells described herein are administered to subjects for the treatment of diabetes.

[0130] In some embodiments, the islet cells disclosed herein (such as primary beta-islet cells isolated from one or more individual donors (e.g., healthy donors)) secrete insulin. In some embodiments, the islet cells exhibit at least two characteristics of endogenous islet cells (e.g., glucose-responsive insulin secretion and expression of beta-islet cell markers, but not limited to the following).

[0131] Exemplary beta-islet cell markers or beta-islet cell precursor markers include, but are not limited to, c-peptide, Pdxl, glucose transporter 2 (Glut2), HNF6, VEGF, glucokinase (GCK), prohormone-converting enzyme (PC1 / 3), Cdcpl, NeuroD, Ngn3, Nkx2.2, Nkx6.l, Nkx6.2, Pax4, Pax6, Ptfla, Isll, Sox9, Soxl7, and FoxA2.

[0132] In some embodiments, primary islet cells may be isolated from primary islets, derived from primary islet cells within primary islets, or as components of primary islets. For example, primary pancreatic beta-islet cells can be edited as single beta-islet cells, as a population of beta-islet cells, or as components of primary islets (e.g., primary pancreatic beta-islet cells present within primary islets along with other cell types). In another example, primary pancreatic beta-islet cells can be administered to a patient as single beta-islet cells, as a population of beta-islet cells, or as components of primary islets (e.g., primary pancreatic beta-islet cells present within primary islets along with other cell types). In embodiments where pancreatic beta-islet cells are present within islets along with other cell types, other cell types may also be edited by the methods described herein.

[0133] In some embodiments, primary islet cells are isolated from the primary islets before or after the manipulation (such as genetic engineering). Such isolated islet cells can be clustered before administration to the patient, and the clusters may include beta islet cells and other cell types (including, but not limited to, those derived from primary islets). The number of islet cells in a cluster may vary, such as about 50, about 100, about 250, about 500, about 750, about 1000, about 1250, about 1500, about 1750, about 2000, about 2250, about 2500, about 2750, about 3000, about 3500, about 4000, about 4500, or about 5000 cells. Patients can be administered clusters of approximately 10, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, or 1000 units.

[0134] In some embodiments, primary islet cells isolated from one or more individual donors (e.g., healthy donors) produce insulin in response to an increase in glucose. In some embodiments, the islet cells are beta islet cells. In some embodiments, the beta islet cells are monitored to assess their glucose regulatory capacity. Assays for monitoring glucose regulation may include, but are not limited to, continuous monitoring of blood glucose levels, monitoring of blood glucose levels after fasting periods, glucose tolerance (e.g., glucose loading) tests, glucose utilization and oxidation, insulin secretion (e.g., by U-PLEX® Meso Scale Discovery (MSD) assays and / or glucose-stimulated insulin secretion (GSIS) assays), measurement of the presence of specific transcription factors and pathways (e.g., homeobox transcription factors SIX2, NKX6-1, and PDX1), measurement of mitochondrial respiration, and measurement of changes in intracellular Ca2+ calcium flow (e.g., glucose-induced Ca2+ elevation, Ca2+-activated exocytosis). In the art, various methods for measuring glucose regulation are known, including those described in Velazco-Cruz et al., Cell Reports, 2020, 31, 107687; Pagliuca et al., Cell, 2014, 159(2):428-439; Davis et al., Cell Reports, 2020, 31(6):107623; and Alcazar et al., Cell Transplantation, 2020, 29, and these disclosures, including figures, figure legends, and method descriptions, are incorporated herein by reference in their entirety. In some embodiments, beta islet cells (e.g., modified beta islet cells) may exhibit GSIS. In some embodiments, GSIs are measured in a perfusion GSIS assay. In some embodiments, GSIs are dynamic GSIS, including dynamic insulin secretion in phases 1 and 2. In some embodiments, GSIs are static GSIS. For example, a static incubation index could be greater than or approximately greater than 1, greater than or approximately greater than 2, greater than or approximately greater than 5, greater than or approximately greater than 10, or greater than or approximately greater than 20.In various embodiments, pancreatic islet cells secrete insulin in response to an increase in glucose. In some embodiments, the cells have distinctly different morphologies (e.g., cobblestone cell morphology and / or a diameter of about 17 pm to about 25 pm).

[0135] In some embodiments, the cells used to generate the manipulated island cells are differentiateable stem cells or progenitor cells (e.g., stem cells are totipotent, pluripotent, or multipotent). In some embodiments, the cells are isolated from embryonic or neonatal tissue. In some embodiments, the cells are embryonic stem cells. In some embodiments, the cells are induced pluripotent stem cells derived from somatic cells (e.g., skin or blood cells) and are reprogrammed into an embryo-like pluripotent state. In some embodiments, the induced pluripotent stem cells are derived from fibroblasts. In some embodiments, the cells modified as provided herein are pluripotent stem cells or cells differentiated from pluripotent stem cells. The cells may be vertebrate cells, mammalian cells such as human or mouse cells. The cells may also be vertebrate stem cells, such as mammalian stem cells (e.g., human or mouse stem cells). In several embodiments, the cells or stem cells are suitable for modification. Cells or stem cells, or cells derived from such stem cells, may have therapeutic value, and as a result, cells or stem cells, or cells obtained from or differentiated from such stem cells, may be used to treat diseases, disorders, defects, or injuries in subjects requiring treatment.

[0136] In some embodiments, island cells containing beta cells modified or manipulated as provided herein are modified pluripotent stem cells (e.g., modified iPSCs). The generation of mammalian (e.g., mouse and human) pluripotent stem cells (generally referred to as iPSCs, and miPSCs for mouse cells or hiPSCs for human cells) is generally known in the art. As will be understood by those skilled in the art, there are a variety of different methods for generating iPSCs. The original induction was performed from mouse embryonic or adult fibroblasts using viral transduction of four transcription factors: Oct3 / 4, Sox2, c-Myc, and Klf4. See Takahashi and Yamanaka Cell 126:663-676 (2006) (the whole, and in particular the methods outlined therein, are incorporated herein by reference). Subsequently, numerous methods have been developed. For an overview, please refer to Seki et al, World J. Stem Cells 7(1):116-125 (2015), and Lakshmipathy and Vermuri, editors, Methods in Molecular Biology: Pluripotent Stem Cells, Methods and Protocols, Springer 2013. Both of these are explicitly incorporated herein by reference, particularly regarding methods for producing hiPSCs (see, for example, Chapter 3 of the latter reference).

[0137] Generally, iPSCs are created by the transient expression of one or more reprogramming factors in host cells, usually introduced using episomatic vectors. Under these conditions, only a small number of cells are induced to become iPSCs (this step is generally less efficient because no selection markers are used). While we do not wish to be bound by theory, it is thought that once a cell is "reprogrammed" and becomes pluripotent, it loses its episomatic vector(s) and uses endogenous genes to produce the factor(s).

[0138] As will also be understood by those skilled in the art, the number of reprogramming factors that can be used or are used can vary. Generally, when the number of reprogramming factors used is reduced, not only does the efficiency of converting cells to a pluripotent state decrease, but "pluripotency" itself also decreases. For example, using fewer reprogramming factors may result in cells that are not fully pluripotent but may only be capable of differentiating into a smaller number of cell types.

[0139] In some embodiments, a single reprogramming factor OCT4 is used. In other embodiments, two reprogramming factors, OCT4 and KLF4, are used. In other embodiments, three reprogramming factors, OCT4, KLF4, and SOX2, are used. In other embodiments, four reprogramming factors, OCT4, KLF4, SOX2, and c-Myc, are used. In other embodiments, five, six, or seven reprogramming factors selected from SOKMNLT, i.e., SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L T antigen, may be used. Generally, these reprogramming factor genes are provided in episomal vectors (such as those known in the art and commercially available ones).

[0140] In some embodiments, the host cells used to transfect one or more reprogramming factors are non-pluripotent stem cells. Generally, as is known in the art, iPSCs are produced from non-pluripotent cells (e.g., blood cells, fibroblasts, etc.) by transiently expressing the reprogramming factors described herein. In some embodiments, the non-pluripotent cells (e.g., fibroblasts) are obtained or isolated from one or more individual subjects or donors before cell reprogramming. In some embodiments, iPSCs are produced from a pool of isolated non-pluripotent stem cells (e.g., fibroblasts) obtained from one or more (e.g., two or more, three or more, four or more, five or more, ten or more, twenty or more, fifty or more, or one hundred or more) different donor subjects. In some embodiments, non-pluripotent cells (such as fibroblasts) are isolated or obtained from multiple different donor subjects (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 50 or more, or 100 or more), pooled together, reprogrammed as iPSCs, and modified according to the provided methods.

[0141] In some embodiments, iPSCs are obtained by transiently transfecting cells derived from a pool of non-pluripotent cells (e.g., fibroblasts) from one or more donor subjects different from the recipient subject (e.g., the patient to whom the cells are administered), such as by transfecting cells with one or more reprogramming factors. The non-pluripotent cells (e.g., fibroblasts) to be induced into iPSCs can be obtained from and pooled together from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or more donor subjects. The non-pluripotent cells (e.g., fibroblasts) can be obtained from and pooled together from 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 50 or more, or 100 or more donor subjects. In some embodiments, non-pluripotent cells (e.g., fibroblasts) are collected from one or more individuals, and in some cases, a pool of non-pluripotent cells (e.g., fibroblasts) is cultured in vitro and transfected with one or more reprogramming factors to induce the generation of iPSCs. In some embodiments, a pool of non-pluripotent cells (e.g., fibroblasts) is modified according to a method provided herein. In some embodiments, the modified iPSCs or pool of modified iPSCs are then subjected to a differentiation process for differentiation into any cell of an organism and tissue.

[0142] PSCs can differentiate into beta cells of organisms and tissues. In one embodiment, modified cells differentiated from iPSCs into beta cells for subsequent administration to a recipient subject are provided herein. Differentiation can be assayed as is known in the art, generally by evaluating the presence of cell-specific markers. As will be understood by those skilled in the art, differentiated modified (e.g., low immunogenic) pluripotent cell derivatives can be transplanted using methods known in the art, depending on both the cell type and the end use of such cells. Exemplary differentiated cell types and methods for obtaining them are described below. In some embodiments, iPSCs can differentiate into beta cells. In some embodiments, iPSCs differentiate into beta islet cells. In some embodiments, host cells (such as non-pluripotent cells (e.g., fibroblasts)) derived from individual donors or a pool of individual donors are isolated or obtained and converted into iPSCs, which are then modified to contain the modifications (e.g., genetic modifications) described herein and subsequently differentiated into the desired cell type.

[0143] In some embodiments, the cells are beta islet cells obtained by differentiating modified iPSCs containing the modifications described herein (e.g., genetic modifications) into beta islet cells. As will be understood by those skilled in the art, the method for differentiation depends on the desired cell type using known techniques. In some embodiments, the cells differentiated into various beta islet cells can be used for subsequent transplantation or engraftment into a subject (e.g., a recipient). In some embodiments, the pancreatic islet cells are derived from the modified pluripotent cells described herein. Useful methods for differentiating pluripotent stem cells into beta islet cells are described, for example, in U.S. Patent No. 9,683,215; U.S. Patent No. 9,157,062; U.S. Patent No. 8,927,280; U.S. Patent Publication No. 2021 / 0207099; Hogrebe et al., “Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cells,” Nat. Biotechnol., 2020, 38:460-470; and Hogrebe et al., “Generation of insulin-producing pancreatic beta cells from multiple human stem cell lines,” Nat. Protoc., 2021, the contents of which are incorporated herein by reference in their entirety.

[0144] In some embodiments, the modified pluripotent cells described herein differentiate into beta-like cells or island organoids for transplantation to address type 1 diabetes mellitus (T1DM). The cell line is a promising approach to addressing T1DM; see, for example, Ellis et al, Nat Rev Gastroenterol Hepatol. 2017 Oct;14(10):612-628 (incorporated herein by reference). Furthermore, Pagliuca et al. (Cell, 2014, 159(2):428-39) reported successful differentiation of hiPSCs into beta cells, the entire report of which is incorporated herein by reference, particularly regarding the methods and reagents outlined therein for the large-scale production of functional human beta cells from human pluripotent stem cells. Furthermore, Vegas et al. have demonstrated the production of human beta cells from human pluripotent stem cells, followed by encapsulation, to avoid host immune rejection; Vegas et al., Nat Med, 2016, 22(3):306-11 (the entire report, in particular the methods and reagents outlined therein for the large-scale production of functional human beta cells from human pluripotent stem cells, is incorporated herein by reference).

[0145] In some embodiments, a method for producing a population of modified islet cells derived from a population of modified pluripotent cells by in vitro differentiation comprises: (a) culturing a population of modified iPSCs in a first culture medium containing one or more factors selected from the group consisting of insulin-like growth factor, transforming growth factor, FGF, EGF, HGF, SHH, VEGF, transforming growth factor-b superfamily, BMP2, BMP7, GSK inhibitor, ALK inhibitor, BMP1 receptor inhibitor, and retinoic acid to produce a population of immature islet cells; and (b) culturing the population of immature islet cells in a second culture medium different from the first culture medium to produce a population of modified islet cells. In some embodiments, the GSK inhibitor is CHIR-99021, its derivatives, or variants thereof. In some examples, the GSK inhibitor is in a concentration range of about 2 mM to about 10 mM. In some embodiments, the ALK inhibitor is SB-431542, its derivatives, or variants thereof. In some examples, the ALK inhibitor is present at concentrations ranging from approximately 1 pM to approximately 10 pM. In some embodiments, the first culture medium and / or the second culture medium do not contain animal serum.

[0146] Differentiation is assayed, as is known in the art, by evaluating the presence of β-cell-related or specific markers, including but not limited to insulin. Differentiation can also be functionally measured, for example, by measuring glucose metabolism. See, in general, Murarō et al., Cell Syst. 2016 Oct 26;3(4):385-394.e3, in particular, the biomarkers outlined therein are incorporated herein by reference in their entirety. Once β-cells are generated, they can be transplanted into the portal vein / liver, reticular, gastrointestinal mucosa, bone marrow, muscle, or subcutaneous capsule (as a cell suspension, as cell clusters, or in a permeable or semipermeable device or gel matrix as discussed herein).

[0147] In some embodiments, islet cells (such as beta islet cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors)) produce insulin in response to an increase in glucose. In various embodiments, islet cells secrete insulin in response to an increase in glucose. In some embodiments, the cells have distinctly different morphologies (such as cobblestone cell morphology and / or a diameter of about 17 pm to about 25 pm).

[0148] Once the manipulated islands are generated, they can be assayed for the retention of their low immunogenicity and / or pluripotency, as described in WO2016183041 and WO2018132783. In some embodiments, low immunogenicity is assayed using several techniques, as illustrated in Figures 13 and 15 of WO2018132783. These techniques include transplantation into allogeneic hosts and monitoring for the proliferation of low immunogenic pluripotent cells that evade the host immune system (e.g., teratomas). In some cases, derivatives of low immunogenic pluripotent cells can be transduced to express luciferase and then tracked using bioluminescence imaging. Similarly, the T cell and / or B cell response of the host animal to such cells is tested to confirm that the cells do not provoke an immune response in the host animal. The T cell response can be assessed by Elispot, ELISA, FACS, PCR, or mass cytometry (CYTOF). B-cell responses or antibody responses are assessed using FACS or Luminex. Additionally or alternatively, cells may be assayed for their ability to evade innate immune responses (e.g., killing by NK cells), as commonly shown in Figures 14 and 15 of WO2018132783.

[0149] In some embodiments, the immunogenicity of cells is evaluated using T cell immunoassays recognized by those skilled in the art (such as T cell proliferation assays, T cell activation assays, and T cell killing assays). In some cases, a T cell proliferation assay includes pre-treating cells with interferon-gamma, co-culturing the cells with labeled T cells, and assaying the presence of a T cell population (or a proliferating T cell population) after a predetermined time. In some cases, a T cell activation assay includes co-culturing T cells with cells outlined herein and determining the expression levels of T cell activation markers in the T cells.

[0150] In vivo assays can be performed to evaluate the immunogenicity of the cells outlined herein. In some embodiments, the viability and immunogenicity of modified iPSCs are determined using allogeneic humanized immunodeficiency mouse models. In some cases, modified iPSCs are transplanted into allogeneic humanized NSG-SGM3 mice and assayed for cell rejection, cell viability, and teratoma formation. In some cases, grafted modified iPSCs or their differentiated cells exhibit long-term survival in the mouse model.

[0151] Additional techniques for determining the immunogenicity of cells, including low immunogenicity, are described, for example, Deuse et al., Nature Biotechnology, 2019, 37, 252-258 and Han et al., Proc Natl Acad Sci USA, 2019, 116(21), 10441-10446, and such disclosure, including figures, figure legends, and method descriptions, is incorporated herein by reference in their entirety.

[0152] Similarly, the retention of pluripotency can be tested in numerous ways. In one embodiment, pluripotency is assayed by the expression of specific pluripotency-specific factors, which are generally described herein and shown in Figure 29 of WO2018132783. Additionally or alternatively, pluripotent cells differentiate into one or more cell types as an indicator of pluripotency.

[0153] Once modified pluripotent stem cells (modified iPSCs) are created, they can be maintained in an undifferentiated state, as is known regarding the maintenance of iPSCs. For example, cells can be cultured on Matrigel using a culture medium that inhibits differentiation and maintains pluripotency. In addition, cells can exist in culture medium under conditions that maintain pluripotency.

[0154] B. Compositions and Formulations In some embodiments, the manipulated beta islands are provided as a pharmaceutical composition for administration to a subject. In some embodiments, the pharmaceutical composition comprises the manipulated islands and a pharmaceutically acceptable carrier.

[0155] Acceptable carriers, pharmaceutical additives, or stabilizers are non-toxic to the recipient at the dosage and concentration used and include buffers (such as phosphates, citrates, and other organic acids); antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens (such as methylparaben or propylparaben); catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than approximately 10 residues) polypeptides; proteins (serum albumin, ze) These include: latin or immunoglobulins; hydrophilic polymers (such as polyvinylpyrrolidone); amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents (such as EDTA); sugars (such as sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (such as sodium); metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants (such as polysorbate (TWEEN®), poloxamer (PLURONICS®), or polyethylene glycol (PEG)). In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable buffer (e.g., neutral buffered saline or phosphate-buffered saline). In some embodiments, the pharmaceutical composition may contain one or more pharmaceutical additives to modify, maintain, or retain, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Those skilled in the art will understand that in some embodiments, a pharmaceutical composition containing cells may differ from a pharmaceutical composition containing proteins.

