Methods of administering and administering engineered islet cells

Abstract

Provided herein are methods of administering engineered islet cells, including functionally modified beta cells containing one or more modifications (such as genetic modifications). In some embodiments, the engineered pancreatic islets are low immunogen 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, while increasing the expression of one or more tolerogenic factors, such as CD47. In some embodiments, the subject has a beta cell related condition, such as diabetes (e.g., type I diabetes).

Description

[0001] Cross-references to related applications This application claims priority to the following provisional patent application: filed May 3, 2023, entitled "Methods of Dosing and Administration of Engineered Islet Cells". The contents of the following provisional patent applications are incorporated herein by reference in their entirety: U.S. Provisional Patent Application No. 63 / 463,885 entitled “Method for Administration and Delivery of Engineered Islet Cells”, filed May 22, 2023; U.S. Provisional Patent Application No. 63 / 468,217 entitled “Method for Administration and Delivery of Engineered Islet Cells”, filed September 6, 2023; U.S. Provisional Patent Application No. 63 / 580,934 entitled “Method for Administration and Delivery of Engineered Islet Cells”, filed October 27, 2023; U.S. Provisional Patent Application No. 63 / 593,944 entitled “Method for Administration and Delivery of Engineered Islet Cells”, filed November 20, 2023; and U.S. Provisional Patent Application No. 63 / 601,142 entitled “Method for Administration and Delivery of Engineered Islet Cells”, filed February 7, 2024.

[0002] The sequence list is incorporated by reference. This application is submitted together with an electronic sequence list. The sequence list is provided as a document named 186152009340SeqList.xml, created on May 2, 2024, and is 99,122 bytes in size. The information in the electronic sequence list is incorporated herein by reference in its entirety. Technical Field

[0003] In some aspects, this disclosure relates to a method of administering engineered islet cells, the cells comprising functionally modified β cells containing one or more modifications, such as genetic modifications. In some embodiments, the engineered islets 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, while simultaneously increasing the expression of one or more tolerogenic factors (such as CD47). In some embodiments, the subject suffers from a β-cell-related condition, such as diabetes (e.g., type 1 diabetes). Summary of the Invention

[0004] In some embodiments, this document provides a method for treating or preventing β-cell disease in a subject of need, the method comprising administering to the subject a dose of engineered, low-immunogenic islets, wherein the dose is administered to the subject via intramuscular injection, and wherein the dose is: A) approximately 1 × 10⁻⁶7 One cell to approximately 3 × 10 8 A) Approximately 1.25 × 10⁻⁶ cells; B) Approximately 1.25 × 10⁻⁶ cells. 5 Cells / kg to approximately 1.2 × 10⁻⁶ 7 C) Approximately 6,500 islet equivalents (IEQ) to approximately 600,000 IEQ; or D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg.

[0005] In some implementations, this document provides a method for reducing exogenous insulin dependence in subjects who have or are at risk of developing β-cell disorders, the method comprising administering to the subject a dose of engineered, low-immunogenic islets, wherein the dose is administered via intramuscular injection, wherein the dose is: A) approximately 1 × 10⁻⁶ 7 One cell to approximately 3 × 10 8 A) Approximately 1.25 × 10⁻⁶ cells; B) Approximately 1.25 × 10⁻⁶ cells. 5 Cells / kg to approximately 1.2 × 10⁻⁶ 7 C) approximately 6,500 islet equivalents (IEQ) to approximately 600,000 IEQ; or D) approximately 80 IEQ / kg to approximately 24,000 IEQ / kg, wherein the amount of exogenous insulin required is less than that required by subjects treated with non-hypoimmunogenic islets or less than that required by untreated subjects with β-cell disease.

[0006] In some embodiments, this document provides a method for promoting insulin independence in subjects who have or are at risk of having β-cell disorders, the method comprising administering to the subject a dose of engineered, low-immunogenic islets, wherein the dose is administered via intramuscular injection, and wherein the dose is: A) approximately 1 × 10⁻⁶ 7 One cell to approximately 3 × 10 8 A) Approximately 1.25 × 10⁻⁶ cells; B) Approximately 1.25 × 10⁻⁶ cells. 5 Cells / kg to approximately 1.2 × 10⁻⁶ 7 C) Approximately 6,500 islet equivalents (IEQ) to approximately 600,000 IEQ; or D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg.

[0007] In some embodiments, this document provides a method for improving graft function in subjects who have or are at risk of having β-cell disease, the method comprising administering to the subject a dose of engineered, low-immunogenic islets, wherein the dose is administered via intramuscular injection, and wherein the dose is: A) approximately 1 × 10⁻⁶ 7 One cell to approximately 3 × 10 8 A) Approximately 1.25 × 10⁻⁶ cells; B) Approximately 1.25 × 10⁻⁶ cells. 5Cells / kg to approximately 1.2 × 10⁻⁶ 7 C) Approximately 6,500 islet equivalents (IEQ) to approximately 600,000 IEQ; or D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg.

[0008] In some embodiments, this document provides a method for enhancing colonization in subjects who have or are at risk of developing β-cell disorders, the method comprising administering to the subject a dose of engineered, low-immunogenic islets, wherein the dose is administered via intramuscular injection, and wherein the dose is: A) approximately 1 × 10⁻⁶ 7 One cell to approximately 3 × 10 8 A) Approximately 1.25 × 10⁻⁶ cells; B) Approximately 1.25 × 10⁻⁶ cells. 5 Cells / kg to approximately 1.2 × 10⁻⁶ 7 D) Approximately 6,500 islet equivalents (IEQ) to approximately 600,000 islet equivalents (IEQ); D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg.

[0009] In some embodiments, this document provides a method for stabilizing blood glucose levels in a subject who has or is at risk of developing β-cell disorders, the method comprising administering to the subject a dose of engineered, low-immunogenic islets, wherein the dose is administered via intramuscular injection, wherein the dose is: A) approximately 1 × 10⁻⁶ 7 One cell to approximately 3 × 10 8 A) Approximately 1.25 × 10⁻⁶ cells; B) Approximately 1.25 × 10⁻⁶ cells. 5 Cells / kg to approximately 1.2 × 10⁻⁶ 7 C) approximately 6,500 islet equivalents (IEQ) to approximately 600,000 IEQ; or D) approximately 80 IEQ / kg to approximately 24,000 IEQ / kg, wherein the blood glucose level is stable compared to subjects receiving alternative islet therapy or compared to untreated subjects.

[0010] In some embodiments, this document provides a method for stabilizing / increasing C-peptide levels in a subject who has or is at risk of having β-cell disease, the method comprising administering to the subject a dose of engineered, low-immunogenic islets, wherein the dose is administered via intramuscular injection, and wherein the dose is: A) approximately 1 × 10⁷ cells to approximately 3 × 10⁷ cells. 8 A) Approximately 1.25 × 10⁻⁶ cells; B) Approximately 1.25 × 10⁻⁶ cells. 5 Cells / kg to approximately 1.2 × 10⁻⁶ 7C) approximately 6,500 islet equivalents (IEQ) to approximately 600,000 IEQ; or D) approximately 80 IEQ / kg to approximately 24,000 IEQ / kg, wherein the C-peptide level is stable or increased compared to subjects receiving alternative islet therapy or compared to untreated subjects.

[0011] In some embodiments, this document provides a method for reducing HbA1c levels in subjects who have or are at risk of having β-cell disorders, the method comprising administering to the subject a dose of engineered, low-immunogenic islets, wherein the dose is administered via intramuscular injection, and wherein the dose is: A) approximately 1 × 10⁻⁶ 7 One cell to approximately 3 × 10 8 A) Approximately 1.25 × 10⁻⁶ cells; B) Approximately 1.25 × 10⁻⁶ cells. 5 Cells / kg to approximately 1.2 × 10⁻⁶ 7 (A) approximately 6,500 islet equivalents (IEQ) to approximately 600,000 IEQ; or (B) approximately 80 IEQ / kg to approximately 24,000 IEQ / kg, wherein the HbA1c level was reduced compared to subjects receiving alternative islet therapy or compared to untreated subjects.

[0012] In some embodiments, this document provides a method for reducing adverse side effects associated with islet cell therapy in subjects who have or are at risk of developing β-cell disease, the method comprising: i) introducing a low-immunogenic modification into a population of islet cells containing β-cells to generate engineered low-immunogenic islets; and ii) administering a dose of the engineered low-immunogenic islets to a subject who has or is at risk of developing β-cell disease, wherein the dose is administered via intramuscular injection, and wherein the dose is: A) about 1 × 10⁷ cells to about 3 × 10⁷ cells. 8 A) Approximately 1.25 × 10⁻⁶ cells; B) Approximately 1.25 × 10⁻⁶ cells. 5 Cells / kg to approximately 1.2 × 10⁻⁶ 7 C) Approximately 6,500 islet equivalents (IEQ) to approximately 600,000 IEQ; or D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg.

[0013] In some implementations, this document provides a method for increasing the time within a target range (TIR) ​​in subjects who have or are at risk of developing β-cell disease, the method comprising administering to the subject a dose of engineered, low-immunogenic islets, wherein the dose is administered via intramuscular injection, and wherein the dose is: A) approximately 1 × 10⁻⁶ 7 One cell to approximately 3 × 10 8 A) Approximately 1.25 × 10⁻⁶ cells; B) Approximately 1.25 × 10⁻⁶ cells. 5Cells / kg to approximately 1.2 × 10⁻⁶ 7 The TIR was increased in subjects receiving alternative islet therapy or in untreated subjects compared to subjects receiving alternative islet therapy or in untreated subjects.

[0014] In some embodiments of any of these implementations, the method results in a reduced need for other medications used to treat β-cell disorders, optionally including insulin as the diabetes medication. In some embodiments of any of these implementations, the subject exhibits reduced insulin dependence.

[0015] In some embodiments of any implementation scheme, the amount of exogenous insulin is reduced by 10% or more compared to the amount of exogenous insulin required by a subject receiving non-low-immunogenic islets to treat β-cell disease or by the amount of exogenous insulin required by an untreated subject with β-cell disease. In some embodiments of any implementation scheme, the amount of insulin is reduced by more than about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, or more.

[0016] In some embodiments of any implementation, the method is characterized by the subject meeting one or more of the following criteria: (i) fasting capillary blood glucose levels exceeding 140 mg / dL (7.8 mmol / L) no more than three times in one week (based on at least seven capillary blood glucose levels measured over seven days); (ii) 2-hour postprandial capillary blood glucose levels exceeding 180 mg / dL (10.0 mmol / L) no more than three times in one week (based on at least 21 capillary blood glucose levels measured over seven days); and (iii) evidence of endogenous insulin production, defined as fasting or post-stimulation C-peptide levels >0.5 ng / mL (0.16 pmol / L).

[0017] In some implementations of any of these implementations, the method results in subjects exhibiting insulin non-dependency. In some implementations of any of these implementations, subjects exhibit insulin non-dependency for periods longer 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. In some implementations of any of these implementations, subjects exhibit insulin non-dependency for periods of at least one year.

[0018] In some implementation schemes of any of these schemes, subjects were able to gradually discontinue insulin therapy for at least one week and meet one or more of the following criteria: (i) fasting capillary blood glucose levels exceeding 140 mg / dL (7.8 mmol / L) no more than three times in one week (based on at least seven capillary blood glucose level measurements over seven days); (ii) 2-hour postprandial capillary blood glucose levels exceeding 180 mg / dL (10.0 mmol / L) no more than three times in one week (based on at least 21 capillary blood glucose level measurements over seven days); and (iii) evidence of endogenous insulin production, defined as fasting or post-stimulation C-peptide levels >0.5 ng / mL (0.16 pmol / L).

[0019] In some implementations of any of the embodiments, the subject is able to gradually discontinue insulin therapy over a period of time and meet one or more of the following criteria: (i) fasting capillary blood glucose levels exceeding 140 mg / dL (7.8 mmol / L) no more than three times in one week (based on at least 7 capillary blood glucose measurements over 7 days); (ii) 2-hour postprandial capillary blood glucose levels exceeding 180 mg / dL (10.0 mmol / L) no more than three times in one week (based on at least 21 capillary blood glucose measurements over 7 days); and (iii) evidence of endogenous insulin production, defined as fasting or post-stimulation C-peptide levels >0.5 ng / mL (0.16 pmol / L). In some implementations of any of the embodiments, the subject is characterized by at least two of (i)-(iii). In some implementations of any of the embodiments, the subject is characterized by each of (i)-(iii).

[0020] In some embodiments of any implementation scheme, the method is characterized in that the subject meets one or more of the following: a) peak C-peptide > 0.20 nmol / L (as assessed by a mixed-meal tolerance test); b) non-fasting C-peptide > 0.10 nmol / L (as assessed by a mixed-meal tolerance test); c) daily exogenous insulin requirement < 0.25 U / kg; d) daily exogenous insulin requirement = 0 U / kg; e) reduced exogenous insulin requirement (per kg body weight); f) reduced HbA1c (per kg body weight); g) reduced glycemic variability (stable); h) shortened duration of hypoglycemia and / or hyperglycemia (improved normoglycemic status); i) glycemic control HbA1c ≤ 6.5% (48 mmol / mol); and j) glycemic control HbA1c < 7.0% (53 mmol / mol). In some embodiments of any implementation scheme, the method is characterized in that the subject meets 2, 3, 4, 5, 6, 7, 8, 9, or 10 of a)-j). In some implementations of any implementation, the method is characterized in that the subject satisfies each of a)-j).

[0021] In some embodiments of any implementation, the engineered low-immunogenic islets comprise the following modifications: (a) inactivating or disrupting one or more alleles of: (i) one or more major histocompatibility complex (MHC) class I molecules or one or more molecules that regulate the expression of such one or more MHC class I molecules, and / or (ii) one or more MHC class II molecules or one or more molecules that regulate the expression of such one or more MHC class II molecules; and / or (b) increasing the expression of one or more tolerogenic factors, wherein such increase is relative to control or wild-type islets without modifications.

[0022] In some embodiments of any implementation, the engineered low-immunogenic islets comprise engineered β islet cells. In some embodiments of any implementation, the engineered low-immunogenic islets further comprise additional engineered islet cells, wherein the additional engineered islet cells include α cells and / or δ cells. In some embodiments of any implementation, the additional engineered islet cells comprise cells comprising the same modifications as the engineered β islet cells.

[0023] In some embodiments of any implementation, 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 engineered low-immunogenic islets comprise engineered β-islet cells. In some embodiments of any implementation, at least 60% of the cells in the engineered low-immunogenic islets comprise engineered β-islet cells. In some embodiments of any implementation, the engineered low-immunogenic islets are islet clusters. In some embodiments of any implementation, the engineered low-immunogenic islets are engineered from primary islets. In some embodiments of any implementation, the primary islets are derived from the pancreas. In some embodiments of any implementation, the primary islets are derived from a human subject. In some embodiments of any implementation, the primary islets are derived from an animal subject. In some embodiments of any implementation, the primary islets are from a pig, cow, or sheep.

[0024] In some embodiments of any implementation, the primary islets are derived from donor subjects who are not suspected of having β-cell-related diseases. In some embodiments of any implementation, the donor is a cadaver. In some embodiments of any implementation, the engineered low-immunogenic islets are ABO blood type O. In some embodiments of any implementation, the engineered low-immunogenic islets are rhesus factor negative (Rh-).

[0025] In some embodiments of any implementation, the engineered, low-immunogenic islets are derived from stem cells. In some embodiments of any implementation, 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 embodiments of any implementation, 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 of any implementation, the stem cells are pluripotent stem cells (PSCs).