[0156] In some embodiments, the pharmaceutical composition contains the manipulated islands described herein in an amount effective for treating or inhibiting a beta cell-related disease or disorder (such as a therapeutically effective amount or a prophylactically effective amount). In some embodiments, the pharmaceutical composition contains the manipulated islands described herein in an amount effective for treating or inhibiting a beta cell-related disease or disorder (such as a therapeutically effective amount or a prophylactically effective amount). Therapeutic or prophylactic effectiveness is monitored in some embodiments by periodic evaluation of the subject being treated. Depending on the condition, if administered repeatedly over several days or more, treatment is repeated until suppression of the desired disease symptoms occurs. However, other dosing regimens may be useful and may be determined. The desired dose can be delivered by a single bolus administration of the composition, multiple bolus administrations of the composition, or sequential infusion administrations of the composition.

[0157] In some embodiments, the manipulated islands are administered using standard administration methods, formulations, and / or devices. In some embodiments, the manipulated islands or compositions or groups described herein are administered using standard administration methods, formulations, and / or devices. Formulations and devices (such as syringes and vials) are provided for the storage and administration of the compositions. The manipulated islands may be administered via local injection (including catheter administration), systemic injection, intravenous injection, or parenteral administration. When a therapeutic composition (such as one containing manipulated islands) is administered, the therapeutic composition is generally formulated in injection unit dosage forms (solutions, suspensions, emulsions).

[0158] Formulations include those for intravenous, intraperitoneal, or subcutaneous administration. In some embodiments, one or more immunosuppressants are administered parenterally. The term "parenteral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, one or more immunosuppressants are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0159] In some embodiments, the composition is provided as a sterile liquid preparation (e.g., an isotonic aqueous solution, suspension, emulsion, or dispersion), which in some embodiments may be buffered to a selected pH. Liquid compositions are somewhat more convenient, especially for administration by injection. Liquid compositions may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof. Sterile injection solutions can be prepared by incorporating one or more immunosuppressants into a solvent, such as by mixing them with a suitable carrier, diluent, or pharmaceutical additive (e.g., sterile water, saline, glucose, dextrose, or similar).

[0160] In some embodiments, the pharmaceutical composition can be formulated for administration by any route known to those skilled in the art, including intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, subcutaneous, intratumoral, epidural, nasal, oral, vaginal, rectal, surface, topical, ear, inhalation, oral cavity (e.g., sublingual), and transdermal administration or any other route. In some embodiments, other modes of administration are also intended. In some embodiments, administration is by bolus injection, injection (e.g., intravenous or subcutaneous injection, intraocular injection, periorbital injection, subretinal injection, intravitreous injection, transseptal injection, subscleral injection, choroidal injection, anterior chamber injection, subconjunctival injection, subtenon's capsule injection, retrobulbar injection, peribulbar injection), or delivery near the posterior sclera. In some embodiments, administration is by parenteral administration, intrapulmonary administration, and intranasal administration, and, if topical treatment is desired, by intralesional administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In some embodiments, administration is via the portal vein. In some embodiments, administration is by injection into the intramuscular lumen of the subject's forearm.

[0161] In some embodiments, the composition may be administered continuously, intermittently, or in combination with other biologically active agents in the same composition. In some embodiments, administration may include controlled-release systems (including controlled-release formulations) and device-controlled release (such as via a pump). In some embodiments, administration is oral. In some embodiments, administration is intravenous.

[0162] In some embodiments, pharmaceutically acceptable carriers may include all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retardants, and similar substances suitable for drug administration (Gennaro, 2000, Remington: The science and practice of pharmacy, Lippincott, Williams & Wilkins, Philadelphia, PA). Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as non-volatile oils, may also be used. Auxiliary active compounds may also be incorporated into the composition. The pharmaceutical carrier should be suitable for one or more immunosuppressants (e.g., a solution containing saline, dextrose, or human serum albumin). In some embodiments, a pharmaceutically acceptable carrier or medium for such a composition is any non-toxic aqueous solution capable of maintaining or keeping viable the manipulated islands for a sufficient time to allow administration of live cells. For example, a pharmaceutically acceptable carrier or medium may be physiological saline or buffered physiological saline.

[0163] This specification also provides compositions suitable for cryopreservation of manipulated islands. In some embodiments, the manipulated islands are cryopreserved in a cryopreservation medium. In some embodiments, the cryopreservation medium is a serum-free cryopreservation medium. In some embodiments, the composition containing the manipulated islands or a population of them contains a cryoprotective substance. In some embodiments, the cryoprotective substance is DMSO and / or glycerol, or contains the same. In some embodiments, the cryopreservation medium is 5% to 10% or about 5% to about 10% DMSO(v / v). In some embodiments, the cryopreservation medium is 5% or about 5% DMSO(v / v). In some embodiments, the cryopreservation medium is 6% or about 6% DMSO(v / v). In some embodiments, the cryopreservation medium is 7% or about 7% DMSO(v / v). In some embodiments, the cryopreservation medium is 7.5% or about 7.5% DMSO(v / v). In some embodiments, the cryopreservation medium is 8% or about 8% DMSO(v / v). In some embodiments, the cryopreservation medium is 9% or about 9% DMSO(v / v). In some embodiments, the cryopreservation medium is 10% or about 10% DMSO(v / v). In some embodiments, the cryopreservation medium contains a commercially available cryopreservation solution (CryoStor® CS10). CryoStor® CS10 is a cryopreservation medium containing 10% dimethyl sulfoxide (DMSO). In some embodiments, the composition formulated for cryopreservation can be stored at low temperatures (such as very low temperatures), for example, in a temperature range of -40°C to -150°C, or at about 80°C ± 6.0°C.

[0164] In some embodiments, cryopreserved manipulated islands are prepared for administration by thawing. In some cases, the manipulated islands can be administered to a subject immediately after thawing. In such embodiments, the composition containing the manipulated islands can be used immediately without any further processing. In other cases, after thawing, the manipulated islands are further processed by resuspension on a pharmaceutically acceptable carrier, incubation with an activator or stimulant, or activated, washed, and resuspended in a pharmaceutically acceptable buffer before administration.

[0165] In some embodiments, the composition, including the pharmaceutical composition, is sterile.

[0166] In some embodiments, the pharmaceutical composition comprises an operated island and a pharmaceutically acceptable carrier comprising 31.25% (v / v) Plasma-Lyte A, 31.25% (v / v) 5% dextrose / 0.45% sodium chloride, 10% dextran 40 (LMD) / 5% dextrose, 20% (v / v) 25% human serum albumin (HSA), and 7.5% (v / v) dimethyl sulfoxide (DMSO).

[0167] C. Medication and Administration In some embodiments, the engineered islets can be administered by any route known to those skilled in the art, including intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, subcutaneous, renal capsule, intratumoral, epidural, nasal, oral, intravaginal, intrarectal, topical, local, ear, inhalation, intraoral (e.g., sublingual), and transdermal administration or any route. In some embodiments, other modes of administration are also contemplated. In some embodiments, the administration is by bolus injection, injection (e.g., intravenous or subcutaneous injection, intravitreal injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subtenon injection, retrobulbar injection, peribulbar injection), or delivery near the posterior sclera. In some embodiments, the administration is by parenteral administration, pulmonary administration, and intranasal administration, and when local treatment is desired, by intralesional administration. Parenteral injection includes intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration, or subcutaneous administration. In some embodiments, the administration is via the portal vein. In some embodiments, the administration is by injection into the intramuscular lumen of the subject's forearm. In some embodiments, the administration is by the renal capsule.

[0168] In some embodiments, the engineered islets can be administered at any suitable location in the subject. For example, in some embodiments, the engineered islets are administered to the subject's kidney, forearm, mouth, anus, nose, upper arm, hip joint, thigh, buttock, liver, spleen, muscle, subcutaneous tissue, or white adipose tissue. In some embodiments, the engineered cells are administered to the subject's liver, muscle, or white adipose tissue. In some embodiments, the white adipose tissue is a network.

[0169] In certain embodiments, the engineered islets are administered by intramuscular injection. In some embodiments, the engineered islets are administered to the subject's forearm. In some embodiments, the engineered islets are administered into the intramuscular lumen of the subject's forearm.

[0170] In some embodiments, early islet loss via the immediate blood-mediated inflammatory reaction (IBMIR), which is known to occur after portal vein injection (Bennet et al., Diabetes (1999) 48:1907-1914), is avoided by injection into muscle. Muscle is well vascularized, and islet transplantation into skeletal muscle has been clinically successful (Christoffersson et al, Diabetes (2010) 59:2569-2578; Rafael et al., Am J Transplant (2008) 8:458-462).

[0171] In some aspects, the administration method involves transplanting the engineered islets into a subject. In some aspects, the engineered islets can be transplanted as dispersed cells or as forming clusters. In some embodiments, the engineered islets are administered as a suspension of a population of islet cells. In some embodiments, the engineered islets are engineered tissue grafts comprising a population of engineered islet cells and a matrix. In some embodiments, the engineered islet cells are present in a composition that is administered as a suspension of a population of engineered islet cells.

[0172] The specific amount / dosage regimen of the engineered islets will vary depending on the subject's weight, gender, age, and health status, the formulation, biochemical properties, biological activity, bioavailability, and side effects of the engineered islets, as well as the number and identity of the engineered cells. The dosage for administration can depend on a number of factors including the patient's condition and response to treatment and can be determined by one of ordinary skill in the art.

[0173] In some embodiments, the dose of the operated island is administered in amounts of approximately 1,000 island equivalent units (IEQ) to 1 × 10⁶ IEQ or approximately 1 × 10⁶ IEQ, such as 1,000 IEG or approximately 1,000 IEG to 500,000 IEQ or approximately 500,000 IEQ, 1,000 IEQ or approximately 1,000 IEQ to 250,000 IEQ or approximately 250,000 IEQ, 1,000 IEQ or approximately 1,000 IEQ to 100,000 IEQ or approximately 100,000 IEQ, 1,000 IEQ or approximately 1,000 IEQ to 50,000 IEQ or approximately 50,000 IEQ, 1 000IEQ or approximately 1000IEQ to 25,000IEQ or approximately 25,000IEQ, 1000IEQ or approximately 1000IEQ to 10000IEQ or approximately 10000IEQ, 1000IEQ or approximately 1000IEQ to 5000IEQ or approximately 5000IEQ, 5000IEQ or approximately 5000IEQ to 1 x 10⁶IEQ or approximately 1 x 10⁶IEQ, 5000IEQ or approximately 5000IEQ to 500,000IEQ or approximately 500,000IEQ, 5000IEQ or approximately 5000IEQ to 250,000IEQ or approximately 25 0,000IEQ, 5,000IEQ or approximately 5,000IEQ ~ 100,000IEQ or approximately 100,000IEQ, 5,000IEQ or approximately 5,000IEQ ~ 50,000IEQ or approximately 50,000IEQ, 5,000IEQ or approximately 5,000IEQ ~ 250,000IEQ or approximately 250,000IEQ, 5,000IEQ or approximately 5,000IEQ ~ 10,000IEQ or approximately 10,000IEQ, 10,000IEQ or approximately 10,000IEQ ~ 1 × 10⁶IEQ or approximately 1 × 10⁶IEQ, 10,000IEQ or approximately 10,000IEQ ~500000IEQ or approximately 500000IEQ, 10000IEQ or approximately 10000IEQ~250000IEQ or approximately 250000IEQ, 10000IEQ or approximately 10000IEQ~100000IEQ or approximately 100000IEQ, 10000IEQ or approximately 10000IEQ~50000IEQ or approximately 50000IEQ, 10000IEQ or approximately 10000IEQ~250000IEQ or approximately 250000IEQ, 25000IEQ or approximately 25000IEQ~1×10⁶IEQ or approximately 1×10⁶IEQ,25000IEQ or approximately 25000IEQ~500000IEQ or approximately 500000IEQ, 25000IEQ or approximately 25000IEQ~250000IEQ or approximately 250000IEQ, 25000IEQ or approximately 25000IEQ~100000IEQ or approximately 100000IEQ, 25000IEQ or approximately 25000IEQ~5000 0IEQ or approximately 50000IEQ, 50000IEQ or approximately 50000IEQ~1×10⁶IEQ or approximately 1×10⁶IEQ, 50000IEQ or approximately 50000IEQ~500000IEQ or approximately 500000IEQ, 50000IEQ or approximately 50000IEQ~150000IEQ or approximately 150000IEQ, 50000IEQ or Approximately 50,000 IEQ to 100,000 IEQ or approximately 100,000 IEQ, 100,000 IEQ or approximately 100,000 IEQ to 1 × 10⁶ IEQ or approximately 1 × 10⁶ IEQ, 100,000 IEQ or approximately 100,000 IEQ to 500,000 IEQ or approximately 500,000 IEQ, 100,000 IEQ or approximately 100,000 IEQ to 250,000 IEQ or It is administered in amounts such as approximately 250,000 IEQ, 250,000 IEQ, or approximately 250,000 IEQ to 1 × 10⁶ IEQ, or approximately 1 × 10⁶ IEQ, 250,000 IEQ, or approximately 250,000 IEQ to 500,000 IEQ, or approximately 500,000 IEQ, or 500,000 IEQ, or approximately 500,000 IEQ to 1 × 10⁶ IEQ, or approximately 1 × 10⁶ IEQ. In some embodiments, modified SB-beta cells are administered in amounts of 50,000 IEQ or approximately 50,000 IEQ, 100,000 IEQ or approximately 100,000 IEQ, 200,000 IEQ or approximately 200,000 IEQ, 300,000 IEQ or approximately 300,000 IEQ, 400,000 IEQ or approximately 400,000 IEQ, or 500,000 IEQ or approximately 500,000 IEQ, or any value between any of the aforementioned. IEQ is a standardized estimate of island volume, where 1 IEQ corresponds to the volume of a perfectly spherical island with a diameter of 150 μm (Ricordi et al. Acta Diabetol. Lat. 27, 185-195 (1990)).

[0174] In some embodiments, the dose of the manipulated island administered to the subject is given per kg of the subject's body weight. In some embodiments, the manipulated island is 500 IEQ / kg body weight or approximately 500 IEQ / kg to 10000 IEQ / kg or approximately 10000 IEQ / kg, 500 IEQ / kg or approximately 500 IEQ / kg to 5000 IEQ / kg or approximately 5000 IEQ / kg, 500 IEQ / kg or approximately 500 IEQ / kg to 2500 IEQ / kg or approximately 2500 IEQ / kg, 500 IEQ / kg or approximately 500 IEQ / kg to 1000 IEQ / kg or approximately 1000 IEQ / kg, 1000 IEQ / kg or approximately 1000 IEQ / kg to 10000 IEQ / kg or approximately 10000 IEQ / kg It is administered in doses of g, 1000 IEQ / kg or approximately 1000 IEQ / kg to 5000 IEQ / kg or approximately 5000 IEQ / kg, 1000 IEQ / kg or approximately 1000 IEQ / kg to 2500 IEQ / kg or approximately 2500 IEQ / kg, 2500 IEQ / kg or approximately 2500 IEQ / kg to 10000 IEQ / kg or approximately 10000 IEQ / kg, 2500 IEQ / kg or approximately 2500 IEQ / kg to 5000 IEQ / kg or approximately 5000 IEQ / kg, or 5000 IEQ / kg or approximately 5000 IEQ / kg to 10000 IEQ / kg or approximately 10000 IEQ / kg.

[0175] Depending on the condition being treated, any therapeutically effective amount of the cells described herein may be included in the pharmaceutical composition. Non-limiting examples of cells include the primary islet cells described (e.g., engineered hypoimmunogenic islet cells). In some embodiments, the pharmaceutical composition contains at least about 1 × 10⁻⁶ cells. 7 , 2×10 7 , 3 x 10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1 x 10 8 , 2×10 8 , 3 x 10 8contains cells. In some embodiments, the pharmaceutical composition comprises up to about 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2×10 8 , 3×10 8 cells. In some embodiments, the pharmaceutical composition comprises up to about 1×10 7 cells. In some embodiments, the pharmaceutical composition comprises up to about 3×10 8 cells. In some embodiments, the pharmaceutical composition comprises at least about 1×10 7 to 3×10 7 , 2×10 7 to 4×10 7 , 3×10 7 to 5×10 7 , 4×10 7 to 6×10 7 , 5×10 7 to 7×10 7 , 6×10 7 to 8×10 7 , 7×10 7 to 9×10 7 , 8×10 7 to 1×10 8 , 9×10 7 to 2×10 8 , or 1×10 8 to 3×10 8 cells. In an exemplary embodiment, the pharmaceutical composition comprises about 1×10 7 to about 3×10 8 cells. In some embodiments, the pharmaceutical composition comprises at least about 25×10 6 to at least about 25×10 7 cells. In some embodiments, the pharmaceutical composition comprises at least about 80×10 6 to at least about 80×10 7 cells. In another exemplary embodiment, the pharmaceutical composition comprises about 25×10 6~About 80×10 6 It contains individual cells. In some embodiments, the pharmaceutical composition contains about 25 × 10 6 ~About 80×10 7 Contains individual cells.

[0176] In some embodiments, the pharmaceutical composition is approximately 1.25 × 10 per kg of body weight. 5 ~Approx. 1.2×10 7 It is administered as a single dose of individual manipulated, low immunogenic island cells. In some embodiments, the pharmaceutical composition is approximately 1.25 × 10¹⁶ cells per kg of body weight. 5 ~Approx. 1.25×10 6 , about 1.5×10 5 ~Approx. 1.5×10 6 , about 2.0×10 5 ~Approx. 2.0×10 6 , about 2.5×10 5 ~Approx. 2.5×10 6 , about 3.0×10 5 ~Approx. 3.0×10 6 , about 3.5×10 5 ~Approx. 3.5×10 6 , about 4.0×10 5 ~Approx. 4.0×10 6 , about 4.5×10 5 ~Approx. 4.5×10 6 , about 5.0×10 5 ~Approx. 5.0×10 6 , about 5.5×10 5 ~Approx. 5.5×10 6 , about 6.0×10 5 ~Approx. 6.0×10 6 , about 6.5×10 5 ~Approx. 6.5×10 6 , about 7.0×10 5 ~Approx. 7.0×10 6 , about 7.5×10 5 ~Approx. 7.5×10 6 , about 8.0×10 5 ~Approx. 8.0×10 6 , about 8.5×10 5 ~Approx. 8.5×10 6 , about 9.0×10 5 ~Approx. 9.0×10 6 , about 1.0×10 6 ~Approx. 1.0×107 , or approximately 1.2 × 10 6 ~Approx. 1.2×10 7 It is administered as a single dose per cell. In many embodiments, the dose is approximately 1.25 × 10⁻⁶ 5 ~Approx. 1.2×10 7 The dose is in the range of individual cells / kg of body weight. In many embodiments, the dose is about 1.25 × 10⁻⁶ 5 ~Approx. 1.2×10 7 The dose is in a range higher than individual cells / kg of body weight. In some embodiments, the dose is administered intravenously.

[0177] In some embodiments, the pharmaceutical composition contains island equivalents (IEQ). In some embodiments, the pharmaceutical composition contains at least about 6,500 IEQ, 50,000 IEQ, 100,500 IEQ, 200,000 IEQ, 300,000 IEQ, 400,000 IEQ, 500,000 IEQ, or 600,000 IEQ. In some embodiments, the pharmaceutical composition contains up to about 6,500 IEQ, 50,000 IEQ, 100,500 IEQ, 200,000 IEQ, 300,000 IEQ, 400,000 IEQ, 500,000 IEQ, or 600,000 IEQ. In some embodiments, the pharmaceutical composition contains up to about 6,500 IEQ. In some embodiments, the pharmaceutical composition contains up to about 600,000 IEQ. In some embodiments, the pharmaceutical composition includes at least about 6,500 IEQ, 50,000 IEQ, 100,500 IEQ, 200,000 IEQ, 300,000 IEQ, 400,000 IEQ, 500,000 IEQ, or 600,000 IEQ. In exemplary embodiments, the pharmaceutical composition includes about 6,500 to about 600,000 IEQ.

[0178] In some embodiments, the pharmaceutical composition is administered as a single dose ranging from approximately 80 IEQ / kg to approximately 24,000 IEQ / kg. In some embodiments, the pharmaceutical composition is administered as a single dose of approximately 80 IEQ / kg to approximately 800 IEQ / kg, approximately 100 IEQ / kg to approximately 1,000 IEQ / kg, approximately 200 IEQ / kg to approximately 2,000 IEQ / kg, approximately 300 IEQ / kg to approximately 3,000 IEQ / kg, approximately 400 IEQ / kg to approximately 4,000 IEQ / kg, approximately 500 IEQ / kg to approximately 5,000 IEQ / kg, approximately 1,000 IEQ / kg to approximately 10,000 IEQ / kg, approximately 5,000 IEQ / kg to approximately 15,000 IEQ / kg, approximately 10,000 IEQ / kg to approximately 20,000 IEQ / kg, or approximately 14,000 IEQ / kg to approximately 24,000 IEQ / kg. In many embodiments, the dose is in the range of approximately 80 IEQ / kg to less than approximately 24,000 IEQ / kg. In many embodiments, the dose is in the range of approximately 80 IEQ / kg to more than approximately 24,000 IEQ / kg. In some embodiments, the dose is administered intravenously.

[0179] In some embodiments, the pharmaceutical composition is administered as a single dose of approximately 500 to approximately 1500 islands / clusters. In some embodiments, the pharmaceutical composition is administered as a single dose of approximately 500, 1000, or 1500 islands / clusters.

[0180] D. Subject 1. Beta cell-related disorders The modified cells provided herein can be administered to any suitable subject (e.g., a patient), including, for example, a candidate cell therapy for the treatment of a beta cell-related disease or disorder. Candidate cell therapies include any subject having a beta cell-related disease or disorder who may benefit from the therapeutic effects of the modified beta cells of the subject provided herein and one or more immunosuppressants. In some embodiments, the subject is an allogeneic recipient of the modified beta cells to be administered. In some embodiments, the modified beta cells and one or more immunosuppressants provided are effective for use in allogeneic cell therapy. Subjects who benefit from the therapeutic effects of the modified beta cells of the subject provided herein and one or more immunosuppressants exhibit the disappearance, reduction, or improvement of a beta cell-related disease or disorder. In some embodiments, the subject has a beta cell-related disorder or an increased risk of developing one.

[0181] In some embodiments, beta-cell-related disorders are metabolic disorders. Metabolic disorders can occur when abnormal chemical reactions in the subject's body disrupt metabolic processes (e.g., processes associated with the metabolism or breakdown of energy into sugars and acids or the storage of such energy). In some embodiments, metabolic disorders affect the breakdown of amino acids, carbohydrates, or lipids in the subject's body. In some embodiments, metabolic disorders affect the mitochondria of the subject (e.g., mitochondrial disease). In some embodiments, metabolic disorders manifest when the organ of the subject (e.g., the liver or pancreas) becomes diseased and / or does not function properly. Exemplary metabolic disorders as described herein may include, but are not limited to, any disease or disorder characterized by elevated blood pressure, hyperglycemia, excess body fat around the waist, and abnormal cholesterol or triglyceride levels. In some embodiments, the metabolic disorder is familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabbe disease, maple syrup urine disease, metachromatic leukodystrophy, mitochondrial encephalopathy, lactic acidosis, stroke-like episodes (MELAS), Niemann-Pick disease, phenylketonuria (PKU), porphyria, Tay-Sachs disease, Wilson's disease, type 1 diabetes mellitus, type 2 diabetes mellitus, obesity, hypertension, dyslipidemia, or carbohydrate intolerance. In some embodiments, the metabolic disorder is type 2 diabetes mellitus. In some embodiments, the metabolic disorder is type 1 diabetes mellitus. In some embodiments, the metabolic disorder is type 1 diabetes mellitus.

[0182] In some embodiments, the subject has been diagnosed with a beta cell-related disease or disorder (e.g., type 1 diabetes) prior to the administration of one or more immunosuppressants and / or modified beta cells or compositions (such as any of the compositions comprising immunosuppressants and / or modified beta cells described herein). In some embodiments, the subject has been diagnosed with a beta cell-related disease or disorder during the period from about 1 to about 5 years prior to the administration of one or more immunosuppressants and / or modified beta cells or compositions. In some embodiments, the subject has been diagnosed with a beta cell-related disease or disorder at least about 1 year prior to the administration of one or more immunosuppressants and / or modified beta cells or compositions (such as at least about 2, 3, 4, 5 years prior, or more prior to the administration of one or more immunosuppressants and / or modified beta cells or compositions). In some embodiments, the subject was diagnosed with a beta cell-related disease or disorder less than 5 years prior to the administration of one or more immunosuppressants and / or modified beta cells or compositions (such as less than 4 years, less than 3 years, less than 2 years, less than 1 year, or any of these less than 4 years prior to the administration of one or more immunosuppressants and / or modified beta cells or compositions). In some embodiments, the subject was diagnosed with type 1 diabetes at least 1 year prior to the administration of one or more immunosuppressants and / or modified beta cells or compositions (such as at least 2 years, 3 years, 4 years, 5 years, or any of these less than 2 years prior to the administration of one or more immunosuppressants and / or modified beta cells or compositions). In some embodiments, the subject was diagnosed with type 1 diabetes less than 5 years prior to the administration of one or more immunosuppressants and / or modified beta cells or compositions (such as less than 4 years, less than 3 years, less than 2 years, less than 1 year, or any of these less than 4 years prior to the administration of one or more immunosuppressants and / or modified beta cells or compositions).

[0183] 2. Selection Criteria In some embodiments, a subject meets one or more selection criteria prior to administration of a dose of the manipulated low immunogenicity island. Where used herein, the term “selection criteria” refers to the clinical phenotype of the subject that makes it eligible for application of the methods and uses provided herein.

[0184] In some embodiments, the subjects are young, teenagers, middle-aged, or elderly. In some embodiments, the subjects are young. In some embodiments, the subjects are between approximately 1 month and 18 years old (e.g., approximately 1 month to 1 year, approximately 6 months to 5 years, approximately 2 years to 10 years, or approximately 8 years to 15 years). In some embodiments, the subjects are older than approximately 1 month old (e.g., approximately 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, or older than any of these ages). In some embodiments, the subjects are younger than approximately 18 years old (e.g., younger than approximately 17 years, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 year, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, or any of the younger ages). In some embodiments, the subjects are between approximately 18 and approximately 90 years old (e.g., approximately 18 to approximately 40 years, approximately 20 to approximately 60 years, approximately 50 to approximately 80 years, or approximately 60 to approximately 90 years). In some embodiments, the subjects are older than approximately 18 years old (e.g., older than approximately 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or any older than that). In some embodiments, the subjects are younger than approximately 90 years old (e.g., younger than approximately 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 18, or any younger than that).

[0185] In some embodiments, the subjects to be treated are characterized by one or more of the following: diagnosed before the age of 18; receiving intensive diabetes management; being between 18 and 45 years of age; and weighing less than 80 kg. In some embodiments, the subjects to be treated are diagnosed before the age of 18. In some embodiments, the subjects to be treated are receiving intensive diabetes management. In some embodiments, intensive diabetes management includes self-monitoring of subcutaneous glucose levels by continuous glucose monitoring or intermittent scanning glucose monitoring at least three times per day on average over each week. In some embodiments, intensive diabetes management includes administration of insulin injections or insulin pump therapy at least three times per day. In some embodiments, intensive diabetes management includes self-monitoring of subcutaneous glucose levels by continuous glucose monitoring or intermittent scanning glucose monitoring at least three times per day on average over each week, and administration of insulin injections or insulin pump therapy at least three times per day. In some embodiments, the subjects to be treated are between 18 and 45 years of age. In some embodiments, the subjects to be treated weigh less than 80 kg.

[0186] 3. Exclusion criteria In some embodiments, the subject does not meet any of the exclusion criteria prior to administration of a dose of the manipulated low immunogenic island. The term “exclusion criteria,” as used herein, refers to the clinical phenotype of the subject that makes the subject unsuitable for application of the methods and uses provided herein.

[0187] In some embodiments, subjects are not characterized by having: any history of organ transplantation; any history of malignancy; use of any investigational drug(s) within 4 weeks of administration of a dose of engineered low immunogenic islets; use of any antidiabetic drug other than insulin within 4 weeks of administration of a dose of engineered low immunogenic islets; active infection including tuberculosis, HIV, HBV, and HCV; liver function test values ​​of AST, ALT, GGT, or ALP exceeding the respective reference ranges; serological evidence of infection by HTLVI or HTLVII; pregnancy, lactation, or intention to become pregnant; grade 3 or higher chronic kidney disease (GFR < 60 ml / min as estimated by creatine measurement); history of heart disease or symptoms consistent with heart disease at screening; HLA immunization, MIC A / B immunization; known autoimmune disease other than type 1 diabetes (e.g., Hashimoto's disease); administration of live attenuated vaccine less than 6 months prior to administration of a dose of engineered low immunogenic islets; island antibodies GADA > 2000 IE / mL or IA2A > 4000 IE / mL, or ZnT8 autoantibodies; untreated proliferative diabetic retinopathy; progressive psychosis; progressive substance abuse, drug or alcohol abuse, or poor treatment adherence; and known hypersensitivity to ciprofloxacin, gentamicin, or amphotericin.

[0188] In some embodiments, the subjects have no history of organ transplantation. In some embodiments, the subjects have no history of malignant tumors. In some embodiments, the subjects have not used any investigational drug(s) within four weeks prior to the administration of the manipulated low-immunogenic islet dose. In some embodiments, the subjects have not used any antidiabetic drugs other than insulin within four weeks prior to the administration of the manipulated low-immunogenic islet dose. In some embodiments, the subjects have never had any active infection, including tuberculosis, HIV, HBV, and HCV. In some embodiments, the subjects' liver function test values ​​for AST, ALT, GGT, and ALP are within their respective reference ranges. In some embodiments, the subjects have no serological evidence of infection by HTLV-VI or HTLV-II. In some embodiments, the subjects are neither pregnant, breastfeeding, nor intend to become pregnant. In some embodiments, subjects do not have grade 3 or higher chronic kidney disease (GFR < 60 ml / min as estimated by creatine measurement). In some embodiments, subjects have no history of heart disease or symptoms consistent with heart disease at screening. In some embodiments, subjects do not have HLA immunization or MIC A / B immunization. In some embodiments, subjects do not have any known autoimmune disease other than type 1 diabetes (e.g., Hashimoto's disease). In some embodiments, subjects have not received live attenuated vaccine less than 6 months prior to administration of the engineered low immunogenicity island dose. In some embodiments, subjects do not have island antibody GADA > 2000 IE / mL, island antibody IA2A > 4000 IE / mL, or ZnT8 autoantibodies. In some embodiments, subjects do not have untreated proliferative diabetic retinopathy. In some embodiments, subjects do not have ongoing psychosis. In some embodiments, subjects do not have ongoing substance abuse, drug or alcohol abuse, or poor treatment adherence. In some embodiments, the subjects do not have known hypersensitivity to ciprofloxacin, gentamicin, or amphotericin.

[0189] E. Outcome of the method This specification provides methods related to administering an engineered islet generally containing engineered beta cells to a subject. In some embodiments, the methods provided are useful for treating beta cell-related disorders (e.g., type I diabetes) in a subject, promoting engraftment or survival of beta cells in a subject, and / or restoring glucose metabolism in a subject.

[0190] In some embodiments, the methods provided can improve glucose tolerance in a subject. Glucose tolerance can be measured by any suitable method (such as those described herein (e.g., insulin secretion assays)). In some embodiments, the engineered islets exhibit glucose-stimulated insulin secretion (GSIS). Thus, in some embodiments, the improvement in glucose tolerance is measured in a GSIS perfusion assay. Glucose intolerance is associated with insulin resistance and can cause diabetes (e.g., type 1 diabetes and type II diabetes). Therefore, in some embodiments, methods for treating beta cell-related disorders (e.g., diabetes) are provided, including administering the engineered islets described herein to a subject. In some embodiments, the subject is a diabetic patient. In some embodiments, the subject has type I diabetes. In some embodiments, the subject has type II diabetes. Specifically, in some embodiments, methods for improving glucose tolerance in a subject are provided, the methods including administering the engineered islets described herein to a subject. In some embodiments, the glucose tolerance is improved as compared to the subject's glucose tolerance prior to administration of the engineered islets. In some embodiments, the engineered islets reduce exogenous insulin use in the subject. In some embodiments, the glucose tolerance is improved when measured by HbA1c levels. In some embodiments, the subject is in a fasting state. In some embodiments, the engineered islets improve insulin secretion in the subject. In some embodiments, the insulin secretion is improved as compared to the subject's insulin secretion prior to administration of the engineered islets.

[0191] In some embodiments, the methods disclosed herein further include monitoring patients for insulin independence. In some embodiments, “insulin independence” or “insulin-independent” is achieved in subjects (e.g., islet cell recipients) who can gradually reduce insulin therapy for at least one week and who meet one or more (e.g., all) of the following criteria: (i) fasting capillary glucose levels do not exceed 140 mg / dL (7.8 mmol / L) more than three times per week (based on measuring capillary glucose levels at least seven times over seven days); (ii) postprandial 2-hour capillary glucose levels do not exceed 180 mg / dL (10.0 mmol / L) more than three times per week (based on measuring capillary glucose levels at least 21 times over seven days); and (iii) evidence of endogenous insulin production, defined as fasting or stimulated C-peptide levels >0.5 ng / mL (0.16 pmol / L). In some embodiments, the subject is characterized by one of (i) to (iii). In some embodiments, the subject is characterized by two of (i) to (iii). In some embodiments, the subject is characterized by each of (i) to (iii).

[0192] In some embodiments, subjects are monitored at approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or more, after administration of any of the cells provided herein (e.g., a dose of engineered low immunogenic island cells). In some embodiments, the methods disclosed herein include monitoring subjects for up to 1 year for insulin-independent purposes after administration of any of the cells provided herein (e.g., a dose of engineered low immunogenic island cells).

[0193] In some embodiments, the subjects are insulin-dependent compared to the amount of exogenous insulin required for subjects administered non-immunogenic islands to treat beta-cell dysfunction, or the amount of exogenous insulin required for untreated subjects with beta-cell dysfunction (e.g., the dose of exogenous insulin is reduced by 10% or more (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more)). In some embodiments, the reduction in insulin dependence is achieved over a period of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months or more following administration of any of the cells provided herein (e.g., dose of engineered immunogenic island cells).

[0194] In some embodiments, the target is insulin-independent. In some embodiments, insulin independence is achieved over a period of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months or longer following administration of any of the cells provided herein (e.g., a dose of manipulated low immunogenic island cells).

[0195] In some embodiments, the methods disclosed herein further include monitoring the patient once or more, or continuously throughout the period for graft survival. In some embodiments, the period of graft survival may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years, or more (e.g., about 1 year or more, about 2 years or more, about 5 years or more, about 7 years or more, or about 10 years or more).

[0196] In some embodiments, the method disclosed herein further includes administering one or more additional doses of the manipulated islands to subjects who are not insulin-independent or are insulin-dependent at the end of the monitoring period. In some embodiments, a subject is “insulin-dependent” if the subject (e.g., an islet cell recipient) does not meet the above criteria for insulin independence. In some embodiments, the method disclosed herein further includes administering one or more additional doses of cells to subjects whose C-peptide levels in a serum sample are less than about 0.2, 0.3, 0.4, or 0.5 ng / ml (e.g., about 0.3 ng / ml) at the end of the monitoring period. In some embodiments, subjects whose C-peptide levels in a serum sample are less than about 0.2, 0.3, 0.4, or 0.5 ng / ml (e.g., about 0.3 ng / ml) are not insulin-independent.

[0197] In some embodiments, administration of the provided engineered island cells does not induce an adaptive immune response in the subject. In some embodiments, the adaptive immune response is assessed using ELISPOT. For example, the adaptive immune response may be assessed by measuring the level of IFNg cytokine secretion by CD8+ T cells. In some embodiments, the level of IFNg produced after administration of engineered islands is lower than that of wild-type primary island cells or lower compared to SC-derived island cells from unmodified pluripotent stem cells (e.g., IFNg levels are approximately 400-fold, 300-fold, 200-fold, 100-fold, 50-fold, 25-fold, or 10-fold lower). In some embodiments, the adaptive immune response is assessed using flow cytometry. For example, in some embodiments, the adaptive immune response is assessed by measuring the level of donor-specific antibodies (DSAs), such as IgG or IgM. In some embodiments, engineered islands exhibit lower DSA levels compared to wild-type primary island cells (e.g., DSA levels are approximately 2-fold, 1.5-fold, and 1-fold lower compared to control or wild-type beta cells).