[0026] In some embodiments of any implementation scheme, the β-cell disorder is a metabolic disorder. In some embodiments of any implementation scheme, 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 encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), Niemann-Pick disease, phenylketonuria (PKU), porphyria, Tay-Sachs disease, Wilson disease, type 1 diabetes, type 2 diabetes, obesity, hypertension, dyslipidemia, and carbohydrate intolerance. In some embodiments of any implementation scheme, the β-cell disorder is diabetes. In some embodiments of any implementation scheme, the β-cell disorder is type 1 diabetes.

[0027] In some implementation schemes of any implementation scheme, the subject to be treated is characterized by one or more of the following: type 1 diabetes for more than 5 years, negative C-peptide (or <0.01 nmol / L) in response to mixed meal tolerance test (MMTT), positive antibody against GAD or IA2, HbA1c ≥70 mmol / mol, and exogenous insulin requirement <1 U / kg.

[0028] In some embodiments of any implementation, a dose of engineered, low-immunogenic islets comprises a pharmaceutically acceptable carrier. In some embodiments of any implementation, the pharmaceutically acceptable carrier is a buffered aqueous solution. In some embodiments of any implementation, the buffered aqueous solution is saline. In some embodiments of any implementation, the dose is administered intravenously to the subject. In some embodiments of any implementation, when the dose is administered intravenously, it is administered via the portal vein. In some embodiments of any implementation, the dose is administered to the subject via the renal capsule. In some embodiments of any implementation, the dose is administered subcutaneously to the subject.

[0029] In some embodiments of any implementation, engineered low-immunogenic islets are administered intramuscularly to the subject. In some embodiments of any implementation, intramuscular administration is via the intermuscular space of the forearm. In some embodiments of any implementation, engineered low-immunogenic islets are administered to the upper arm, hip, thigh, or buttock.

[0030] In some embodiments of any implementation scheme, the dose is administered to the liver, kidney, spleen, muscle, subcutaneous tissue, or white adipose tissue of the subject. In some embodiments of any implementation scheme, the dose is administered to the liver, muscle, or white adipose tissue of the subject. In some embodiments of any implementation scheme, the white adipose tissue is the omentum.

[0031] In some embodiments of any implementation, administration includes the administration of one or more additional doses of low-immunogenic engineered cells.

[0032] In some embodiments of any implementation scheme, one or more additional doses of low-immunogenic engineered cells are administered to the subject when, after the initial dose, the subject does not exhibit a reduced requirement for other medications used to treat β-cell disease, optionally wherein the β-cell disease medication is insulin; and / or (b) the administered low-immunogenic engineered cells are not detected by imaging. In some embodiments of any implementation scheme, the subject does not exhibit a reduction in insulin dependence after the initial dose.

[0033] In some implementation schemes of any implementation scheme, if a subject does not meet one or more of the following criteria after the initial dose, one or more additional doses of low-immunogenic engineered cells are administered to the subject: (i) a fasting capillary blood glucose level greater than 140 mg / dL (7.8 mmol / L) no more than three times in one week (based on at least 7 measurements of capillary blood glucose levels over seven days); (ii) a 2-hour postprandial capillary blood glucose level greater than 180 mg / dL (10.0 mmol / L) no more than three times in one week (based on at least 21 measurements of capillary blood glucose levels over seven days); and (iii) evidence of endogenous insulin production, defined as a fasting or post-stimulation C-peptide level >0.5 ng / mL (0.16 pmol / L).

[0034] In some embodiments of any implementation scheme, one or more additional doses of low-immunogenic engineered cells are administered to the subject when: (a) the subject does not achieve insulin independence for a period of time after the initial dose; and / or (b) the subject does not exhibit a reduced demand for other medications used to treat β-cell disease for a period of time, optionally wherein the β-cell disease medication is insulin.

[0035] In some implementation schemes of any scheme, subjects did not achieve insulin non-dependency for periods longer 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, optionally including periods of two weeks. In some implementation schemes of any scheme, subjects did not achieve insulin non-dependency for periods of at least one year.

[0036] In some implementation schemes of any implementation scheme, when a subject is unable to gradually discontinue insulin therapy for at least 1 week after the initial dose and meets one or more of the following criteria, one or more additional doses of low-immunogenic engineered cells are administered to the subject: (i) fasting capillary blood glucose levels exceeding 140 mg / dL (7.8 mmol / L) no more than three times within 1 week (based on at least 7 capillary blood glucose level measurements over 7 days); (ii) 2-hour postprandial capillary blood glucose levels exceeding 180 mg / dL (10.0 mmol / L) no more than three times within 1 week (based on at least 21 capillary blood glucose level measurements over 7 days); and (iii) evidence of endogenous insulin production, defined as fasting or post-stimulation C-peptide levels >0.5 ng / mL (0.16 pmol / L).

[0037] In some embodiments of any implementation scheme, when a subject is unable to gradually discontinue insulin therapy over a period of time after the initial dose and meets one or more of the following criteria, one or more additional doses of low-immunogenic engineered cells are administered to the subject: (i) a fasting capillary blood glucose level exceeding 140 mg / dL (7.8 mmol / L) no more than three times in one week (based on at least 7 capillary blood glucose level measurements over seven days); (ii) a 2-hour postprandial capillary blood glucose level exceeding 180 mg / dL (10.0 mmol / L) no more than three times in one week (based on at least 21 capillary blood glucose level measurements over seven days); and (iii) evidence of endogenous insulin production, defined as a fasting or post-stimulation C-peptide level >0.5 ng / mL (0.16 pmol / L). In some embodiments of any implementation scheme, the subject is characterized by not meeting at least two or each of (i)-(iii).

[0038] In some embodiments of any implementation scheme, if a subject does not meet one or more of the following criteria after the initial dose, one or more additional doses of low-immunogenic engineered cells are administered to the subject: a) peak C-peptide > 0.20 nmol / L (as assessed by a mixed-meal tolerance test); b) non-fasting C-peptide > 0.10 nmol / L (as assessed by a mixed-meal tolerance test); c) daily exogenous insulin requirement < 0.25 U / kg; d) daily exogenous insulin requirement = 0 U / kg; e) reduced exogenous insulin requirement (per kg body weight); f) reduced HbA1c (per kg body weight); g) reduced glycemic variability (stable); h) shortened duration of hypoglycemia and / or hyperglycemia (improved normoglycemic status); i) glycemic control HbA1c ≤ 6.5% (48 mmol / mol); and j) glycemic control HbA1c < 7.0% (53 mmol / mol). In some embodiments of any implementation scheme, if the subject does not meet 2, 3, 4, 5, 6, 7, 8, 9, or 10 of a)-j), one or more additional doses are administered to the subject after the initial dose. In some embodiments of any implementation scheme, if the subject does not meet each of a)-j), one or more additional doses are administered to the subject after the initial dose.

[0039] In some embodiments of any implementation, the number of engineered low-immunogenic islets in the subject's body derived from the initial dose is cleared or reduced prior to administration of one or more additional doses of engineered low-immunogenic islets. In some embodiments of any implementation, the number of engineered low-immunogenic islets in the subject's body is reduced following administration of an exogenously administered agent to guide targeted death of the engineered low-immunogenic islets. In some embodiments of any implementation, the exogenously administered agent activates a suicide gene or safety switch in the engineered cells, or recognizes one or more tolerogenic factors on the surface of the engineered low-immunogenic islets.