[0198] In some embodiments, the manipulated island cells are less immunogenic and exhibit a reduced or lower immune response compared to island cells not manipulated with the modification. In some embodiments, the immune response to the manipulated cells is reduced or decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% compared to the level of immune response resulting from administration of immunogenic cells (e.g., a population of cells of the same or similar cell type or phenotype but without the modification of the modified cells (e.g., genetic modification)). In some embodiments, the administered manipulated islands do not induce an immune response to the modified cells in the subject.

[0199] In some embodiments, the level of systemic TH1 activation induced in a subject by the administered manipulated island is reduced or decreased. In some cases, the level of systemic TH1 activation induced by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower than the level of systemic TH1 activation produced by the administration of immunogenic cells (e.g., a population of cells of the same or similar cell type or phenotype but without modifications to the modified cells (e.g., genetic modification)). In some embodiments, the administered manipulated island does not induce systemic TH1 activation in a subject.

[0200] In some embodiments, the level of immune activation of peripheral blood mononuclear cells (PBMCs) induced by the administered manipulated islands in a subject is reduced or decreased. In some cases, the level of immune activation of PBMCs induced by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower than the level of immune activation of PBMCs induced by the administration of immunogenic cells (e.g., a population of cells of the same or similar cell type or phenotype but without modifications to the modified cells (e.g., genetic modification)). In some embodiments, the administered manipulated islands do not induce immune activation of PBMCs in a subject.

[0201] In some embodiments, the level of donor-specific IgG antibodies induced in a subject by the administered modified cells is reduced or decreased. In some cases, the level of donor-specific IgG antibodies induced by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower than the level of donor-specific IgG antibodies produced by the administration of immunogenic cells (e.g., a population of cells that are the same or similar cell type or phenotype but do not contain modifications of the modified cells (e.g., genetic modification)). In some embodiments, the population of modified cells administered does not induce donor-specific IgG antibodies in the subject.

[0202] In some embodiments, the levels of IgM and IgG antibody production induced in a subject by the administered manipulated islands are reduced or decreased. In some cases, the levels of IgM and IgG antibody production induced by the cells are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower than the levels of IgM and IgG antibody production produced by the administration of immunogenic cells (e.g., a population of cells of the same or similar cell type or phenotype but without modifications to the modified cells (e.g., genetic modification)). In some embodiments, the administered manipulated islands do not induce IgM and IgG antibody production in a subject.

[0203] In some embodiments, the level of cytotoxic T cell killing induced by the administered manipulated island in a subject is reduced or decreased. In some cases, the level of cytotoxic T cell killing induced by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower than the level of cytotoxic T cell killing produced by the administration of immunogenic cells (e.g., a population of cells of the same or similar cell type or phenotype but without modifications to the modified cells (e.g., genetic modification)). In some embodiments, the administered manipulated island does not induce cytotoxic T cell killing in a subject.

[0204] Upon administration of the manipulated islands described herein, subjects exhibit no systemic immune response or a reduced level of systemic immune response compared to the response to non-hypoimmunogenic cells. In some embodiments, subjects exhibit no adaptive immune response or a reduced level of adaptive immune response compared to the response to non-hypoimmunogenic cells. In some embodiments, subjects exhibit no innate immune response or a reduced level of innate immune response compared to the response to non-hypoimmunogenic cells. In some embodiments, subjects exhibit no T cell response or a reduced level of T cell response compared to the response to non-hypoimmunogenic cells. In some embodiments, subjects exhibit no B cell response or a reduced level of B cell response compared to the response to non-hypoimmunogenic cells.

[0205] In some embodiments, no adverse events occur in subjects upon administration of the manipulated islands described herein. In some embodiments, subjects experience fewer adverse events compared to subjects who do not receive one or more immunosuppressants. In some embodiments, adverse events are evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE) v5.0. Adverse events may include, but are not limited to, hypoglycemic and hyperglycemic thresholds for blood glucose-related risks, muscle soreness during administration of the manipulated islands, local bleeding during administration of the manipulated islands and / or one or more immunosuppressants, and / or cytokine release syndrome.

[0206] In some embodiments, the administered manipulated islands evade the subject's immune system when assessed by PBMC and serum. In some embodiments, the manipulated islands evade the subject's immune system at 0, 2, 4, 8, 12, 18, 26, and 52 weeks after administration of the manipulated islands to the subject. In some embodiments, the administered manipulated islands survive in the subject when assessed by MRI. In some embodiments, the manipulated islands survive for up to 48 hours after administration of the manipulated islands to the subject. In some embodiments, the manipulated islands survive at 2, 4, 6, 8, 12, 26, and 52 weeks after administration of the manipulated islands to the subject. In some embodiments, upon administration of the manipulated islands, the subject exhibits a peak c-peptide level greater than 0.01 nmol / l in response to a mixed food challenge test (MMTT). In some embodiments, the peak c-peptide level is greater than 0.01 nmol / l in response to MMTT at 4, 8, 12, 18, 26, and 52 weeks after administration of the manipulated islands to the subject. In some embodiments, peak c-peptide is measured by area under the curve (AUC). In some embodiments, upon administration of the manipulated island, the subject exhibits a non-fasting c-peptide concentration greater than 0.01 nmol / l. In some embodiments, the non-fasting c-peptide concentration is greater than 0.01 nmol / l at 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 52 weeks after administration of the manipulated island to the subject. In some embodiments, upon administration of the manipulated island, the subject exhibits a decrease in insulin requirements per kilogram of body weight (BW). In some embodiments, the insulin requirements per kilogram of BW decrease at 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 52 weeks after administration of the manipulated island to the subject. In some embodiments, upon administration of the manipulated island, the subject exhibits a decrease in HbA1c. In some embodiments, HbA1c decreases 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 26, and 52 weeks after administration of the manipulated island to the subjects. In some embodiments, upon administration of the manipulated island, the subjects show reduced glucose variability.In some embodiments, glucose fluctuations are reduced at 4, 8, 12, 18, 26, and 52 weeks following administration of the manipulated islands to the subjects. In some embodiments, subjects show a reduction in hypoglycemia at the time of administration of the manipulated islands. In some embodiments, hypoglycemia is reduced at 4, 8, 12, 18, 26, and 52 weeks following administration of the manipulated islands to the subjects. In some embodiments, subjects show a reduction in hyperglycemia at the time of administration of the manipulated islands. In some embodiments, hyperglycemia is reduced at 4, 8, 12, 18, 26, and 52 weeks following administration of the manipulated islands to the subjects.

[0207] II. Methods and administration of beta-cell therapy in combination with immunosuppressants This specification provides methods and uses for combination therapy comprising engineered islands (such as doses of engineered low immunogenic islands) and one or more immunosuppressants.

[0208] A. Immunosuppressants and their regimens In some embodiments of the methods, combinations, kits, and uses provided herein, one or more immunosuppressants are administered to a subject. In some embodiments, the goal of immunosuppression may include promoting the engraftment and / or survival of modified beta cells or compositions (e.g., compositions comprising modified beta cells) in the subject, while simultaneously minimizing drug toxicity, infection, and malignancy in the subject. In some embodiments, one or more immunosuppressants are administered to a subject in combination with compositions comprising modified beta cells for use in methods of treating beta cell-related disorders, including diabetes (e.g., type 1 diabetes). In some embodiments, the provided methods of administering one or more immunosuppressants and compositions comprising modified beta cells are useful for restoring or imparting glucose metabolism to a subject in need.

[0209] 1. Administration In some embodiments, the provided method involves administering a composition comprising one or more immunosuppressants and modified beta cells to a target.

[0210] In some embodiments, the method provided involves administering at least one regimen of one or more immunosuppressants before, after, during, concurrently with, sequentially with, and / or intermittently with, the administration of modified beta cells or a composition. In some embodiments, the method provided involves administering a first dose of one or more immunosuppressants before, after (after), during, concurrently with, sequentially with, or intermittently with, the administration of modified beta cells or a composition. In some embodiments, "concurrently" indicates that the administration of one or more immunosuppressants overlaps with the administration of the modified beta cells or a composition in such a way that the at least one regimen of one or more immunosuppressants overlaps with the administration of the composition containing modified beta, and / or the administration of one or more immunosuppressants takes place at the same time (e.g., on the same day and / or at the same point in time) as the administration of modified beta cells or a composition.

[0211] In some embodiments, the method includes administering one or more immunosuppressants (e.g., one or more regimens of one or more immunosuppressants) before, simultaneously with, and / or after, the administration of modified beta cells or compositions to a subject.

[0212] In some embodiments, administering one or more immunosuppressants involves implementing at least one regimen of one or more immunosuppressants prior to administering modified beta cells or compositions to the subject. In some embodiments, one or more immunosuppressants are administered to the subject only prior to the implementation of the first and / or second regimen of modified beta cells or compositions. In some embodiments, one or more immunosuppressants are administered about 30 seconds to about 10 weeks before administering modified beta cells or compositions to the subject (e.g., about 30 seconds to about 1 hour, about 30 minutes to about 12 hours, about 6 hours to about 1 day, about 10 hours to about 5 days, about 2 days to about 7 days, about 5 days to about 14 days, about 7 days to about 4 weeks, about 2 weeks to about 10 weeks). In some embodiments, one or more immunosuppressants are administered at least about 30 seconds before administration of modified beta cells or compositions to the subject (such as at least about 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more before). In some embodiments, one or more immunosuppressants are administered less than approximately 10 weeks before administration of modified beta cells or compositions to the subject (such as less than 9 weeks, less than 8 weeks, less than 7 weeks, less than 6 weeks, less than 5 weeks, less than 4 weeks, less than 3 weeks, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, less than 1 day, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, less than 5 minutes, less than 1 minute, or less than 30 seconds).In some embodiments, one or more immunosuppressants are administered to the subject approximately 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 5 minutes, 1 minute, or 30 seconds before administration of modified beta cells or composition to the subject. In some embodiments, one or more immunosuppressants are administered to the subject before administration of modified beta cells or composition and are administered continuously throughout the subject's lifetime. In some embodiments, one or more immunosuppressants are administered to the subject before each administration of modified beta cells or composition. In some embodiments, one or more immunosuppressants are administered to the subject before each administration of modified beta cells or compositions and are administered continuously throughout the subject's lifetime.

[0213] In some embodiments, administering one or more immunosuppressants includes administering one or more immunosuppressants on the same day as the administration of modified beta cells or compositions to the subject. In some embodiments, administering one or more immunosuppressants includes administering one or more immunosuppressants concurrently with the administration of modified beta cells or compositions to the subject. In some embodiments, administering one or more immunosuppressants includes administering one or more immunosuppressants on the same day as the administration of modified beta cells or compositions to the subject and continuing administration throughout the subject's lifetime. In some embodiments, administering one or more immunosuppressants includes administering one or more immunosuppressants concurrently with the administration of modified beta cells or compositions to the subject and continuing administration throughout the subject's lifetime. In some embodiments, a first regimen of one or more immunosuppressants is implemented in the subject concurrently with the administration of modified beta cells or compositions to the subject. In some embodiments, a second regimen of one or more immunosuppressants is implemented in the subject concurrently with the administration of modified beta cells or compositions to the subject. In some embodiments, one or more immunosuppressants are administered to the subject on the same day as each administration of modified beta cells or compositions. In some embodiments, one or more immunosuppressants are administered to the subject on the same day as each administration of modified beta cells or the composition and are administered continuously throughout the subject's lifetime. In some embodiments, one or more immunosuppressants are administered to the subject concurrently with each administration of modified beta cells or the composition. In some embodiments, one or more immunosuppressants are administered to the subject concurrently with each administration of modified beta cells or the composition and are administered continuously throughout the subject's lifetime.

[0214] In some embodiments, administering one or more immunosuppressants involves implementing a regimen of one or more immunosuppressants (e.g., at least one regimen) after administering modified beta cells or compositions to a subject. In some embodiments, one or more immunosuppressants are administered to the subject only after implementing a first and / or second regimen of modified beta cells or compositions. In some embodiments, one or more immunosuppressants are administered approximately 30 seconds to approximately 10 weeks after administering modified beta cells or compositions to a subject (e.g., approximately 30 seconds to approximately 1 hour, approximately 30 minutes to approximately 12 hours, approximately 6 hours to approximately 1 day, approximately 10 hours to approximately 5 days, approximately 2 days to approximately 7 days, approximately 5 days to approximately 14 days, approximately 7 days to approximately 4 weeks, approximately 2 weeks to approximately 10 weeks, etc.). In some embodiments, one or more immunosuppressants are administered at least about 30 seconds after administration of the modified beta cells or composition to the subject (such as at least about 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more). In some embodiments, one or more immunosuppressants are administered less than approximately 10 weeks after administration of the modified beta cells or composition to the subject (such as less than 9 weeks, less than 8 weeks, less than 7 weeks, less than 6 weeks, less than 5 weeks, less than 4 weeks, less than 3 weeks, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, less than 1 day, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, less than 5 minutes, less than 1 minute, or less than 30 seconds before administration of the modified beta cells or composition to the subject).In some embodiments, one or more immunosuppressants are administered to the subject approximately 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 5 minutes, 1 minute, or 30 seconds after administration of the modified beta cells or composition to the subject. In some embodiments, one or more immunosuppressants are administered to the subject after administration of the modified beta cells or composition and are administered continuously throughout the subject's lifetime. In some embodiments, one or more immunosuppressants are administered to the subject after each administration of the modified beta cells or composition. In some embodiments, one or more immunosuppressants are administered to the subject after each administration of modified beta cells or compositions and are administered continuously throughout the subject's lifetime.

[0215] In some embodiments, one or more immunosuppressants are administered to a subject at a lower dose compared to the dose of one or more immunosuppressants administered to the subject to reduce the immune rejection of immunogenic cells that do not contain the modified beta cells.

[0216] In some embodiments, one or more immunosuppressants are administered to a subject in a single regimen (e.g., dose). In some embodiments, one or more immunosuppressants are administered to a subject in multiple regimens. In some embodiments, one or more immunosuppressants are administered daily. In some embodiments, one or more immunosuppressants are administered at least once a day. In some embodiments, the total daily dose of one or more immunosuppressants is provided as a single regimen per day. In some embodiments, one or more immunosuppressants are administered as divided regimens.

[0217] In some embodiments, the total daily dose of one or more immunosuppressants is divided into two, three, or four regimens per day. In some embodiments, the total daily dose of one or more immunosuppressants is divided into two regimens per day. In some embodiments, the regimens of one or more immunosuppressants are administered approximately every 12 hours. In some embodiments, the total daily dose of one or more immunosuppressants is divided into three regimens per day. In some embodiments, the total daily dose of one or more immunosuppressants is divided into four regimens per day.

[0218] In some embodiments, one or more immunosuppressants are administered for approximately 3 months, 6 months, 12 months, 24 months, 36 months, 48 ​​months, 60 months, or longer, following administration of modified beta cells or compositions to the subject (e.g., daily). In some embodiments, one or more immunosuppressants are administered for approximately 3 months following administration of modified beta cells or compositions to the subject. In some embodiments, one or more immunosuppressants are administered for approximately 6 months following administration of modified beta cells or compositions to the subject. In some embodiments, one or more immunosuppressants are administered for approximately 9 months following administration of modified beta cells or compositions to the subject. In some embodiments, one or more immunosuppressants are administered for approximately 12 months following administration of modified beta cells or compositions to the subject. In some embodiments, one or more immunosuppressants are administered for approximately 24 months following administration of modified beta cells or compositions to the subject. In some embodiments, one or more immunosuppressants are administered for approximately 48 months following administration of modified beta cells or compositions to the subject. In some embodiments, one or more immunosuppressants are administered to the subject over a period of approximately 60 months following the administration of modified beta cells or compositions.

[0219] In some embodiments, one or more immunosuppressants are administered over the lifetime of the modified beta cells or composition in the subject (e.g., daily). In some embodiments, one or more immunosuppressants are administered over the lifetime of the subject (e.g., daily). In some embodiments, one or more immunosuppressants can be formulated for administration by any route known to those skilled in the art, including intramuscular, intravenous, intradermal, intrafocal, intraperitoneal injection, subcutaneous, intratumoral, epidural, nasal, oral, vaginal, rectal, surface, topical, ear, inhalation, oral cavity (e.g., sublingual), and transdermal administration or any other route. In some embodiments, other modes of administration are also intended. In some embodiments, administration is by bolus injection, injection (e.g., intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, choroidal injection, anterior chamber injection, subconjunctival injection, sub-Tenon's capsule injection, retrobulbar injection, peribulbar injection), or delivery near the posterior sclera. In some embodiments, administration is by parenteral administration, intrapulmonary administration, intranasal administration, and, if local treatment is desired, intralesional administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In some embodiments, administration is via the portal vein. In some embodiments, administration is by injection into the intramuscular lumen of the forearm of the subject.

[0220] In some embodiments, one or more immunosuppressants may be administered at any preferred location in the subject. For example, in some embodiments, one or more immunosuppressants may be administered to the kidney, forearm, mouth, anus, nose, upper arm, hip joint, thigh, buttocks, liver, spleen, muscle, subcutaneous tissue, or white adipose tissue of the subject. In some embodiments, one or more immunosuppressants may be administered to the forearm of the subject. In some embodiments, one or more immunosuppressants may be administered into the intramuscular lumen of the forearm of the subject. In some embodiments, one or more immunosuppressants may be administered to the liver, muscle, or white adipose tissue of the subject. In some embodiments, the white adipose tissue is a reticular formation.

[0221] 4. Exemplary immunosuppressants Most immunosuppressive regimens used clinically consist of a combination of one or more immunosuppressants used according to the selected regimen. Immunosuppressive regimens can be classified as induction, maintenance, or anti-rejection regimens. Induction regimens may provide potent early postoperative immunosuppression (e.g., before, concurrently with, and / or after, administration of modified beta cells or compositions to the subject), while maintenance regimens are used throughout the subject's life to prevent both acute and chronic rejection of modified beta cells or compositions. In some exemplary embodiments, the immunosuppressive regimens provided herein use the highest intensity of immunosuppression (e.g., induction immunosuppression) immediately before, concurrently with, and / or immediately after administration of modified beta cells or compositions to the subject, and gradually decrease in intensity over a period of about one year after administration of modified beta cells or compositions to the subject (e.g., maintenance immunosuppression), because the probability of immunoreactivity and rejection is highest early after administration of modified beta cells or compositions and decreases over time. In such embodiments, a minimum maintenance level of immunosuppression is reached over time that is compatible with preventing rejection while minimizing drug toxicity. According to some embodiments, maintenance immunosuppression may be gradually reduced, and in some cases, discontinued completely.

[0222] This specification provides exemplary immunosuppressants and administration regimens for subjects. It should be understood that the specific immunosuppressants and administration regimens described herein may be modified and optimized depending on the specific subject and / or the state of disease or disorder associated with beta cells. Various immunosuppressive regimens are known in the art, and each of them may be applicable to the methods and uses provided herein. For example, see the following references: Markmann et al. “Phase 3 trial of human islet-after-kidney transplantation in type 1 diabetes.” Am J Transplant. 2021;21(4):1477-1492; Shapiro et al. “Clinical pancreatic islet transplantation.” Nature Reviews. Endocrinology. 2017;13(5):268-277; Hering et al. “Phase 3 trial of transplantation of human islets in type 1 diabetes complicated by severe hypoglycemia.” Diabetes Care. 2016;39(7):1230-1240; Foster et al. “Clinical Islet Transplantation Consortium. Improved health-related quality of life in a phase 3 islet transplantation trial in type 1 diabetes complicated by severe hypoglycemia.” Diabetes Care.2018.Pii:dc171779.Doi:10.2337 / dc17-1779;Korsgren et al. “Current status of clinical islet transplantation.” Transplantation 2005;79:1289-1293;Shapiro et al.“Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.” N.Engl.J.Med.2000;343:230-238; and NIAID. “Islet transplantation in type 1 diabetes” [ClinicalTrials.gov study NCT00434811]. .

[0223] In some embodiments, one or more immunosuppressants are small molecules. In some embodiments, the small molecules are chemical substances. In some embodiments, the small molecules are nucleic acids. In some embodiments, one or more immunosuppressants are biological products. In some embodiments, the biological products are proteins. In some embodiments, the biological products are antibodies. In some embodiments, one or more immunosuppressants are their pharmaceutically acceptable salts and / or alternative forms.

[0224] In some embodiments, one or more immunosuppressants are one or more immunomodulators. In some embodiments, one or more immunomodulators are small molecules. In some embodiments, the small molecules are chemical substances. In some embodiments, the small molecules are nucleic acids. In some embodiments, one or more immunomodulators are biological products. In some embodiments, the biological products are proteins. In some embodiments, the biological products are antibodies. In some embodiments, one or more immunomodulators are their pharmaceutically acceptable salts and / or alternative forms.

[0225] Non-limiting examples of immunosuppressants include calcineurin inhibitors, steroids, alkylating agents, antibiotics, analgesics, anti-inflammatory agents, antihistamines, antivirals, antifungals, anticoagulants, DNA synthesis inhibitors, anticoagulants, blood rheology modifiers, inosine monophosphate dehydrogenase (IMDH) inhibitors, Janus kinase inhibitors, mTOR inhibitors, TNF inhibitors, and anti-CD25 inhibitors. In some embodiments, one or more immunosuppressants are, but are not limited to, antithymocyte globulin (ATG), corticosteroids, prednisone, cortisone, prednisolone methylprednisolone, dexamethasone, betamethasone, hydrocortisone, methotrexate, acetaminophen, diphenhydramine, sirolimus (rapamycin), one or more immunosuppressants (FK-506), mycophenolic acid (MPA), mycophenophenate The composition includes mofetil nolate (MMF), sodium mycophenolate, cyclosporine, etanercept (TNFR-Fc), azathioprine, gold salt, sulfasalazine, antimalarial agents, brequinal, leflunomide, mizoribine, 15-deoxysperguarin, 6-mercaptopurine, cyclophosphamide, OKT3, antithymocyte globulin, thymopentin (thymosin-α), fludarabine, cyclophosphamide, and immunosuppressive antibodies. In the methods and uses provided herein, any preferred combination of immunosuppressants, regimens, and dosages described herein may be used in combination with the composition containing modified beta cells.

[0226] Anti-thymocyte globulin (ATG) In some embodiments, one or more immunosuppressants include anti-thymocyte globulin (ATG). In some embodiments, ATG is administered to a subject (e.g., one or more regimens of ATG are administered to the subject). In some embodiments, ATG is administered to a subject in one or more compositions (e.g., pharmaceutical compositions containing ATG). In some embodiments, ATG is administered to the subject before, concurrently with, and / or after, the administration of modified beta cells or compositions to the subject. In some embodiments, at least one regimen of ATG is administered to the subject before, concurrently with, and / or after, the administration of modified beta cells or compositions to the subject.

[0227] In some embodiments, ATG is administered to the subject before administration of modified beta cells or compositions to the subject. In some embodiments, ATG is administered to the subject only before administration of modified beta cells or compositions to the subject. In some embodiments, at least one regimen of ATG is performed on the subject before administration of modified beta cells or compositions to the subject. In some embodiments, more than one regimen of ATG is performed on the subject before administration of modified beta cells or compositions to the subject. In some embodiments, ATG (e.g., at least one regimen of ATG) is performed about 30 seconds to about 10 weeks before administration of modified beta cells or compositions to the subject (about 30 seconds to about 1 hour before, about 30 minutes to about 12 hours before, about 6 hours to about 1 day before, about 10 hours to about 5 days before, about 2 days to about 7 days before, about 5 days to about 14 days before, about 7 days to about 4 weeks before, about 2 weeks to about 10 weeks before, etc.). In some embodiments, ATG is administered at least about 30 seconds before administration of modified beta cells or compositions to the subject (such as at least about 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more before). In some embodiments, ATG is administered less than approximately 10 weeks before administration of the modified beta cells or composition to the subject (such as less than 9 weeks, less than 8 weeks, less than 7 weeks, less than 6 weeks, less than 5 weeks, less than 4 weeks, less than 3 weeks, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, less than 1 day, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, less than 5 minutes, less than 1 minute, or less than 30 seconds).In some embodiments, ATG is administered approximately 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 5 minutes, 1 minute, or 30 seconds before administration of modified beta cells or composition to the subject. In some embodiments, the first regimen of ATG is administered to the subject approximately 2 days before administration of modified beta cells or composition to the subject. In some embodiments, the first regimen of ATG is administered to the subject approximately 1 day before administration of modified beta cells or composition to the subject. In some embodiments, the first regimen of ATG is administered to the subject about two days before administration of the modified beta cells or composition to the subject, and the second regimen of ATG is administered to the subject about one day before administration of the modified beta cells or composition to the subject.

[0228] In some embodiments, ATG is administered to the subject before administration of modified beta cells or the composition and is administered continuously throughout the subject's lifetime. In some embodiments, ATG is administered to the subject before each administration of modified beta cells or the composition. In some embodiments, ATG is administered to the subject before each administration of modified beta cells or the composition and is administered continuously throughout the subject's lifetime.

[0229] In some embodiments, ATG (e.g., an ATG regimen) is administered to the subject on the same day as the administration of modified beta cells or a composition to the subject. In some embodiments, at least one regimen of ATG is administered to the subject on the same day as the administration of modified beta cells or a composition to the subject. In some embodiments, ATG is administered to the subject simultaneously with the administration of modified beta cells or a composition to the subject. In some embodiments, at least one regimen of ATG is administered to the subject simultaneously with the administration of modified beta cells or a composition to the subject. In some embodiments, ATG is administered to the subject on the same day as the administration of modified beta cells or a composition to the subject and is administered continuously throughout the subject's lifetime. In some embodiments, ATG is administered to the subject simultaneously with the administration of modified beta cells or a composition to the subject and is administered continuously throughout the subject's lifetime. In some embodiments, a first regimen of ATG is administered to the subject simultaneously with the administration of modified beta cells or a composition to the subject. In some embodiments, a second regimen of ATG is administered to the subject simultaneously with the administration of modified beta cells or a composition to the subject. In some embodiments, ATG is administered to the subject on the same day as each administration of modified beta cells or a composition. In some embodiments, ATG is administered to the subject on the same day as each administration of modified beta cells or the composition and is administered continuously throughout the subject's lifetime. In some embodiments, ATG is administered to the subject simultaneously with each administration of modified beta cells or the composition. In some embodiments, ATG is administered to the subject simultaneously with each administration of modified beta cells or the composition and is administered continuously throughout the subject's lifetime.

[0230] In some embodiments, ATG (e.g., an ATG regimen) is administered to the subject after administration of modified beta cells or a composition to the subject. In some embodiments, at least one regimen of ATG is implemented on the subject after administration of modified beta cells or a composition to the subject. In some embodiments, more than one regimen of ATG is implemented on the subject after administration of modified beta cells or a composition to the subject. In some embodiments, ATG is administered to the subject only after implementation of a first and / or second regimen of modified beta cells or a composition to the subject. In some embodiments, ATG is administered about 30 seconds to about 10 weeks after administration of modified beta cells or a composition to the subject (e.g., about 30 seconds to about 1 hour, about 30 minutes to about 12 hours, about 6 hours to about 1 day, about 10 hours to about 5 days, about 2 days to about 7 days, about 5 days to about 14 days, about 7 days to about 4 weeks, about 2 weeks to about 10 weeks, etc.). In some embodiments, ATG is administered at least about 30 seconds after administration of the modified beta cells or composition to the subject (such as at least about 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more after administration of the modified beta cells or composition to the subject). In some embodiments, ATG is administered less than approximately 10 weeks after administration of the modified beta cells or composition to the subject (such as less than 9 weeks, less than 8 weeks, less than 7 weeks, less than 6 weeks, less than 5 weeks, less than 4 weeks, less than 3 weeks, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, less than 1 day, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, less than 5 minutes, less than 1 minute, or less than 30 seconds before administration of the modified beta cells or composition to the subject).In some embodiments, ATG is administered approximately 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 5 minutes, 1 minute, or 30 seconds after administration of the modified beta cells or composition to the subject. In some embodiments, ATG is administered to the subject 48 hours after administration of the modified beta cells or composition. In some embodiments, ATG is administered to the subject after administration of the modified beta cells or composition and is administered continuously throughout the subject's lifetime. In some embodiments, ATG is administered to the subject after each administration of the modified beta cells or composition. In some embodiments, ATG is administered to the subject after each administration of modified beta cells or composition and is administered continuously throughout the subject's lifetime.

[0231] In some embodiments, ATG is administered to the subject before and after the administration of modified beta cells or the composition to the subject. In some embodiments, ATG is administered to the subject before, on the same day as, and after the administration of modified beta cells or the composition to the subject. In some embodiments, ATG is administered to the subject before, simultaneously with, and after the administration of modified beta cells or the composition to the subject. In some embodiments, ATG is administered to the subject i) about two days before, ii) about one day before, iii) on the same day, iv) about one day after, and / or v) about two days before, the administration of a composition containing modified beta cells to the subject.

[0232] In some embodiments, regimens of ATG ranging from approximately 0.05 mg / kg to approximately 4.0 mg / kg are administered to the subject, and / or a total daily dose (such as a regimen of approximately 0.05 mg / kg to approximately 1.0 mg / kg of ATG, approximately 0.1 mg / kg to approximately 2.0 mg / kg, approximately 1.0 mg / kg to approximately 3.0 mg / kg, or approximately 2.0 mg / kg to approximately 4.0 mg / kg) is administered to the subject. In some embodiments, regimens of ATG ranging from approximately 0.1 mg / kg to approximately 2.0 mg / kg are administered to the subject. In some embodiments, the regimen targets ATG concentrations greater than approximately 0.05 mg / kg (such as ATG regimens greater than approximately 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, or any of these). In some embodiments, the regimen is implemented with an ATG of less than approximately 4.0 mg / kg (such as an ATG regimen of less than approximately 3.5 mg / kg, less than 3.0 mg / kg, less than 2.5 mg / kg, less than 2.0 mg / kg, less than 1.5 mg / kg, less than 1.0 mg / kg, less than 0.5 mg / kg, less than 0.4 mg / kg, less than 0.3 mg / kg, less than 0.2 mg / kg, less than 0.1 mg / kg, less than 0.05 mg / kg, or any of these less than the above). In some embodiments, the regimen is implemented with an ATG of approximately 0.5 mg / kg. In some embodiments, the regimen is implemented with an ATG of approximately 0.1 mg / kg. In some embodiments, the regimen is implemented with an ATG of approximately 1.5 mg / kg.

[0233] In some embodiments, a regimen of approximately 0.5 mg / kg of ATG is administered to the subject about two days before administration of the modified beta cells or composition. In some embodiments, a regimen of approximately 1.0 mg / kg of ATG is administered to the subject about one day before administration of the modified beta cells or composition. In some embodiments, a regimen of approximately 1.5 mg / kg of ATG is administered to the subject on the same day as administration of the modified beta cells or composition. In some embodiments, a regimen of approximately 1.5 mg / kg of ATG is administered to the subject one day after administration of the modified beta cells or composition. In some embodiments, a regimen of approximately 1.5 mg / kg of ATG is administered to the subject about two days after administration of the modified beta cells or composition. In some embodiments, i) a regimen of approximately 0.5 mg / kg of ATG is administered to the subject about two days before administration of the modified beta cells or composition to the subject; ii) a regimen of approximately 1.0 mg / kg of ATG is administered to the subject about one day before administration of the modified beta cells or composition to the subject; and iii) a regimen of approximately 1.5 mg / kg of ATG is administered to the subject on the same day as administration of the modified beta cells or composition to the subject, about one day after administration of the modified beta cells or composition to the subject, and about two days after administration of the modified beta cells or composition to the subject. In some embodiments, ATG is administered to the subject at a lower dose.

[0234] a. Steroids In some embodiments, one or more immunosuppressants include steroids (e.g., one or more steroids). Steroids may be used to reduce inflammation in a subject. In some embodiments, the steroids are corticosteroids. In some embodiments, one or more immunosuppressants include prednisone, cortisone, prednisolone-methylprednisolone, dexamethasone, betamethasone, and / or hydrocortisone. In some embodiments, the steroids are administered to a subject (e.g., one or more regimens of one or more steroids are administered to the subject). In some embodiments, one or more steroids are administered to a subject in one or more compositions (e.g., pharmaceutical compositions containing one or more steroids). In some embodiments, one or more steroids are administered to a subject before, concurrently with, and / or after, the administration of modified beta cells or compositions (e.g., compositions containing modified beta cells) to the subject. In some embodiments, at least one regimen of steroids is administered to a subject before, concurrently with, and / or after, the administration of modified beta cells or compositions to the subject.

[0235] In some embodiments, one or more immunosuppressants do not contain steroids. In some embodiments, subjects who have previously received or are currently receiving steroid treatment are not suitable for treatment in any of the methods or uses provided herein.

[0236] In some embodiments, one or more immunosuppressants include methylprednisolone. In some embodiments, methylprednisolone (e.g., a regimen of methylprednisolone) is administered to the subject before, concurrently with, and / or after, the administration of modified beta cells or a composition to the subject. In some embodiments, at least one regimen of methylprednisolone is administered to the subject before, concurrently with, and / or after, the administration of modified beta cells or a composition to the subject.

[0237] In some embodiments, methylprednisolone (e.g., a regimen of methylprednisolone) is administered to the subject prior to administration of modified beta cells or a composition to the subject. In some embodiments, methylprednisolone is administered to the subject only prior to administration of modified beta cells or a composition to the subject. In some embodiments, at least one regimen of methylprednisolone is administered to the subject prior to administration of modified beta cells or a composition to the subject. In some embodiments, one or more regimens of methylprednisolone are administered to the subject prior to administration of modified beta cells or a composition to the subject. In some embodiments, methylprednisolone (e.g., at least one regimen of methylprednisolone) is administered about 30 seconds to about 10 weeks before administration of modified beta cells or compositions to the subject (e.g., about 30 seconds to about 1 hour, about 30 minutes to about 12 hours, about 6 hours to about 1 day, about 10 hours to about 5 days, about 2 days to about 7 days, about 5 days to about 14 days, about 7 days to about 4 weeks, about 2 weeks to about 10 weeks before administration of modified beta cells or compositions to the subject). In some embodiments, methylprednisolone is administered at least about 30 seconds before administration of the modified beta cells or composition to the subject (such as at least about 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more before).In some embodiments, methylprednisolone is administered less than approximately 10 weeks before administration of the modified beta cells or composition to the subject (such as less than 9 weeks, less than 8 weeks, less than 7 weeks, less than 6 weeks, less than 5 weeks, less than 4 weeks, less than 3 weeks, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, less than 1 day, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, less than 5 minutes, less than 1 minute, or less than 30 seconds). In some embodiments, methylprednisolone is administered to the subject approximately 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 5 minutes, 1 minute, or 30 seconds before administration of modified beta cells or composition to the subject. In some embodiments, the methylprednisolone regimen is administered to the subject approximately 2 days before administration of modified beta cells or composition to the subject.

[0238] In some embodiments, a methylprednisolone regimen is administered to the subject before the administration of an ATG regimen to the subject. In some embodiments, the methylprednisolone regimen is administered to the subject only before the administration of an ATG regimen to the subject. In some embodiments, the methylprednisolone regimen is administered to the subject before the administration of a first ATG regimen to the subject. In some embodiments, the methylprednisolone regimen is administered to the subject only before the administration of a first ATG regimen to the subject. In some embodiments, the methylprednisolone regimen is administered to the subject about 30 minutes to about 24 hours before the administration of an ATG regimen to the subject (e.g., a first regimen) (e.g., about 30 minutes to about 5 hours, about 1 hour to about 3 hours, about 4 hours to about 10 hours, or about 8 hours to about 24 hours before the administration of an ATG regimen to the subject). In some embodiments, the methylprednisolone regimen is administered to the subject about one hour before the administration of the ATG regimen to the subject. In some embodiments, the methylprednisolone regimen is administered to the subject about one hour before the administration of the first ATG regimen to the subject. In some embodiments, the methylprednisolone regimen is administered to the subject simultaneously with the administration of the ATG regimen to the subject. In some embodiments, the methylprednisolone regimen is administered to the subject simultaneously with the administration of the first ATG regimen to the subject. In some embodiments, the methylprednisolone regimen is administered to the subject approximately midway through the administration of the ATG regimen (e.g., the first regimen) to the subject. In some embodiments, the methylprednisolone regimen is administered to the subject before the administration of the methylprednisolone regimen, before the administration of ATG to the subject, and before the administration of modified beta cells or compositions to the subject. In some embodiments, both the methylprednisolone regimen and the ATG regimen are administered to the subject prior to administration of the modified beta cells or composition to the subject.