[0040] In some embodiments of any implementation scheme, an immunosuppressive regimen is administered to the subject. In some embodiments of any implementation scheme, the immunosuppressive regimen comprises one or more of mycophenolate mofetil (MMF), an anti-CD25 antibody (e.g., bailiximab), and a calcineurin inhibitor (e.g., tacrolimus; FK-506). In some embodiments of any implementation scheme, the immunosuppressive regimen comprises administration of bailiximab (e.g., 2 x 20 mg IV), followed by administration of tacrolimus (starting dose 0.1 mg / kg / 24h; target concentration 10-12) and MMF immunosuppression (500 mg 2 x 2, subsequently adjusted based on AUC). In some implementations of any of these implementations, the subject is also given one or more of the following: valganciclovir (e.g., 450 mg 2x1) for CMV prevention, omeprazole (e.g., 20 mg 1x1) for ulcer prevention, etanercept (e.g., 50 mg IV, followed by 25 mg SC on days 3, 7 and 10) for TNF-α inhibition, and a standard antibiotic.

[0041] In some embodiments of any implementation scheme, the immunosuppressive regimen is administered to the subject only before the administration of a certain dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, the immunosuppressive regimen is administered to the subject only for days 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 prior to the administration of a certain dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, an immunosuppressive regimen is administered to subjects 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 administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, an immunosuppressive regimen is administered to subjects only 1, 2, 3, 4, or 5 weeks prior to administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, an immunosuppressive regimen is administered to subjects only 1, 2, 3, or 4 weeks prior to administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, an immunosuppressive regimen is administered to subjects only after administration of a dose of engineered low-immunogenic islets. In some implementation schemes of any of these schemes, an immunosuppressive regimen is administered to subjects only for 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 a dose of engineered, low-immunogenic islets. In some embodiments of any implementation scheme, an immunosuppressive regimen is administered to subjects only for 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 a dose of engineered, low-immunogenic islets. In some embodiments of any implementation scheme, an immunosuppressive regimen is administered to subjects only for 1, 2, 3, 4, or 5 weeks after administration of a dose of engineered, low-immunogenic islets.In some implementation schemes of any of these schemes, an immunosuppressive regimen is administered to subjects only 1, 2, 3, or 4 weeks after administration of a dose of engineered, low-immunogenic islets.

[0042] In some embodiments of any implementation, the immunosuppressive regimen is administered intravenously to the subject. In some embodiments of any implementation, the immunosuppressive regimen is administered via the renal sac to the subject. In some embodiments of any implementation, the immunosuppressive regimen is administered orally to the subject. In some embodiments of any implementation, the immunosuppressive regimen is administered rectally to the subject. In some embodiments of any implementation, the immunosuppressive regimen is administered subcutaneously to the subject. In some embodiments of any implementation, the immunosuppressive regimen is administered intramuscularly to the subject. In some embodiments of any implementation, the immunosuppressive regimen is administered to the forearm of the subject. In some embodiments of any implementation, the immunosuppressive regimen is administered to the upper arm, hip, thigh, or buttock. In some embodiments of any implementation, the immunosuppressive regimen is administered at least once daily. In some embodiments of any implementation, the immunosuppressive regimen is administered as a single daily dose. In some embodiments of any implementation, the immunosuppressive regimen is administered as a divided dose. In some embodiments of any implementation, the immunosuppressive regimen is divided into 2-dose or 3-dose regimens. In some embodiments of any implementation scheme, the immunosuppressive regimen includes one or more immunosuppressants. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject prior to administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject only prior to the first and / or second administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, 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 administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or earlier prior to administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject only after the first and / or second administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, 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 engineered low-immunogenic islets. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or later, following the administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, 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 later, following the administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or later, following the administration of a dose of engineered low-immunogenic islets.In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject on the same day as a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject concurrently with a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject only for 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 administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject only for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or earlier prior to the first and / or second administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject only for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the first and / or second administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject only for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after the first and / or second administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject only for at least 2, 4, 6, 8, 10, 12, or 16 weeks after the administration of a dose of engineered low-immunogenic islets. In some implementation schemes of any of these schemes, one or more immunosuppressants are administered to the subject only for at least 1, 2, 3, 4, 5, or 6 months after administration of a dose of engineered, low-immunogenic islets.

[0043] In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject prior to administration of a dose of engineered low-immunogenic islets and are continued throughout the subject's lifespan. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject on the same day and / or concurrently with administration of a dose of engineered low-immunogenic islets and are continued throughout the subject's lifespan. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject after administration of a dose of engineered low-immunogenic islets and are continued throughout the subject's lifespan. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject prior to each round of administration of a dose of engineered low-immunogenic islets and optionally are continued throughout the subject's lifespan. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject on the same day and / or concurrently with each round of administration of a dose of engineered low-immunogenic islets and optionally are continued throughout the subject's lifespan. In some embodiments of any implementation scheme, one or more immunosuppressants are administered to the subject after each round of administration of a dose of engineered, low-immunogenic islets, and optionally continued throughout the subject's lifespan.

[0044] In some embodiments of any implementation, one or more immunosuppressants are administered to the subject at a lower dose than the dose of one or more immunosuppressants administered to the subject, to reduce immune rejection of modified immunogenic cells that do not contain a dose of engineered, low-immunogenic islets. In some embodiments of any implementation, one or more immunosuppressants comprise small molecules or biological products. In some embodiments of any implementation, the biological product is a protein and / or antibody. In some embodiments of any implementation, the small molecule is a chemical compound or nucleic acid. In some embodiments of any implementation, one or more immunosuppressants comprise one or more immunomodulators. In some embodiments of any implementation, one or more immunomodulators are small molecules or biological products. In some embodiments of any implementation, the biological product is a protein or a peptide thereof and / or an antibody. In some embodiments of any implementation, the small molecule is a chemical compound or nucleic acid. In some embodiments of any implementation, one or more immunosuppressants are pharmaceutical salts of themselves, their prodrug forms, and / or derivatives thereof. In some embodiments of any implementation, one or more immunomodulators are pharmaceutical salts of themselves, their prodrug forms, and / or derivatives thereof.

[0045] In some embodiments of any implementation scheme, 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, blood rheology modifiers, inosine monophosphate dehydrogenase (IMDH) inhibitors, Janus kinase inhibitors, mTOR inhibitors, TNF inhibitors, and antiCD25 inhibitors. In some embodiments of any implementation scheme, one or more immunosuppressants are selected from the group consisting of: anti-thymocyte globulin (ATG), corticosteroids, prednisone, cortisone, prednisolone methylprednisolone, dexamethasone, betamethasone, hydrocortisone, methotrexate, acetaminophen, diphenhydramine, sirolimus (rapamycin), tacrolimus (FK-506), mycophenolic acid (MPA), mycophenolic acid ethyl ester (MMF), mycophenolic acid sodium, cyclosporine, etanercept (TNFR-Fc), azathioprine, gold salts, sulfasalazine, antimalarial drugs, buquina, leflunomide, imidazolidin, 15-deoxyguanidine, 6-mercaptopurine, cyclophosphamide, OKT3, anti-thymocyte globulin, thymopentin (thymosin-α), fludarabine, cyclophosphamide, and immunosuppressive antibodies.