[0239] In some embodiments, regimens of methylprednisolone ranging from approximately 0.05 mg / kg to approximately 4.0 mg / kg are administered to the subject, and / or a total daily dose (such as regimens of approximately 0.05 mg / kg to approximately 1.0 mg / kg of methylprednisolone, approximately 0.1 mg / kg to approximately 2.0 mg / kg, approximately 1.0 mg / kg to approximately 3.0 mg / kg, or approximately 2.0 mg / kg to approximately 4.0 mg / kg) is administered to the subject. In some embodiments, regimens of methylprednisolone ranging from approximately 0.1 mg / kg to approximately 2.0 mg / kg are administered to the subject. In some embodiments, regimens of methylprednisolone at concentrations greater than approximately 0.05 mg / kg (such as regimens of methylprednisolone at concentrations greater than approximately 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, or any of these) are used. In some embodiments, regimens of methylprednisolone at doses less than approximately 4.0 mg / kg are used (for example, regimens of methylprednisolone at doses less than approximately 3.5 mg / kg, less than 3.0 mg / kg, less than 2.5 mg / kg, less than 2.0 mg / kg, less than 1.5 mg / kg, less than 1.0 mg / kg, less than 0.5 mg / kg, less than 0.4 mg / kg, less than 0.3 mg / kg, less than 0.2 mg / kg, less than 0.1 mg / kg, less than 0.05 mg / kg, or any of these). In some embodiments, regimens of methylprednisolone at approximately 1.0 mg / kg are used. In some embodiments, methylprednisolone is administered intravenously to the subject.

[0240] In some embodiments, a regimen of methylprednisolone at approximately 1.0 mg / kg is administered to the subject about 1 hour before the administration of the first regimen of ATG to the subject. In some embodiments, a regimen of methylprednisolone at approximately 1.0 mg / kg is administered to the subject about midway through the administration of the first regimen of ATG to the subject. In some embodiments, a first regimen of ATG at approximately 0.5 mg / kg is administered to the subject about 2 days before the administration of modified beta cells or the composition to the subject. In some embodiments, a regimen of ATG at approximately 1.0 mg / kg is administered to the subject about 1 day before the administration of modified beta cells or the composition to the subject. In some embodiments, a regimen of ATG at approximately 1.5 mg / kg is administered to the subject on the same day as the administration of modified beta cells or the composition to the subject. In some embodiments, a regimen of ATG at approximately 1.5 mg / kg is administered to the subject one day after the administration of modified beta cells or the composition to the subject. In some embodiments, a regimen of approximately 1.5 mg / kg of ATG is administered to subjects approximately two days after administration of the modified beta cells or composition. In some embodiments, i) a regimen of methylprednisolone at approximately 1.0 mg / kg is administered to the subject about 1 hour before the administration of the first regimen of ATG to the subject; ii) a regimen of methylprednisolone at approximately the middle of the administration of the first regimen of ATG to the subject; iii) a regimen of ATG at approximately 0.5 mg / kg is administered to the subject about 2 days before the administration of modified beta cells or composition to the subject; iv) a regimen of ATG at approximately 1.0 mg / kg is administered to the subject about 1 day before the administration of modified beta cells or composition to the subject, and / or v) a regimen of ATG at approximately 1.5 mg / kg is administered to the subject on the same day as the administration of modified beta cells or composition to the subject, about 1 day after the administration of the composition containing modified beta cells to the subject, and about 2 days after the administration of modified beta cells or composition to the subject. In some embodiments, the regimen of methylprednisolone is administered at a lower dose. In some embodiments, the ATG regimen is implemented at lower doses.

[0241] b. Pain relievers In some embodiments, one or more immunosuppressants include analgesics (e.g., one or more analgesics). Analgesics are drugs that can be used to relieve pain. In some embodiments, the analgesics are acetaminophen, opioids, or nonsteroidal anti-inflammatory drugs (NSAIDs). In some embodiments, the analgesics are administered to a subject (e.g., one or more regimens of analgesics are administered to a subject). In some embodiments, the analgesics are administered to a subject in one or more compositions (e.g., pharmaceutical compositions containing analgesics). In some embodiments, the analgesics are administered to a subject before, concurrently with, and / or after, the administration of modified beta cells or compositions (e.g., compositions containing modified beta cells) to the subject. In some embodiments, at least one regimen of analgesics is administered to a subject before, concurrently with, and / or after, the administration of modified beta cells or compositions to the subject.

[0242] In some embodiments, one or more immunosuppressants include acetaminophen. In some embodiments, acetaminophen is administered to the subject before, concurrently with, and / or after, the administration of modified beta cells or compositions to the subject. In some embodiments, at least one regimen of acetaminophen is administered to the subject before, concurrently with, and / or after, the administration of modified beta cells or compositions to the subject.

[0243] In some embodiments, acetaminophen is administered to the subject prior to administration of the modified beta cells or composition to the subject. In some embodiments, acetaminophen is administered to the subject only prior to administration of the modified beta cells or composition to the subject. In some embodiments, at least one regimen of acetaminophen is administered to the subject prior to administration of the modified beta cells or composition to the subject. In some embodiments, more than one regimen of acetaminophen is administered to the subject prior to administration of the modified beta cells or composition to the subject. In some embodiments, acetaminophen (e.g., at least one regimen of acetaminophen) is administered about 30 seconds to about 10 weeks before administration of the modified beta cells or composition to the subject (about 30 seconds to about 1 hour before, about 30 minutes to about 12 hours before, about 6 hours to about 1 day before, about 10 hours to about 5 days before, about 2 days to about 7 days before, about 5 days to about 14 days before, about 7 days to about 4 weeks before, about 2 weeks to about 10 weeks before, etc.). In some embodiments, acetaminophen is administered at least about 30 seconds before administration of the modified beta cells or composition to the subject (such as at least about 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more before).In some embodiments, acetaminophen is administered less than approximately 10 weeks before administration of the modified beta cells or composition to the subject (such as less than 9 weeks, less than 8 weeks, less than 7 weeks, less than 6 weeks, less than 5 weeks, less than 4 weeks, less than 3 weeks, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, less than 1 day, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, less than 5 minutes, less than 1 minute, or less than 30 seconds). In some embodiments, acetaminophen is administered approximately 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 5 minutes, 1 minute, or 30 seconds before administration of modified beta cells or composition to the subject. In some embodiments, the acetaminophen regimen is administered to the subject approximately 2 days before administration of modified beta cells or composition to the subject.

[0244] In some embodiments, an acetaminophen regimen is administered to the subject before the administration of the ATG regimen to the subject. In some embodiments, an acetaminophen regimen is administered to the subject only before the administration of the ATG regimen to the subject. In some embodiments, an acetaminophen regimen is administered to the subject before the administration of the first ATG regimen to the subject. In some embodiments, an acetaminophen regimen is administered to the subject only before the administration of the first ATG regimen to the subject. In some embodiments, the acetaminophen regimen is administered to the subject approximately 30 minutes to 24 hours before the administration of the ATG regimen to the subject (e.g., the first regimen) (e.g., approximately 30 minutes to 5 hours, 1 hour to 3 hours, 4 hours to 10 hours, or 8 hours to 24 hours before the administration of the ATG regimen to the subject). In some embodiments, the acetaminophen regimen is administered to the subject approximately 30 minutes before the administration of the ATG regimen to the subject. In some embodiments, the acetaminophen regimen is administered to the subject about 30 minutes before the administration of the first ATG regimen to the subject. In some embodiments, the acetaminophen regimen is administered to the subject simultaneously with the administration of the ATG regimen to the subject. In some embodiments, the acetaminophen regimen is administered to the subject simultaneously with the administration of the first ATG regimen to the subject. In some embodiments, the acetaminophen regimen is administered to the subject approximately midway through the administration of the ATG regimen (e.g., the first regimen) to the subject. In some embodiments, the acetaminophen regimen is administered to the subject before the administration of the acetaminophen regimen, before the administration of ATG to the subject, and before the administration of modified beta cells or compositions to the subject. In some embodiments, both the acetaminophen regimen and the ATG regimen are administered to the subject before the administration of modified beta cells or compositions to the subject.

[0245] In some embodiments, regimens of acetaminophen ranging from approximately 10 mg to approximately 5,000 mg are administered to the subject, and / or a total daily dose (such as regimens of approximately 10 mg to approximately 100 mg of acetaminophen, approximately 100 mg to approximately 1,000 mg, or approximately 500 mg to approximately 5,000 mg). In some embodiments, regimens of acetaminophen ranging from approximately 100 to approximately 10,000 mg are administered to the subject. In some embodiments, regimens of acetaminophen exceeding approximately 10 mg are administered to the subject (such as regimens of acetaminophen exceeding approximately 20 mg, exceeding 30 mg, exceeding 40 mg, exceeding 50 mg, exceeding 100 mg, exceeding 500 mg, exceeding 1,000 mg, exceeding 2,000 mg, exceeding 3,000 mg, exceeding 4,000 mg, exceeding 5,000 mg, or any of these amounts). In some embodiments, regimens of acetaminophen less than approximately 5,000 mg (such as regimens of acetaminophen less than approximately 4,000 mg, less than 3,000 mg, less than 2,000 mg, less than 1,000 mg, less than 500 mg, less than 100 mg, less than 50 mg, less than 40 mg, less than 30 mg, less than 20 mg, less than 10 mg, or any of these less) are administered to the subject. In some embodiments, regimens of acetaminophen about 650 mg are administered to the subject. In some embodiments, acetaminophen is administered orally or rectally to the subject.

[0246] In some embodiments, a regimen of approximately 650 mg of acetaminophen is administered to the subject about 30 minutes before the administration of the first regimen of ATG to the subject. In some embodiments, a regimen of approximately 650 mg of acetaminophen is administered to the subject approximately midway through the administration of the first regimen of ATG to the subject. In some embodiments, a regimen of approximately 0.5 mg / kg of ATG is administered to the subject about 2 days before the administration of modified beta cells or composition to the subject. In some embodiments, a regimen of approximately 1.0 mg / kg of ATG is administered to the subject about 1 day before the administration of modified beta cells or composition to the subject. In some embodiments, a regimen of approximately 1.5 mg / kg of ATG is administered to the subject on the same day as the administration of modified beta cells or composition to the subject. In some embodiments, a regimen of approximately 1.0 mg / kg of ATG is administered to the subject about 1 day after the administration of modified beta cells or composition to the subject. In some embodiments, a regimen of approximately 1.0 mg / kg of ATG is administered to the subject approximately two days after administration of the modified beta cells or composition to the subject. In some embodiments, i) a regimen of approximately 650 mg of acetaminophen is administered to the subject approximately 30 minutes before administration of the first regimen of ATG to the subject; ii) a regimen of approximately 650 mg of acetaminophen is administered to the subject approximately midway through the administration of the first regimen of ATG to the subject; iii) a regimen of approximately 0.5 mg / kg of ATG is administered to the subject approximately two days before administration of the modified beta cells or composition to the subject; iv) a regimen of approximately 1.0 mg / kg of ATG is administered to the subject approximately one day before administration of the modified beta cells or composition to the subject; and / or v) a regimen of approximately 1.5 mg / kg of ATG is administered to the subject on the same day as administration of the modified beta cells or composition to the subject, approximately one day after administration of the modified beta cells or composition to the subject, and approximately two days after administration of the modified beta cells or composition to the subject. In some embodiments, acetaminophen is administered in a lower dose. In some embodiments, ATG is administered in a lower dose.

[0247] c. Antihistamines In some embodiments, one or more immunosuppressants include antihistamines (e.g., one or more antihistamines). Antihistamines are drugs that can be used to alleviate allergic symptoms (such as runny nose, sneezing, and congestion). In some embodiments, the antihistamines are H1-antihistamines, H2-antihistamines, H3-antihistamines, H4-antihistamines, histidine decarboxylase inhibitors, or mast cell inhibitors. In some embodiments, the antihistamines may be, but are not limited to, diphenhydramine, doxylamine, hydroxyzine, promethazine, phenyltroxamine, orphenadrine, triperenamine, cimetidine, clobenpropit, thioperamide, sodium cromoglycate, or catechin. In some embodiments, the antihistamines are administered to the subject (e.g., one or more regimens of antihistamines are administered to the subject). In some embodiments, the antihistamine is administered to the subject in one or more compositions (e.g., a pharmaceutical composition containing the antihistamine). In some embodiments, the antihistamine is administered to the subject before, simultaneously with, and / or after, the administration of modified beta cells or compositions to the subject. In some embodiments, at least one regimen of antihistamines is administered to the subject before, simultaneously with, and / or after, the administration of modified beta cells or compositions to the subject.

[0248] In some embodiments, one or more immunosuppressants include diphenhydramine. In some embodiments, diphenhydramine (e.g., a regimen of diphenhydramine) is administered to the subject before, concurrently with, and / or after, the administration of modified beta cells or a composition to the subject. In some embodiments, at least one regimen of diphenhydramine is administered to the subject before, concurrently with, and / or after, the administration of modified beta cells or a composition to the subject.

[0249] In some embodiments, diphenhydramine (e.g., a diphenhydramine regimen) is administered to the subject prior to administration of modified beta cells or a composition to the subject. In some embodiments, diphenhydramine is administered to the subject only prior to administration of modified beta cells or a composition to the subject. In some embodiments, at least one diphenhydramine regimen is administered to the subject prior to administration of modified beta cells or a composition to the subject. In some embodiments, one or more diphenhydramine regimens are administered to the subject prior to administration of modified beta cells or a composition to the subject. In some embodiments, diphenhydramine (e.g., at least one regimen of diphenhydramine) is administered about 30 seconds to about 10 weeks before administration of modified beta cells or compositions to the subject (e.g., about 30 seconds to about 1 hour, about 30 minutes to about 12 hours, about 6 hours to about 1 day, about 10 hours to about 5 days, about 2 days to about 7 days, about 5 days to about 14 days, about 7 days to about 4 weeks, about 2 weeks to about 10 weeks before administration of modified beta cells or compositions to the subject). In some embodiments, diphenhydramine is administered at least about 30 seconds before administration of the modified beta cells or composition to the subject (such as at least about 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more before).In some embodiments, diphenhydramine is administered less than approximately 10 weeks before administration of the modified beta cells or composition to the subject (such as less than 9 weeks, less than 8 weeks, less than 7 weeks, less than 6 weeks, less than 5 weeks, less than 4 weeks, less than 3 weeks, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, less than 1 day, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, less than 5 minutes, less than 1 minute, or less than 30 seconds). In some embodiments, diphenhydramine is administered to the subject approximately 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 5 minutes, 1 minute, or 30 seconds before administration of modified beta cells or composition to the subject. In some embodiments, the diphenhydramine regimen is administered to the subject approximately 2 days before administration of modified beta cells or composition to the subject.

[0250] In some embodiments, a diphenhydramine regimen is administered to the subject before the administration of the ATG regimen to the subject. In some embodiments, a diphenhydramine regimen is administered to the subject only before the administration of the ATG regimen to the subject. In some embodiments, a diphenhydramine regimen is administered to the subject before the administration of the first ATG regimen to the subject. In some embodiments, a diphenhydramine regimen is administered to the subject only before the administration of the first ATG regimen to the subject. In some embodiments, the diphenhydramine regimen is administered to the subject approximately 30 minutes to 24 hours before the administration of the ATG regimen to the subject (e.g., the first regimen) (e.g., approximately 30 minutes to 5 hours, 1 hour to 3 hours, 4 hours to 10 hours, or 8 hours to 24 hours before the administration of the ATG regimen to the subject). In some embodiments, the diphenhydramine regimen is administered to the subject approximately 30 minutes before the administration of the ATG regimen to the subject. In some embodiments, the diphenhydramine regimen is administered to the subject approximately 30 minutes before the administration of the first ATG regimen to the subject. In some embodiments, the diphenhydramine regimen is administered to the subject simultaneously with the administration of the ATG regimen to the subject. In some embodiments, the diphenhydramine regimen is administered to the subject simultaneously with the administration of the first ATG regimen to the subject. In some embodiments, the diphenhydramine regimen is administered to the subject approximately midway through the administration of the ATG regimen (e.g., the first regimen) to the subject. In some embodiments, the diphenhydramine regimen is administered to the subject before the administration of the diphenhydramine regimen, before the administration of ATG to the subject, and before the administration of modified beta cells or compositions to the subject. In some embodiments, both the diphenhydramine regimen and the ATG regimen are administered to the subject before the administration of modified beta cells or compositions to the subject.

[0251] In some embodiments, regimens of diphenhydramine ranging from approximately 1 mg to approximately 1,000 mg are administered to the patient, and / or a total daily dose (such as a regimen of approximately 1 mg to approximately 100 mg of diphenhydramine, approximately 50 mg to approximately 500 mg, or approximately 500 mg to approximately 1,000 mg). In some embodiments, regimens of diphenhydramine ranging from approximately 10 to approximately 100 mg are administered to the patient. In some embodiments, regimens of diphenhydramine exceeding approximately 1 mg are administered to the patient (such as a regimen of diphenhydramine exceeding approximately 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 1,000 mg, or any of these amounts). In some embodiments, a diphenhydramine regimen of less than approximately 1,000 mg is administered to the subject (e.g., a diphenhydramine regimen of less than approximately 500 mg, less than 400 mg, less than 300 mg, less than 200 mg, less than 100 mg, less than 50 mg, less than 10 mg, less than 5 mg, less than 4 mg, less than 3 mg, less than 2 mg, less than 1 mg, or any of these less than the target dose). In some embodiments, a diphenhydramine regimen of approximately 50 mg is administered to the subject. In some embodiments, diphenhydramine is administered orally or rectally to the subject.

[0252] In some embodiments, a regimen of approximately 50 mg of diphenhydramine is administered to the subject about 30 minutes before the administration of the first regimen of ATG to the subject. In some embodiments, a regimen of approximately 50 mg of diphenhydramine is administered to the subject approximately midway through the administration of the first regimen of ATG to the subject. In some embodiments, a regimen of approximately 0.5 mg / kg of ATG is administered to the subject about 2 days before the administration of modified beta cells or composition to the subject. In some embodiments, a regimen of approximately 1.0 mg / kg of ATG is administered to the subject about 1 day before the administration of modified beta cells or composition to the subject. In some embodiments, a regimen of approximately 1.5 mg / kg of ATG is administered to the subject on the same day as the administration of modified beta cells or composition to the subject. In some embodiments, a regimen of approximately 1.5 mg / kg of ATG is administered to the subject about 1 day after the administration of modified beta cells or composition to the subject. In some embodiments, a regimen of approximately 1.5 mg / kg of ATG is administered to subjects two days after administration of the modified beta cells or composition. In some embodiments, i) a regimen of approximately 50 mg of diphenhydramine is administered to the subject about 30 minutes before the administration of the first regimen of ATG to the subject; ii) a regimen of approximately 50 mg of diphenhydramine is administered to the subject about midway through the administration of the first regimen of ATG to the subject; iii) a regimen of approximately 0.5 mg / kg of ATG is administered to the subject about 2 days before the administration of modified beta cells or composition to the subject; iv) a regimen of approximately 1.0 mg / kg of ATG is administered to the subject about 1 day before the administration of modified beta cells or composition to the subject; and / or v) a regimen of approximately 1.5 mg / kg of ATG is administered to the subject on the same day as the administration of modified beta cells or composition to the subject, about 1 day after the administration of modified beta cells or composition to the subject, and about 2 days after the administration of modified beta cells or composition to the subject. In some embodiments, diphenhydramine is administered in a lower dose. In some embodiments, ATG is administered in a lower dose.

[0253] d. Anti-inflammatory drugs In some embodiments, one or more immunosuppressants include an anti-inflammatory agent (e.g., one or more anti-inflammatory agents). The anti-inflammatory agent is a drug that can be used to reduce inflammation (redness, swelling, and pain) in a subject. In some embodiments, the anti-inflammatory agent is dexamethasone. In some embodiments, the anti-inflammatory agent is a tumor necrosis factor (TNF) inhibitor. The TNF inhibitor may be infliximab, adalimumab, etanercept (TNFR-Fc), golimumab, and certolizumab, but is not limited to the following. In some embodiments, the TNF inhibitor is etanercept. In some embodiments, the anti-inflammatory agent is administered to the subject (e.g., one or more regimens of anti-inflammatory agents are administered to the subject). In some embodiments, the anti-inflammatory agent is administered to the subject in one or more compositions (e.g., pharmaceutical compositions containing anti-inflammatory agents). In some embodiments, the anti-inflammatory agent is administered to the subject before, concurrently with, and / or after, the administration of modified beta cells or a composition (e.g., a composition containing modified beta cells) to the subject. In some embodiments, at least one regimen of anti-inflammatory agents is administered to the subject before, concurrently with, and / or after, the administration of the modified beta cells or composition to the subject.

[0254] In some embodiments, one or more immunosuppressants include dexamethasone. In some embodiments, dexamethasone (e.g., a regimen of dexamethasone) is administered to the subject before, concurrently with, and / or after, the administration of modified beta cells or compositions to the subject. In some embodiments, at least one regimen of dexamethasone is administered to the subject before, concurrently with, and / or after, the administration of modified beta cells or compositions to the subject.

[0255] In some embodiments, one or more immunosuppressants include etanercept. In some embodiments, etanercept (e.g., a regimen of etanercept) is administered to the subject before, concurrently with, and / or after, the administration of modified beta cells or compositions to the subject. In some embodiments, at least one regimen of etanercept is administered to the subject before, concurrently with, and / or after, the administration of modified beta cells or compositions to the subject.

[0256] In some embodiments, etanercept (e.g., a regimen of etanercept) is administered to the subject prior to administration of modified beta cells or a composition to the subject. In some embodiments, etanercept is administered to the subject only prior to administration of modified beta cells or a composition to the subject. In some embodiments, at least one regimen of etanercept is administered to the subject prior to administration of modified beta cells or a composition to the subject. In some embodiments, one or more regimens of etanercept are administered to the subject prior to administration of modified beta cells or a composition to the subject. In some embodiments, etanercept (e.g., at least one regimen of etanercept) is administered about 30 seconds to about 10 weeks before administration of modified beta cells or compositions to the subject (e.g., about 30 seconds to about 1 hour, about 30 minutes to about 12 hours, about 6 hours to about 1 day, about 10 hours to about 5 days, about 2 days to about 7 days, about 5 days to about 14 days, about 7 days to about 4 weeks, about 2 weeks to about 10 weeks before administration of modified beta cells or compositions to the subject). In some embodiments, etanercept is administered at least about 30 seconds before administration of modified beta cells or compositions to a subject (such as at least about 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more before).In some embodiments, etanercept is administered less than approximately 10 weeks before administration of modified beta cells or compositions to the subject (such as less than 9 weeks, less than 8 weeks, less than 7 weeks, less than 6 weeks, less than 5 weeks, less than 4 weeks, less than 3 weeks, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, less than 1 day, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, less than 5 minutes, less than 1 minute, or less than 30 seconds). In some embodiments, etanercept is administered approximately 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 5 minutes, 1 minute, or 30 seconds before administration of modified beta cells or compositions to a subject.

[0257] In some embodiments, etanercept (e.g., a regimen of etanercept (e.g., a first regimen)) is administered to the subject on the same day as the administration of modified beta cells or a composition to the subject. In some embodiments, at least one regimen of etanercept is administered to the subject on the same day as the administration of modified beta cells or a composition to the subject. In some embodiments, etanercept is administered to the subject simultaneously with the administration of modified beta cells or a composition to the subject. In some embodiments, at least one regimen of etanercept is administered to the subject simultaneously with the administration of modified beta cells or a composition to the subject. In some embodiments, etanercept is administered to the subject on the same day as the administration of modified beta cells or a composition to the subject and is administered continuously throughout the subject's lifetime. In some embodiments, etanercept is administered to the subject simultaneously with the administration of modified beta cells or a composition to the subject and is administered continuously throughout the subject's lifetime. In some embodiments, a first regimen of etanercept is administered to the subject simultaneously with the administration of modified beta cells or a composition to the subject. In some embodiments, a second regimen of etanercept is administered to the subject concurrently with the administration of modified beta cells or the composition. In some embodiments, etanercept is administered to the subject on the same day as each administration of modified beta cells or the composition. In some embodiments, etanercept is administered to the subject on the same day as each administration of modified beta cells or the composition and is administered continuously throughout the subject's lifetime. In some embodiments, etanercept is administered to the subject concurrently with each administration of modified beta cells or the composition. In some embodiments, etanercept is administered to the subject concurrently with each administration of modified beta cells or the composition and is administered continuously throughout the subject's lifetime.

[0258] In some embodiments, etanercept (e.g., a regimen of etanercept) is administered to the subject after administration of modified beta cells or a composition to the subject. In some embodiments, at least one regimen of etanercept is administered to the subject after administration of modified beta cells or a composition to the subject. In some embodiments, one or more regimens of etanercept are administered to the subject after administration of modified beta cells or a composition to the subject. In some embodiments, etanercept is administered to the subject only after administration of a first and / or second regimen of modified beta cells or a composition to the subject. In some embodiments, etanercept is administered approximately 30 seconds to approximately 10 weeks after administration of the modified beta cells or composition to the subject (e.g., approximately 30 seconds to approximately 1 hour, approximately 30 minutes to approximately 12 hours, approximately 6 hours to approximately 1 day, approximately 10 hours to approximately 5 days, approximately 2 days to approximately 7 days, approximately 5 days to approximately 14 days, approximately 7 days to approximately 4 weeks, approximately 2 weeks to approximately 10 weeks). In some embodiments, etanercept is administered at least about 30 seconds after administration of the modified beta cells or composition to the subject (such as at least about 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more after administration of the modified beta cells or composition to the subject).In some embodiments, etanercept is administered less than approximately 10 weeks after administration of modified beta cells or the composition to the subject (such as less than 9 weeks, less than 8 weeks, less than 7 weeks, less than 6 weeks, less than 5 weeks, less than 4 weeks, less than 3 weeks, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, less than 1 day, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, less than 5 minutes, less than 1 minute, or less than 30 seconds before administration of modified beta cells or the composition to the subject). In some embodiments, etanercept is administered to subjects approximately 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 5 minutes, 1 minute, or 30 seconds after administration of modified beta cells or composition to subjects. In some embodiments, etanercept is administered to subjects approximately 3 days after administration of modified beta cells or composition. In some embodiments, etanercept is administered to subjects approximately 7 days after administration of modified beta cells or composition. In some embodiments, etanercept is administered to subjects approximately 10 days after administration of modified beta cells or composition. In some embodiments, etanercept is administered to the subject approximately 3 days, 7 days, and 10 days after administration of the modified beta cells or composition. In some embodiments, etanercept is administered to the subject after administration of the modified beta cells or composition and is administered continuously throughout the subject's lifetime. In some embodiments, etanercept is administered to the subject after each administration of the modified beta cells or composition. In some embodiments, etanercept is administered to the subject after each administration of the modified beta cells or composition and is administered continuously throughout the subject's lifetime.

[0259] In some embodiments, etanercept is administered to the subject on the same day as and thereafter to the administration of modified beta cells or the composition to the subject. In some embodiments, etanercept is administered to the subject simultaneously with and thereafter to the administration of modified beta cells or the composition to the subject. In some embodiments, etanercept is administered to the subject i) on the same day as the administration of the composition containing modified beta cells to the subject, ii) about 3 days prior thereto, iii) about 7 days later thereto, and / or iv) about 10 days later thereto.

[0260] In some embodiments, regimens of etanercept ranging from approximately 1 mg to approximately 1,000 mg are administered to the patient, and / or a total daily dose (such as a regimen of approximately 1 mg to approximately 100 mg of etanercept, approximately 50 mg to approximately 500 mg, or approximately 500 mg to approximately 1,000 mg). In some embodiments, regimens of etanercept ranging from approximately 10 to approximately 100 mg are administered to the patient. In some embodiments, regimens of etanercept exceeding approximately 1 mg are administered to the patient (such as regimens of etanercept exceeding approximately 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 1,000 mg, or any of these). In some embodiments, the regimen is administered with less than approximately 1,000 mg of etanercept (such as regimens of less than approximately 500 mg, less than 400 mg, less than 300 mg, less than 200 mg, less than 100 mg, less than 50 mg, less than 10 mg, less than 5 mg, less than 4 mg, less than 3 mg, less than 2 mg, less than 1 mg, or any of these amounts of etanercept). In some embodiments, the regimen is administered with approximately 50 mg of etanercept. In some embodiments, the regimen is administered with approximately 25 mg of etanercept.

[0261] In some embodiments, a regimen of approximately 50 mg of etanercept is administered to the subject on the same day as the administration of the modified beta cells or composition. In some embodiments, a regimen of approximately 25 mg of etanercept is administered to the subject approximately 3 days after the administration of the modified beta cells or composition. In some embodiments, a regimen of approximately 25 mg of etanercept is administered to the subject approximately 7 days after the administration of the modified beta cells or composition. In some embodiments, a regimen of approximately 25 mg of etanercept is administered to the subject approximately 10 days after the administration of the modified beta cells or composition. In some embodiments, i) a regimen of approximately 50 mg of etanercept is administered to the subject on the same day as the administration of the modified beta cells or composition, and ii) a regimen of approximately 25 mg of etanercept is administered to the subject approximately 3 days, 7 days, and 10 days after the administration of the modified beta cells or composition. In some embodiments, the etanercept regimen is administered at a lower dose.

[0262] In some embodiments, subjects undergo an etanercept regimen and an ATG regimen. In some embodiments, subjects undergo at least one etanercept regimen and at least one ATG regimen. In some embodiments, subjects undergo at least one ATG regimen before, on the same day as, concurrently with, and / or after, at least one etanercept regimen. In some embodiments, subjects undergo at least one ATG regimen before at least one etanercept regimen. In some embodiments, an ATG regimen of approximately 40 mg / kg is administered to subjects daily for four consecutive days. In some embodiments, a first regimen of approximately 25 mg of etanercept is administered to subjects twice a week for two consecutive weeks after an ATG regimen. In some embodiments, an etanercept regimen of approximately 25 mg is administered to subjects once a month for approximately four months after a first regimen of etanercept. In some embodiments, the etanercept regimen is administered at a lower dose. In some embodiments, the ATG regimen is implemented at lower doses.

[0263] In some embodiments, the subject undergoes an etanercept regimen and an IL-1 receptor antagonist regimen. In some embodiments, the subject undergoes at least one etanercept regimen and at least one IL-1 receptor antagonist regimen. In some embodiments, the subject undergoes at least one IL-1 receptor antagonist regimen before, on the same day as, concurrently with, and / or after, at least one etanercept regimen.

[0264] e.mTOR inhibitors In some embodiments, one or more immunosuppressants include a mechanistic target (mTOR) inhibitor of rapamycin (e.g., one or more mTOR inhibitors). An mTOR inhibitor is a drug that inhibits mTOR, a serine / threonine-specific protein kinase belonging to the phosphatidylinositol-3 kinase (PI3K)-related kinase (PIKK) family. In some embodiments, the mTOR inhibitor is rapamycin or an analogue (but not limited to, sirolimus, temsirolimus, everolimus, ridaflorimus, umilolimus, or zotarolimus). In some embodiments, the mTOR inhibitor is sirolimus. In some embodiments, the mTOR inhibitor is administered to a subject (e.g., one or more regimens of mTOR inhibitors are administered to the subject). In some embodiments, the mTOR inhibitor is administered to a subject in one or more compositions (e.g., pharmaceutical compositions containing mTOR inhibitors). In some embodiments, the mTOR inhibitor is administered to a subject before, concurrently with, and / or after, the administration of modified beta cells or compositions to the subject. In some embodiments, at least one regimen of mTOR inhibitors is administered to the subject before, concurrently with, and / or after, the administration of the modified beta cells or composition to the subject.

[0265] In some embodiments, one or more immunosuppressants include sirolimus. In some embodiments, sirolimus (e.g., a sirolimus regimen) is administered to the subject before, concurrently with, and / or after, the administration of modified beta cells or a composition to t...

Claims

1. A method for treating or preventing beta-cell dysfunction in a subject requiring treatment or prevention of beta-cell dysfunction, the method comprising administering a dose of engineered low-immunogenic islands to the subject, The aforementioned dose is administered to the subject via intramuscular injection. The aforementioned dose is A) Approximately 1 × 10 ７ Each cell ~ approximately 3 × 10 ８ individual cells, B) Approximately 1.25 × 10 ５ Cells / kg ~ approximately 1.2 × 10⁻⁶ ７ Cells / kg C) Approximately 6,500 island equivalents (IEQ) to approximately 600,000 IEQ, D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg The dosage is The aforementioned method.

2. A method for reducing exogenous insulin dependence in subjects having or at risk of having beta cell dysfunction, the method comprising administering a dose of engineered low immunogenic islands to the subjects, The aforementioned dose is administered via intramuscular injection. The aforementioned dose is A) Approximately 1 × 10 ７ Each cell ~ approximately 3 × 10 ８ individual cells, B) Approximately 1.25 × 10 ５ Cells / kg ~ approximately 1.2 × 10⁻⁶ ７ Cells / kg C) Approximately 6,500 island equivalents (IEQ) to approximately 600,000 IEQ, D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg This is the dosage, The required amount of exogenous insulin is less than the amount of exogenous insulin required for subjects treated with non-low immunogenic islands, or less than the amount of exogenous insulin required for untreated subjects with the aforementioned beta cell dysfunction. The aforementioned method.

3. A method for stabilizing glucose levels in subjects having or at risk of having beta cell dysfunction, the method comprising administering a dose of engineered low immunogenic islands to the subjects, The aforementioned dose is administered via intramuscular injection. The aforementioned dose is A) Approximately 1 × 10 ７ Each cell ~ approximately 3 × 10 ８ individual cells, B) Approximately 1.25 × 10 ５ Cells / kg ~ approximately 1.2 × 10⁻⁶ ７ Cells / kg C) Approximately 6,500 island equivalents (IEQ) to approximately 600,000 IEQ, D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg This is the dosage, The glucose levels are stabilized compared to subjects who received alternative island therapy or compared to untreated subjects. The aforementioned method.

4. A method for stabilizing / increasing c-peptide levels in subjects having or at risk of having beta cell dysfunction, wherein the method comprises administering a dose of engineered low immunogenic islands to the subjects. The aforementioned dose is administered via intramuscular injection. The aforementioned dose is A) Approximately 1 × 10 ７ Each cell ~ approximately 3 × 10 ８ individual cells, B) Approximately 1.25 × 10 ５ Cells / kg ~ approximately 1.2 × 10⁻⁶ ７ Cells / kg C) Approximately 6,500 island equivalents (IEQ) to approximately 600,000 IEQ, D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg This is the dosage, The c-peptide levels are stabilized or increased compared to subjects who received alternative island therapy or compared to untreated subjects. The aforementioned method.

5. A method for reducing HbA1c levels in subjects having or at risk of having beta cell dysfunction, the method comprising administering a dose of engineered low immunogenic islands to the subjects, The aforementioned dose is administered via intramuscular injection. The aforementioned dose is A) Approximately 1 × 10 ７ Each cell ~ approximately 3 × 10 ８ individual cells, B) Approximately 1.25 × 10 ５ Cells / kg ~ approximately 1.2 × 10⁻⁶ ７ Cells / kg C) Approximately 6,500 island equivalents (IEQ) to approximately 600,000 IEQ, D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg This is the dosage, The aforementioned HbA1c levels are reduced compared to subjects who received alternative island therapy or compared to untreated subjects. The aforementioned method.

6. A method for reducing adverse side effects associated with islet cell therapy in subjects with or at risk of beta cell dysfunction, wherein the method is i) Introducing low immunogenicity modifications to a population of island cells containing beta cells in order to generate manipulated low immunogenic islands, ii) Administering the manipulated low immunogenicity islets to subjects who have or are at risk of having beta cell damage. Includes, The aforementioned dose is administered via intramuscular injection. The aforementioned dose is A) Approximately 1 × 10 ７ Each cell ~ approximately 3 × 10 ８ individual cells, B) Approximately 1.25 × 10 ５ Cells / kg ~ approximately 1.2 × 10⁻⁶ ７ Cells / kg C) Approximately 6,500 island equivalents (IEQ) to approximately 600,000 IEQ, D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg The dosage is The aforementioned method.