[0046] In some embodiments of any implementation, one or more immunosuppressants include anti-thymocyte globulin (ATG). In some embodiments of any implementation, at least one regimen of ATG is administered to the subject before, concurrently with, and / or after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of ATG is administered to the subject before administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of ATG is administered to the subject before each administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of ATG is administered to the subject approximately 7 days, approximately 6 days, approximately 5 days, approximately 4 days, approximately 3 days, approximately 2 days, approximately 1 day, approximately 12 hours, approximately 10 hours, approximately 8 hours, approximately 6 hours, approximately 4 hours, approximately 2 hours, or approximately 1 hour before administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of ATG is administered to the subject approximately 2 days and / or approximately 1 day prior to the administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of ATG is administered to the subject on the same day and / or concurrently with the administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, 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 engineered low-immunogenic islets. In some embodiments of any implementation, a first or second regimen of ATG is administered to the subject on the same day and / or concurrently with the administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of ATG is administered to the subject after the administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, an ATG regimen is administered to the subject after each administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, at least one regimen of ATG is administered to the subject approximately 1 hour, approximately 2 hours, approximately 4 hours, approximately 6 hours, approximately 8 hours, approximately 10 hours, approximately 12 hours, approximately 24 hours, or approximately 48 hours after administration of a dose of engineered, low-immunogenic islets. In some embodiments of any implementation scheme, at least one regimen of ATG is administered to the subject approximately 48 hours after administration of a dose of engineered, low-immunogenic islets.In some embodiments of any implementation scheme, at least one regimen of ATG is administered to the subject approximately 2 days prior to: i) administration of a dose of engineered, low-immunogenic islet; ii) approximately 1 day prior to: iii) administration of a dose of ATG on: iv) approximately 1 day thereafter; and / or, v) approximately 2 days thereafter. In some embodiments of any implementation scheme, at least one or at least two regimens of ATG comprise administering ATG to the subject at doses between approximately 0.1 mg / kg and approximately 2.0 mg / kg. In some embodiments of any implementation scheme, the ATG regimen is administered at a lower dose. In some embodiments of any implementation, the method includes a regimen in which: i) at least one or more of the ATG regimens include administering an ATG dose of approximately 0.5 mg / kg to the subject approximately 2 days prior to administering a dose of engineered low-immunogenic islets; ii) at least one or more of the ATG regimens include administering an ATG dose of approximately 1.0 mg / kg to the subject approximately 1 day prior to administering a dose of engineered low-immunogenic islets; and / or iii) at least one or more of the ATG regimens include administering an ATG dose of approximately 1.5 mg / kg to the subject on the same day as administering a dose of engineered low-immunogenic islets, approximately 1 day after administering a dose of engineered low-immunogenic islets, and approximately 2 days after administering a dose of engineered low-immunogenic islets. In some embodiments of any implementation, the ATG regimen is administered at a lower dose.

[0047] In some embodiments of any implementation, one or more immunosuppressants include corticosteroids. In some embodiments of any implementation, one or more immunosuppressants include prednisone, cortisone, prednisolone methylprednisolone, dexamethasone, betamethasone, or hydrocortisone. In some embodiments of any implementation, one or more immunosuppressants include methylprednisolone. In some embodiments of any implementation, at least one regimen of methylprednisolone is administered to the subject prior to administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of methylprednisolone is administered to the subject approximately 7 days, approximately 6 days, approximately 5 days, approximately 4 days, approximately 3 days, approximately 2 days, approximately 1 day, approximately 12 hours, approximately 10 hours, approximately 8 hours, approximately 6 hours, approximately 4 hours, approximately 2 hours, or approximately 1 hour prior to administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of methylprednisolone is administered to the subject approximately 2 days prior to administration of a dose of engineered, low-immunogenic islets. In some embodiments of any implementation, at least one regimen of methylprednisolone is administered to the subject prior to administration of a first regimen of ATG, wherein both the methylprednisolone regimen and the first ATG regimen are administered to the subject prior to administration of a dose of engineered, low-immunogenic islets. In some embodiments of any implementation, at least one regimen of methylprednisolone is administered to the subject approximately 1 hour prior to administration of the first ATG regimen. In some embodiments of any implementation, at least one regimen of methylprednisolone is administered to the subject approximately midway through administration of the first ATG regimen. In some embodiments of any implementation, at least one regimen of methylprednisolone comprises a dose between approximately 0.1 mg / kg and approximately 2.0 mg / kg. In some embodiments of any implementation, the methylprednisolone regimen is administered at a lower dose. In some embodiments of any implementation, at least one regimen of methylprednisolone comprises about 1.0 mg / kg of methylprednisolone. In some embodiments of any implementation, the methylprednisolone regimen is administered at a lower dose. In some embodiments of any implementation, methylprednisolone is administered intravenously to the subject. In some embodiments of any implementation, the method includes a regimen in which: i) at least one regimen of methylprednisolone comprises administering about 1.0 mg / kg of methylprednisolone to the subject about 1 hour prior to administration of the first regimen ATG; and / or ii) at least one regimen of methylprednisolone comprises administering about 1.0 mg / kg of methylprednisolone to the subject approximately midway through administration of the first regimen ATG.In some embodiments of any implementation, the methylprednisolone regimen and / or ATG regimen are administered at a lower dose.

[0048] In some embodiments of any implementation, one or more immunosuppressants include analgesics. In some embodiments of any implementation, the analgesic is acetaminophen, an opioid, or a nonsteroidal anti-inflammatory drug (NSAID). In some embodiments of any implementation, at least one regimen of acetaminophen is administered to the subject prior to administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of acetaminophen is administered to the subject approximately 7 days, approximately 6 days, approximately 5 days, approximately 4 days, approximately 3 days, approximately 2 days, approximately 1 day, approximately 12 hours, approximately 10 hours, approximately 8 hours, approximately 6 hours, approximately 4 hours, approximately 2 hours, or approximately 1 hour prior to administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of acetaminophen is administered to the subject approximately 2 days prior to administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of acetaminophen is administered to the subject prior to administration of the first regimen of ATG, wherein both the acetaminophen regimen and the first regimen of ATG are administered to the subject prior to administration of a dose of engineered, low-immunogenic islets. In some embodiments of any implementation, at least one regimen of acetaminophen is administered to the subject approximately 30 minutes prior to administration of the first regimen of ATG. In some embodiments of any implementation, at least one regimen of acetaminophen is administered to the subject approximately midway through administration of the first regimen of ATG. In some embodiments of any implementation, at least one regimen of acetaminophen comprises administering acetaminophen to the subject at a dose between approximately 100 mg and approximately 1,000 mg. In some embodiments of any implementation, the acetaminophen regimen is administered at a lower dose. In some embodiments of any implementation, at least one regimen of approximately 650 mg of acetaminophen is administered to the subject. In some embodiments of any implementation, the acetaminophen regimen is administered at a lower dose. In some embodiments of any implementation, acetaminophen is administered to the subject orally or rectally. In some embodiments of any implementation, the method includes a regimen in which: i) approximately 30 minutes prior to administration of a first regimen of ATG to the subject, at least one regimen of approximately 650 mg of acetaminophen is administered; and / or ii) approximately midway through administration of the first regimen of ATG to the subject, at least one regimen of approximately 650 mg of acetaminophen is administered. In some embodiments of any implementation, the acetaminophen regimen and / or the ATG regimen are administered at a lower dose.

[0049] In some embodiments of any implementation, one or more immunosuppressants include an antihistamine. In some embodiments of any implementation, the antihistamine is diphenhydramine. In some embodiments of any implementation, at least one regimen of diphenhydramine is administered to the subject prior to administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of diphenhydramine is administered to the subject approximately 7 days, approximately 6 days, approximately 5 days, approximately 4 days, approximately 3 days, approximately 2 days, approximately 1 day, approximately 12 hours, approximately 10 hours, approximately 8 hours, approximately 6 hours, approximately 4 hours, approximately 2 hours, or approximately 1 hour prior to administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of diphenhydramine is administered to the subject approximately 2 days prior to administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of diphenhydramine is administered to the subject prior to administration of the first regimen of ATG, wherein both the diphenhydramine regimen and the first regimen of ATG are administered to the subject prior to administration of a dose of engineered, low-immunogenic islet. In some embodiments of any implementation, at least one regimen of diphenhydramine is administered to the subject approximately 30 minutes prior to administration of the first regimen of ATG. In some embodiments of any implementation, at least one regimen of diphenhydramine is administered to the subject approximately midway through administration of the first regimen of ATG. In some embodiments of any implementation, at least one regimen of diphenhydramine comprises administering diphenhydramine between approximately 10 mg and approximately 100 mg to the subject. In some embodiments of any implementation, the diphenhydramine regimen is administered at a lower dose. In some embodiments of any implementation, at least one regimen of approximately 50 mg of diphenhydramine is administered to the subject. In some embodiments of any implementation, the diphenhydramine regimen is administered at a lower dose. In some embodiments of any implementation, diphenhydramine is administered to the subject orally or rectally. In some embodiments of any implementation, the method includes a regimen in which: i) at least one regimen of diphenhydramine includes administering approximately 50 mg of diphenhydramine to the subject approximately 30 minutes prior to administering the first regimen ATG; and / or ii) at least one regimen of diphenhydramine includes administering approximately 50 mg of diphenhydramine to the subject approximately midway through administering the first regimen ATG; in some embodiments of any implementation, the diphenhydramine regimen and / or the ATG regimen are administered at a lower dose.

[0050] In some embodiments of any implementation scheme, one or more immunosuppressants include an anti-inflammatory agent. In some embodiments of any implementation scheme, the anti-inflammatory agent is a TNF inhibitor. In some embodiments of any implementation scheme, the TNF inhibitor is selected from the group consisting of infliximab, adalimumab, etanercept, golimumab, and cetuzumab. In some embodiments of any implementation scheme, the TNF inhibitor is etanercept (TNFR-Fc). In some embodiments of any implementation scheme, at least one regimen of etanercept is administered to the subject before, concurrently with, and / or after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, at least one regimen of etanercept is administered to the subject before administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, etanercept is administered to the subject approximately 7 days, approximately 6 days, approximately 5 days, approximately 4 days, approximately 3 days, approximately 2 days, approximately 1 day, approximately 12 hours, approximately 10 hours, approximately 8 hours, approximately 6 hours, approximately 4 hours, approximately 2 hours, or approximately 1 hour prior to the administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, etanercept is administered to the subject on the same day as and / or concurrently with the administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, a first regimen of etanercept is administered to the subject on the same day as and / or concurrently with the administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, etanercept is administered to the subject after the administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, etanercept is administered to the subject approximately 1 hour, approximately 5 hours, approximately 10 hours, approximately 24 hours, approximately 2 days, approximately 3 days, approximately 5 days, approximately 7 days, or approximately 10 days after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, etanercept is administered to the subject approximately 3 days, approximately 7 days, and / or approximately 10 days after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, etanercept is administered to the subject approximately i) on the same day; ii) approximately 3 days thereafter; iii) approximately 7 days thereafter; and / or iv) approximately 10 days thereafter. In some embodiments of any implementation, at least one regimen of etanercept comprises between approximately 10 mg and approximately 100 mg of etanercept. In some embodiments of any implementation, the etanercept regimen is administered at a lower dose.In some embodiments of any implementation, at least one regimen of etanercept comprises about 50 mg of etanercept. In some embodiments of any implementation, the etanercept regimen is administered at a lower dose. In some embodiments of any implementation, at least one regimen of etanercept comprises about 25 mg of etanercept. In some embodiments of any implementation, the etanercept regimen is administered at a lower dose. In some embodiments of any implementation, etanercept is administered intravenously and / or subcutaneously to the subject. In some embodiments of any implementation, the method comprises a method wherein: i) on the same day as administration of a dose of engineered low-immunogenic islets to the subject, at least one regimen of about 50 mg of etanercept is administered to the subject; and / or, ii) about 3 days, about 7 days, and / or about 10 days after administration of a dose of engineered low-immunogenic islets to the subject, at least one regimen of about 25 mg of etanercept is administered to the subject. In some embodiments of any implementation, the etanercept regimen is administered at a lower dose. In some embodiments of any implementation, at least one regimen of etanercept and at least one regimen of ATG are administered to the subject. In some embodiments of any implementation scheme, at least one regimen of ATG is administered to the subject prior to at least one regimen of etanercept. In some embodiments of any implementation scheme, i) at least one regimen of ATG comprises administering approximately 40 mg / kg of ATG to the subject daily for four consecutive days; ii) at least one regimen of etanercept comprises, after i), administering approximately 25 mg of etanercept twice weekly to the subject for two consecutive weeks; and iii) at least one regimen of etanercept comprises, after ii), administering approximately 25 mg of etanercept once monthly to the subject for approximately four months. In some embodiments of any implementation scheme, at least one etanercept regimen and / or at least one ATG regimen are administered at a lower dose. In some embodiments of any implementation scheme, at least one regimen of etanercept and at least one regimen of an IL-1 receptor antagonist are administered to the subject.

[0051] In some embodiments of any implementation scheme, one or more immunosuppressants include an mTOR inhibitor. In some embodiments of any implementation scheme, the mTOR inhibitor is sirolimus (rapamycin). In some embodiments of any implementation scheme, at least one regimen of sirolimus is administered to the subject before, concurrently with, and / or after administration of a dose of engineered, low-immunogenic islets. In some embodiments of any implementation scheme, at least one regimen of sirolimus is administered to the subject before administration of a dose of engineered, low-immunogenic islets.

[0052] In some embodiments of any implementation, sirolimus is administered to the subject at least 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 administration of a dose of engineered, low-immunogenic islets.

[0053] In some embodiments of any implementation scheme, sirolimus is administered to the subject on the same day and / or concurrently with the administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, sirolimus is administered to the subject following the administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, sirolimus 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 later, after the administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, the total daily dose of sirolimus administered to the subject produces a trough plasma concentration between about 1 ng / mL and about 30 ng / mL, between about 2 ng / mL and about 25 ng / mL, between about 5 ng / mL and about 20 ng / mL, or between about 10 ng / mL and about 15 ng / mL, including the endpoints of each interval. In some embodiments of any implementation, a regimen of sirolimus between about 0.1 mg / kg and about 0.2 mg / kg is administered to the subject. In some embodiments of any implementation, a sirolimus regimen is administered at a lower dose. In some embodiments of any implementation, sirolimus is administered orally to the subject.

[0054] In some embodiments of any implementation scheme, i) on the same day as administration of a dose of engineered, low-immunogenic islet to the subject, a regimen of approximately 0.2 mg / kg of sirolimus is administered; ii) following administration of a dose of engineered, low-immunogenic islet to the subject, a regimen of approximately 0 / 1 mg / kg of sirolimus is administered daily to the subject for up to approximately 3 months, wherein the total daily dose of sirolimus administered to the subject produces a trough plasma concentration between approximately 12 ng / mL and approximately 15 ng / mL for approximately 3 months after administration of the composition and thereafter between approximately 7 ng / mL and approximately 10 ng / mL. In some embodiments of any implementation scheme, the sirolimus regimen is administered at a lower dose.

[0055] In some embodiments of any implementation scheme, one or more immunosuppressants include calcineurin inhibitors. In some embodiments of any implementation scheme, the calcineurin inhibitor is tacrolimus (FK-506). In some embodiments of any implementation scheme, tacrolimus is administered to the subject in at least one regimen before, concurrently with, and / or after administration of a dose of engineered, hypoimmunogenic islets.

[0056] In some embodiments of any implementation scheme, at least one regimen of tacrolimus is administered to the subject prior to administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, at least one regimen of tacrolimus is administered to the subject approximately 7 days, approximately 6 days, approximately 5 days, approximately 4 days, approximately 3 days, approximately 2 days, approximately 1 day, approximately 12 hours, approximately 10 hours, approximately 8 hours, approximately 6 hours, approximately 4 hours, approximately 2 hours, or approximately 1 hour prior to administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, at least one regimen of tacrolimus is administered to the subject on the same day and / or concurrently with administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, a first regimen of tacrolimus is administered to the subject on the same day and / or concurrently with administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, tacrolimus is administered to the subject at least one regimen after administration of a dose of engineered, low-immunogenic islets. In some embodiments of any implementation scheme, tacrolimus 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 later, after administration of a dose of engineered, low-immunogenic islets. In some embodiments of any implementation scheme, the total daily dose of tacrolimus administered to the subject produces a trough plasma concentration between approximately 1 ng / mL and approximately 30 ng / mL, between approximately 2 ng / mL and approximately 25 ng / mL, between approximately 5 ng / mL and approximately 20 ng / mL, or between approximately 10 ng / mL and approximately 15 ng / mL, including the endpoints of each interval. In some embodiments of any implementation scheme, the total daily dose of tacrolimus administered to the subject produced a trough plasma concentration between about 5 ng / mL and about 10 ng / mL, including the endpoints of the range. In some embodiments of any implementation scheme, the total daily dose of tacrolimus administered to the subject produced a trough plasma concentration between about 10 ng / mL and about 15 ng / mL, including the endpoints of the range.