7. A method for extending the time-in-range (TIR) ​​in subjects having or at risk of having beta cell dysfunction, the method comprising administering a dose of engineered low immunogenic islands to the subjects, The aforementioned dose is administered via intramuscular injection. The aforementioned dose is A) Approximately 1 × 10 ７ Each cell ~ approximately 3 × 10 ８ individual cells, B) Approximately 1.25 × 10 ５ Cells / kg ~ approximately 1.2 × 10⁻⁶ ７ Cells / kg C) Approximately 6,500 island equivalents (IEQ) to approximately 600,000 IEQ, D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg This is the dosage, The aforementioned TIR is extended compared to subjects who received alternative island therapy or compared to untreated subjects. The aforementioned method.

8. The method according to any one of claims 1 to 7, wherein the method results in a reduction of the amount of other drugs required to treat the beta-cell damage, and optionally the beta-cell damaging agent is insulin.

9. The method according to any one of claims 1 to 8, wherein the subject exhibits a reduction in insulin dependence.

10. The method according to claim 2 or 9, wherein the amount of exogenous insulin is reduced by 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, or more compared to the amount of exogenous insulin required for a subject administered a non-immunogenic island for treating the beta cell damage, or the amount of exogenous insulin required for an untreated subject having the beta cell damage.

11. The method according to any one of claims 2, 9, and 10, characterized in that the subject satisfies one or more of the following criteria: (i) fasting capillary glucose levels not exceeding 140 mg / dL (7.8 mmol / L) more than three times per week (based on measuring capillary glucose levels at least seven times in seven days); (ii) postprandial 2-hour capillary glucose not exceeding 180 mg / dL (10.0 mmol / L) more than three times per week (based on measuring capillary glucose levels at least 21 times in seven days); and (iii) evidence of endogenous insulin production defined as fasting or stimulated C-peptide levels >0.5 ng / mL (0.16 pmol / L).

12. The method according to any one of claims 1 to 6, wherein the subject exhibits insulin independence by the method described above.

13. The method according to claim 11, wherein the subject exhibits insulin independence over a period of more than one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months.

14. The method according to any one of claims 1 to 13, characterized in that the subject satisfies one or more of the following conditions: a) Peak c-peptide > 0.20 nmol / l (as evaluated by mixed food challenge test), b) Non-fasting c-peptide > 0.10 nmol / l (as assessed by mixed food challenge test), c) Daily exogenous insulin requirement < 0.25 U / kg d) Daily exogenous insulin requirement = 0 U / kg e) Reduction in exogenous insulin requirements (per kg of body weight), f) Reduction in HbA1c (per kg of body weight), g) Reduction (stabilization) of glucose fluctuations, h) Reduction in the duration of hypoglycemia and / or hyperglycemia (improvement of normal blood glucose levels), i) Blood glucose management HbA1c ≤ 6.5% (48 mmol / mol), and j) Blood sugar management HbA1c<7.0% (53 mmol / mol).

15. The manipulated low immunogenicity islands (a) (i) inactivating or destroying one or more alleles of one or more major histocompatibility complex (MHC) class I molecules or one or more molecules that modulate the expression of said one or more MHC class I molecules, and / or (ii) one or more MHC class II molecules or one or more molecules that modulate the expression of said one or more MHC class II molecules, and / or (b) Increase the expression of one or more tolerance-causing factors The method according to any one of claims 1 to 14, comprising modifications, wherein the increase in expression is compared to a control or wild-type island without the modifications.

16. The method according to any one of claims 1 to 15, wherein the manipulated low immunogenicity islands comprise manipulated beta islet cells.

17. The method according to claim 16, wherein the manipulated low immunogenicity island further comprises additional manipulated island cells, and optionally, the additional manipulated island cells comprise alpha cells and / or delta cells.

18. The method according to claim 17, wherein the additional manipulated island cells include cells having the same modifications as those of the manipulated beta island cells.

19. The method according to any one of claims 1 to 18, wherein at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the cells in the manipulated low immunogenic island comprises manipulated beta island cells.

20. The method according to any one of claims 1 to 19, wherein the manipulated low immunogenicity island is an island cluster.

21. The method according to any one of claims 1 to 20, wherein the manipulated low immunogenicity islands are manipulated from primary islands.

22. The method according to claim 21, wherein the aforementioned first island is derived from the pancreas.

23. The method according to claim 21 or claim 22, wherein the first island is derived from a human subject or an animal subject, and optionally the first island is derived from a pig, a cow, or a sheep.

24. The method according to either claim 22 or 23, wherein the aforementioned first island is derived from a donor subject not suspected of having beta cell-related disorder.

25. The method according to claim 24, wherein the donor is a corpse.

26. The method according to any one of claims 1 to 25, wherein the manipulated low immunogenicity island is of ABO blood type O.

27. The method according to any one of claims 1 to 26, wherein the manipulated low immunogenicity island is Rh factor negative (Rh-).

28. The method according to any one of claims 1 to 27, wherein the manipulated low immunogenicity islands are differentiated from stem cells.

29. The method according to any one of claims 1 to 28, wherein the beta cell disorder is diabetes mellitus.

30. The method according to any one of claims 1 to 29, wherein the beta cell dysfunction is type 1 diabetes.

31. The method according to any one of claims 1 to 30, wherein the subject to be treated is characterized by one or more of the following: type 1 diabetes for more than 5 years, negative (or <0.01 nmol / l) for C-peptide in response to a mixed meal challenge test (MMTT), positive for an antibody against either GAD or IA2, and HbA １ｃ ≥70 mmol / mol, and exogenous insulin requirement <1 U / kg.

32. The method according to any one of claims 1 to 31, wherein the dose of the manipulated low immunogenic island comprises a pharmaceutically acceptable carrier.

33. The method according to any one of claims 1 to 32, wherein the intramuscular administration is via the intramuscular cavity of the forearm, upper arm, hip joint, thigh, or buttocks.

34. The method according to any one of claims 1 to 33, wherein the dose comprises administering one or more further doses of the low immunogenic engineered cells.

35. After the initial dose (a) The subject does not show a reduction in the amount of other drugs required to treat the beta-cell damage, and, at the discretion of the subject, the beta-cell damaging agent is insulin, and / or (b) The administered low immunogenic manipulated cells are not detectable by imaging. The method according to claim 34, wherein, in the case, one or more further doses of the low immunogenic manipulated cells are administered to the subject.

36. The method according to claim 34, wherein if the subject does not meet one or more of the following criteria after an initial dose, one or more further doses of the low immunogenic engineered cells are administered to the subject: (i) fasting capillary glucose levels not exceeding 140 mg / dL (7.8 mmol / L) more than three times per week (based on measuring capillary glucose levels at least seven times over seven days); (ii) postprandial 2-hour capillary glucose not exceeding 180 mg / dL (10.0 mmol / L) more than three times per week (based on measuring capillary glucose levels at least 21 times over seven days); and (iii) evidence of endogenous insulin production defined as fasting or stimulated C-peptide levels >0.5 ng / mL (0.16 pmol / L).

37. (a) The subject does not achieve insulin independence within a certain period after the initial dose, and / or (b) The subject does not show a reduction in the amount of other drugs required to treat the beta-cell damage within a certain period, and the beta-cell damaging agent is insulin, Optionally, the subject fails to achieve insulin independence for a period of more than 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months; and optionally, the subject fails to achieve insulin independence for a period of 2 weeks. The method according to claim 34, wherein, in the case, one or more further doses of the low immunogenic manipulated cells are administered to the subject.

38. The method according to claim 34, wherein if the subject does not meet one or more of the following criteria after the initial dose, one or more further doses of the low immunogenic manipulated cells are administered to the subject: a) Peak c-peptide > 0.20 nmol / l (as evaluated by mixed food challenge test), b) Non-fasting c-peptide > 0.10 nmol / l (as assessed by mixed food challenge test), c) Daily exogenous insulin requirement < 0.25 U / kg d) Daily exogenous insulin requirement = 0 U / kg e) Reduction in exogenous insulin requirements (per kg of body weight), f) Reduction in HbA1c (per kg of body weight), g) Reduction (stabilization) of glucose fluctuations, h) Reduction in the duration of hypoglycemia and / or hyperglycemia (improvement of normal blood glucose levels), i) Blood glucose management HbA1c ≤ 6.5% (48 mmol / mol), and j) Blood sugar management HbA1c<7.0% (53 mmol / mol).

39. The method according to any one of claims 35 to 38, wherein the number of manipulated hypoimmunogenic islands derived from the initial dose is eliminated or reduced in the subject before administering one or more further doses of the manipulated hypoimmunogenic islands.

40. The method according to claim 39, wherein the number of the manipulated low immunogenic islands is reduced in the subject after administration of an exogenously administered agent to induce targeted death of the manipulated low immunogenic islands, and optionally the exogenously administered agent activates a suicide gene or safety switch in the manipulated cells or recognizes one or more tolerance factors on the surface of the manipulated low immunogenic islands.

41. The method according to any one of claims 1 to 40, wherein the subject is subjected to an immunosuppressive regimen.

42. The method according to claim 41, wherein the immunosuppressive regimen is administered to the subject only before the administration of the dose of the manipulated low immunogenic islets.

43. The method according to claim 41 or 42, wherein the immunosuppressive regimen is administered to the subject only after the administration of the dose of the manipulated low immunogenic islets.

44. The method according to any one of claims 41 to 43, wherein the immunosuppressive regimen comprises one or more immunosuppressants.

45. The method according to claim 44, wherein one or more immunosuppressants comprise small molecules or biological products.

46. The method according to claim 45, wherein the biological product is a protein and / or an antibody.

47. The method according to claim 45, wherein the small molecule is a chemical substance or nucleic acid.

48. The method according to any one of claims 44 to 47, wherein the one or more immunosuppressants are selected from the group consisting of calcineurin inhibitors, steroids, alkylating agents, antibiotics, analgesics, anti-inflammatory agents, antihistamines, antiviral agents, antifungal agents, anticoagulants, DNA synthesis inhibitors, anticoagulants, hemorrhological agents, inosine monophosphate dehydrogenase (IMPDH) inhibitors, Janus kinase inhibitors, mTOR inhibitors, TNF inhibitors, and anti-CD25 inhibitors.

49. The method according to claim 48, wherein the one or more immunosuppressants are selected from the group consisting of antithymocyte globulin (ATG), corticosteroids, prednisone, cortisone, prednisolone-methylprednisolone, dexamethasone, betamethasone, hydrocortisone, methotrexate, acetaminophen, diphenhydramine, sirolimus (rapamycin), tacrolimus (FK-506), mycophenolate (MPA), mycophenolate mofetil (MMF), sodium mycophenolate, cyclosporine, etanercept (TNFR-Fc), azathioprine, gold salt, sulfasalazine, antimalarial agents, brequinal, leflunomide, mizoribine, 15-deoxysperguarin, 6-mercaptopurine, cyclophosphamide, OKT3, antithymocyte globulin, thymopentin (thymosin-α), fludarabine, and immunosuppressive antibodies.

50. The method according to any one of claims 44 to 47, wherein the one or more immunosuppressants comprises antibodies for binding to MHC, CD2, CD3, CD4, CD7, CD28, B7, CD25, CD40, CD45, CD95, IFN-gamma, TNF-alpha, IL-2R-alpha, IL-4, IL-5, IL-6R, IL-6, IGF, IGFR1, IL-7, IL-8, IL-10, CD11-alpha, or CD58, and antibodies for binding to any of their ligands; soluble IL-15R, IL-10, B7 molecules, e.g., B7-1, B7-2, their variants and fragments, ICOS, and OX40; and inhibitors of negative T cell regulators, e.g., antibodies against CTLA-4 or similar agents.

51. The method according to any one of claims 1 to 50, further comprising gradually reducing the administration of one or more immunosuppressants.

52. The method according to any one of claims 15 to 51, wherein the one or more tolerogenic factors are selected from the group consisting of CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, C1 inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc receptor, IL15-RF, H2-M3 (HLA-G), A20 / TNFAIP3, CR1, HLA-F, and MANF.

53. The method according to any one of claims 15 to 52, wherein at least one of the one or more tolerance factors is CD47.

54. The method according to claim 53, wherein the CD47 is an engineered CD47 protein.

55. The manipulated CD47 protein (a) one or more extracellular domains, and (b) One or more membrane tethers Includes, The one or more extracellular domains include a signal regulatory protein alpha (SIRPα) interaction motif, The manipulated protein does not contain one or more full-length CD47 intracellular domains. The method according to claim 54.

56. The method according to claim 55, wherein the SIRPα interaction motif is the CD47 extracellular domain or a part thereof, or comprises the same.

57. The method according to claim 55, wherein the SIRPα interaction motif is a SIRPα antibody or a part thereof, or comprises the same.

58. The manipulated low immunogenicity islands are B2M インデル／インデル CIITA インデル／インデル The method according to any one of claims 1 to 56, having the phenotype CD47tg.

59. The method according to any one of claims 1 to 58, wherein the manipulated low immunogenic islets exhibit one or more functions of wild-type or control beta islet cells, and optionally, the one or more functions are selected from the group consisting of glucose-stimulated insulin secretion (GSIS) in vitro, glucose metabolism, maintenance of fasting blood glucose levels, insulin secretion in response to glucose injection in vivo, and glucose clearance after glucose injection in vivo.

60. The method according to claim 59, wherein the GSIS is a dynamic GSIS including dynamic insulin secretion in phases 1 and 2.

61. The method according to claim 59, wherein the GSIS is a static GSIS, and optionally the static incubation index is greater than or about 1, greater than or about 2, greater than or about 5, greater than or about 10, or greater than or about 20.

62. The method according to any one of claims 1 to 61, wherein the level of insulin secretion from the manipulated low immunogenic islets is at least 20% of the level of insulin secretion observed in primary islets, optionally in corpse islets.

63. The method according to any one of claims 1 to 62, wherein the total insulin content of the manipulated low immunogenic islands is greater than or about 500 μIU insulin per 5,000 cells, greater than or about 1,000 μIU insulin per 5,000 cells, greater than or about 2,000 μIU insulin per 5,000 cells, greater than or about 3,000 μIU insulin per 5,000 cells, or greater than or about 4,000 μIU insulin per 5,000 cells.

64. The method according to any one of claims 1 to 63, wherein the manipulated low immunogenicity islands exhibit functionality for more than two weeks after administration to the subject.

65. The aforementioned dose is approximately 1 x 10 ７ Individual cells ~ approx. 3 x 10 ８ Individual cells, approximately 25 x 10 ６ Individual cells ~ approx. 80 x 10 ７ Individual cells, approximately 25 x 10 ６ Individual cells ~ approx. 25 x 10 ７ Individual cells, approximately 80 x 10 ６ Individual cells ~ approx. 80 x 10 ７ Individual cells, approximately 25 x 10 ６ Individual cells ~ approx. 80 x 10 ６ Individual cells, or approximately 1.25 × 10⁻⁶ ５ Individual cells / kg ~ approx. 1.2 x 10 ７ The method according to any one of claims 1 to 64, selected from individual cells / kg.

66. The method according to any one of claims 1 to 65, wherein the dose is selected from about 6,500 island equivalents (IEQ) to about 600,000 IEQ, or about 80 IEQ / kg to about 24,000 IEQ / kg.

67. The method according to any one of claims 1 to 66, characterized in that the subject satisfies one or more of the following conditions: a) Immunodeficiency of manipulated low immunogenicity islands when evaluated in whole-body PBMCs and serum, b) The peak c-peptide in response to the mixed meal challenge test (MMTT) is greater than 0.01 nmol / l. c) The c-peptide concentration in the non-fasting state is greater than 0.01 nmol / l. d) Survival of manipulated hypoimmunogenic islets as assessed by MRI, e) Insulin requirement / kg decrease in BW, f) A decrease in HbA1c, and g) Reduction of glucose fluctuations, hypoglycemia, and hyperglycemia.

68. The method according to claim 67, wherein the manipulated low immunogenicity islands exhibit immune evasion at 0, 2, 4, 8, 12, 18, 26, and 52 weeks after administration to the subject.

69. The method according to claim 67, wherein the manipulated low immunogenicity islands are viable 2, 4, 6, 8, 12, 26, and 52 weeks after administration to the subject.

70. The method according to claim 67, wherein the insulin requirement / kg body weight decreases at 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 52 weeks after administration of the manipulated low immunogenic island to the subject.

71. The method according to claim 67, wherein the HbA1c decreases at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 26, and 52 weeks after administration of the manipulated low immunogenicity island to the subject.

72. The method according to claim 67, wherein glucose fluctuations, hypoglycemia, and hyperglycemia are reduced at 4, 8, 12, 18, 26, and 52 weeks after administration of the manipulated low immunogenic islands to the subject.

73. The method according to any one of claims 1 to 72, wherein the subject is not characterized by having the following: any history of organ transplantation; any history of malignant tumor; use of any investigational drug(s) within four weeks of administration of the dose of the manipulated low immunogenic islets; use of any antidiabetic drug other than insulin within four weeks of administration of the dose of the manipulated low immunogenic islets; active infection including tuberculosis, HIV, HBV, and HCV; liver function test values ​​of AST, ALT, GGT, or ALP exceeding the respective reference ranges; serological evidence of infection by HTLVI or HTLVII; pregnancy, lactation, or intention to become pregnant; grade 3 or higher chronic kidney disease (GFR < 60 ml / min as estimated by creatine measurement); history of heart disease or symptoms consistent with heart disease at screening; HLA immunization, MIC A / B immunization; known autoimmune diseases other than type 1 diabetes (e.g., Hashimoto's disease); administration of live attenuated vaccine less than 6 months prior to administration of the manipulated low immunogenic island dose; island antibody GADA > 2000 IE / mL or IA2A > 4000 IE / mL, or ZnT8 autoantibody; untreated proliferative diabetic retinopathy; progressive psychosis; progressive substance abuse, drug or alcohol abuse, or poor treatment adherence; and known hypersensitivity to ciprofloxacin, gentamicin, or amphotericin.

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