[0057] In some embodiments of any implementation, a tacrolimus regimen between about 0.1 mg and about 5 mg is administered to the subject. In some embodiments of any implementation, the tacrolimus regimen is administered at a lower dose. In some embodiments of any implementation, one or more immunosuppressants include inosine-1''-monophosphate dehydrogenase (IMPDH) inhibitors. In some embodiments of any implementation, the IMPDH inhibitor is MPA, MMF, or MS. In some embodiments of any implementation, the IMPDH inhibitor is mycophenolic acid (MPA). In some embodiments of any implementation, at least one regimen of MPA is administered to the subject before, concurrently with, and / or after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of MPA is administered to the subject before administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, at least one regimen of MPA is administered to the subject approximately 7 days, approximately 6 days, approximately 5 days, approximately 4 days, approximately 3 days, approximately 2 days, approximately 1 day, approximately 12 hours, approximately 10 hours, approximately 8 hours, approximately 6 hours, approximately 4 hours, approximately 2 hours, or approximately 1 hour prior to the administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, 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 engineered low-immunogenic islets. In some embodiments of any implementation scheme, a first regimen of MPA is administered to the subject on the same day and / or concurrently with the administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, at least one regimen of MPA is administered to the subject after the administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, at least one regimen of MPA 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 later, after administering a dose of engineered low-immunogenic islets to the subject.

[0058] In some embodiments of any implementation, the MPA is mycophenolate mofetil (MMF). In some embodiments of any implementation, the total daily dose of MMF is between about 10 mg and about 3000 mg, about 500 mg and about 3000 mg, about 1000 mg and about 2500 mg, or about 1500 mg and about 2000 mg, including the endpoints of each interval. In some embodiments of any implementation, the total daily dose of MMF is about 100 mg, 500 mg, 1000 mg, about 1500 mg, about 2000 mg, or about 2500 mg. In some embodiments of any implementation, the total daily dose of MMF is lower.

[0059] In some embodiments of any implementation, the MPA is sodium mycophenolate (MS). In some embodiments of any implementation, the total daily dose of MS is between about 10 mg and about 2700 mg, about 360 mg and about 2700 mg, about 720 mg and about 2160 mg, or about 720 mg and about 1620 mg, including the endpoints of each interval. In some embodiments of any implementation, the total daily dose of MS is about 100 mg, about 360 mg, about 720 mg, about 1080 mg, or about 1440 mg. In some embodiments of any implementation, the total daily dose of MS is lower.

[0060] In some embodiments of any implementation scheme, at least one regimen of administering tacrolimus to the subject and at least one regimen of administering MPA.

[0061] In some embodiments of any implementation, one or more immunosuppressants include cyclosporine. In some embodiments of any implementation, at least one regimen of cyclosporine is administered to the subject before, concurrently with, and / or after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of cyclosporine is administered to the subject when the subject exhibits intolerance to a tacrolimus regimen. In some embodiments of any implementation, at least one regimen of cyclosporine is administered to the subject on the same day as and / or concurrently with administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, a first regimen of cyclosporine is administered to the subject on the same day as and / or concurrently with administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of cyclosporine is administered to the subject after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, cyclosporine is administered to the subject at least one regimen approximately 1 hour, 5 hours, 10 hours, 24 hours, 3 months, 6 months, 12 months, 24 months, 36 months, 48 ​​months, 60 months, or later, after administration of a dose of engineered, low-immunogenic islets. In some embodiments of any implementation, the total daily dose of cyclosporine administered to the subject produces a trough plasma concentration between approximately 50 ng / mL and approximately 300 ng / mL, between approximately 100 ng / mL and approximately 250 ng / mL, between approximately 200 ng / mL and approximately 300 ng / mL, or between approximately 150 ng / mL and approximately 200 ng / mL, including the endpoints of each interval.

[0062] In some embodiments of any implementation, a cyclosporine regimen between about 2 mg / kg and about 10 mg / kg is administered to the subject daily. In some embodiments of any implementation, a cyclosporine regimen is administered at a lower dose. In some embodiments of any implementation, a cyclosporine regimen of about 6 mg / kg is administered to the subject daily. In some embodiments of any implementation, a cyclosporine regimen is administered at a lower dose.

[0063] In some embodiments of any implementation scheme, at least one regimen of cyclosporine and at least one regimen of MPA are administered to the subject. In some embodiments of any implementation scheme, at least one regimen of cyclosporine and at least one regimen of ATG are administered to the subject. In some embodiments of any implementation scheme, at least one regimen of ATG is administered to the subject prior to at least one regimen of cyclosporine. In some embodiments of any implementation scheme, wherein: i) a regimen in which an ATG dose of approximately 40 mg / kg is administered to the subject daily for four consecutive days; and ii) after i), a regimen in which cyclosporine doses between approximately 10 mg / kg and approximately 12 mg / kg are administered to the subject daily for six months. In some embodiments of any implementation scheme, the cyclosporine regimen and / or the ATG regimen are administered at a lower dose.

[0064] In some embodiments of any implementation, one or more immunosuppressants include antibodies that bind to MHC, CD2, CD3, CD4, CD7, CD28, B7, CD25, CD40, CD45, CD95, IFN-γ, TNF-α, IL-2Rα, IL-4, IL-5, IL-6R, IL-6, IGF, IGFR1, IL-7, IL-8, IL-10, CD11α, or CD58, and antibodies that bind to any of their ligands. In some embodiments of any implementation, one or more immunosuppressants include soluble IL-15R, IL-10, B7 molecules (such as B7-1, B7-2), their variants and fragments, ICOS, and OX40. In some embodiments of any implementation, one or more immunosuppressants include inhibitors of negative T-cell regulators, such as anti-CTLA-4 antibodies, or similar agents. In some embodiments of any implementation, one or more immunosuppressants include anti-CD25 antibodies or anti-IL-2Rα antibodies. In some embodiments of any implementation scheme, the anti-CD25 antibody or anti-IL-2Rα antibody is selected from the group consisting of baliximab, daclizumab, and alemtuzumab.

[0065] In some embodiments of any implementation scheme, one or more immunosuppressants include bailiximab. In some embodiments of any implementation scheme, at least one regimen of bailiximab is administered to the subject on the same day, concurrently with, and / or after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, at least one regimen of bailiximab 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 later, after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, at least one regimen of bailiximab is administered to the subject after administration of at least one regimen of ATG. In some embodiments of any implementation scheme, at least one of bailiximab is administered to the subject after administration of at least one regimen of ATG and after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of bailiximab is administered to the subject approximately 4 days after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, a regimen of bailiximab between approximately 10 mg and approximately 30 mg is administered to the subject. In some embodiments of any implementation, a regimen of approximately 20 mg of bailiximab is administered to the subject. In some embodiments of any implementation, a lower dose of bailiximab is administered. In some embodiments of any implementation, wherein; i) on the same day as administration of a dose of engineered low-immunogenic islets, a regimen of approximately 20 mg of bailiximab is administered to the subject; and / or, ii) approximately 4 days after administration of a dose of engineered low-immunogenic islets, a regimen of approximately 20 mg of bailiximab is administered to the subject.

[0066] In some embodiments of any implementation scheme, one or more immunosuppressants include dalizumab. In some embodiments of any implementation scheme, at least one regimen of dalizumab is administered to the subject on the same day, concurrently with, and / or after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, at least one regimen of dalizumab 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 later, after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, at least one regimen of dalizumab is administered to the subject approximately every 14 days after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, a regimen of dalizumab between approximately 0.5 mg / kg and approximately 2 mg / kg is administered to the subject. In some embodiments of any implementation scheme, the daclizumab regimen is administered at a lower dose. In some embodiments of any implementation scheme, a regimen of approximately 1 mg / kg of daclizumab is administered to the subject. In some embodiments of any implementation scheme, the daclizumab regimen is administered at a lower dose.

[0067] In some embodiments of any implementation scheme, the subject is administered at least one regimen of tacrolimus and at least one regimen of sirolimus. In some embodiments of any implementation scheme, the subject is administered at least one regimen of tacrolimus and at least one regimen of dalizumab. In some embodiments of any implementation scheme, the subject is administered at least one regimen of sirolimus and at least one regimen of dalizumab. In some embodiments of any implementation scheme, the subject is administered at least one regimen of tacrolimus, at least one regimen of sirolimus, and at least one regimen of dalizumab.

[0068] In some embodiments of any implementation scheme, wherein: i) on the same day as administration of a dose of engineered low-immunogenic islets to the subject, a regimen of approximately 0.2 mg / kg of sirolimus is administered to the subject; ii) following administration of a dose of engineered low-immunogenic islets to the subject, a regimen of approximately 0.1 mg / kg of sirolimus is administered to the subject daily, wherein in the first three months following administration of the composition to the subject, the total daily dose of sirolimus administered to the subject produced a trough plasma concentration between approximately 12 ng / mL and approximately 15 ng / mL, including the endpoints of the interval, and wherein after the first three months, the total daily dose of sirolimus administered to the subject produced a trough plasma concentration between approximately 7 ng / mL and... The following are possible interpretations of the drug administration regimens: iii) administering approximately 1 mg of tacrolimus to the subject on the same day as administering a dose of engineered low-immunogenic islets; iv) administering approximately 1 mg of tacrolimus twice daily to the subject approximately 12 hours after administering a dose of engineered low-immunogenic islets, wherein the total daily dose of tacrolimus administered to the subject produces a trough blood concentration between approximately 3 ng / mL and approximately 6 ng / mL, including the endpoints of the interval; and / or v) administering approximately 1 mg / kg of dalizumab to the subject approximately every 14 days after administering a dose of engineered low-immunogenic islets.

[0069] In some embodiments of any implementation scheme, the sirolimus, tacrolimus, and / or dalizumab regimens are administered at lower doses. In some embodiments of any implementation scheme, no glucocorticoids are administered to the subject. In some embodiments of any implementation scheme, one or more immunosuppressants include alemtuzumab. In some embodiments of any implementation scheme, at least one regimen of alemtuzumab is administered to the subject before, on the same day, concurrently with, and / or after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, at least one regimen of alemtuzumab is administered to the subject before administration of at least one regimen of tacrolimus and / or MPA. In some embodiments of any implementation scheme, at least one regimen of alemtuzumab and at least one regimen of tacrolimus and / or MPA are administered to the subject after administration of a dose of engineered low-immunogenic islets.

[0070] In some embodiments of any implementation, one or more immunosuppressants include an anti-CD3 antibody. In some embodiments of any implementation, the anti-CD3 antibody is an anti-CD3 antibody. εAntibodies. In some embodiments of any implementation, the anti-CD3 antibody is OKT3. In some embodiments of any implementation, one or more immunosuppressants include an anti-IL-33 antibody. In some embodiments of any implementation, one or more immunosuppressants include an anti-CD95 antibody. In some embodiments of any implementation, one or more immunosuppressants include fingolimod hydrochloride. In some embodiments of any implementation, one or more immunosuppressants include liposomal clophosphonate. In some embodiments of any implementation, one or more immunosuppressants include CTLA4-Ig. In some embodiments of any implementation, one or more immunosuppressants include the methyl 2-(1′H-indole-3′-carbonyl)-thiazolyl-4-carboxylate (ITE) ligand of the aryl hydrocarbon receptor (AhR). In some embodiments of any implementation, one or more immunosuppressants include the T1D autoantigen proinsulin. In some embodiments of any implementation, one or more immunosuppressants include TGF-β-glucan-β-carboxylic acid. β 1. In some embodiments of any implementation, one or more immunosuppressants include dexamethasone. In some embodiments of any implementation, one or more immunosuppressants include methotrexate. In some embodiments of any implementation, one or more immunosuppressants include gold salts. In some embodiments of any implementation, one or more immunosuppressants include sulfasalazine. In some embodiments of any implementation, one or more immunosuppressants include one or more antimalarial drugs. In some embodiments of any implementation, one or more immunosuppressants include buquina. In some embodiments of any implementation, one or more immunosuppressants include leflunomide. In some embodiments of any implementation, one or more immunosuppressants include imidazolidin. In some embodiments of any implementation, one or more immunosuppressants include 15-deoxyguanidin. In some embodiments of any implementation, one or more immunosuppressants include 6-mercaptopurine. In some embodiments of any implementation, one or more immunosuppressants include cyclophosphamide. In some embodiments of any implementation, one or more immunosuppressants include antithymocyte globulin. In some embodiments of any implementation, one or more immunosuppressants include antibacterial agents.

[0071] In some embodiments of any implementation scheme, the antimicrobial agent is selected from the group consisting of: trimethoprim / sulfamethoxazole, penicillin, amoxicillin, cephalexin, erythromycin (E-Mycin), clarithromycin (Biaxin), azithromycin (Zithromax), ciprofloxacin (Cipro), levofloxacin (Levaquin), ofloxacin (Floxin), bactrim and trimethoprim (Proloprim), tetracycline (Sumycin) and doxycycline (Vibramycin), gentamicin (Garamycin) and tobramycin (Tobrex). In some embodiments of any implementation scheme, the antimicrobial agent is trimethoprim / sulfamethoxazole. In some embodiments of any implementation scheme, at least one regimen of trimethoprim / sulfamethoxazole is administered to the subject after administration of a dose of engineered, low-immunogenic islet insulin.

[0072] In some embodiments of any implementation scheme, at least one regimen of trimethoprim / sulfamethoxazole 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 later, after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, at least one regimen of trimethoprim / sulfamethoxazole is administered to the subject daily for approximately 6 months, following administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, a trimethoprim / sulfamethoxazole regimen between approximately 50 mg and approximately 500 mg is administered to the subject. In some embodiments of any implementation scheme, a trimethoprim / sulfamethoxazole regimen is administered at a lower dose. In some embodiments of any implementation scheme, a trimethoprim / sulfamethoxazole regimen between approximately 80 mg and approximately 400 mg is administered to the subject. In some embodiments of any implementation, the trimethoprim / sulfamethoxazole regimen is administered at a lower dose. In some embodiments of any implementation, one or more immunosuppressants include an antifungal agent. In some embodiments of any implementation, the antifungal agent is selected from the group consisting of clotrimazole, miconazole, ketoconazole, itraconazole, and fluconazole. In some embodiments of any implementation, the antifungal agent is clotrimazole. In some embodiments of any implementation, at least one regimen of clotrimazole is administered to the subject before, on the same day, concurrently with, and / or after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, a regimen of clotrimazole is administered to the subject approximately four times daily. In some embodiments of any implementation, at least one regimen of clotrimazole is administered to the subject daily for up to approximately three months after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, one or more immunosuppressants include an antiviral agent. In some embodiments of any implementation, the antiviral agent is selected from the group consisting of darunavir, atazanavir, ritonavir, acyclovir, valacyclovir, valganciclovir, tenofovir, and raltegravir. In some embodiments of any implementation, the antiviral agent is an anticytomegalovirus agent. In some embodiments of any implementation, the antiviral agent is valganciclovir. In some embodiments of any implementation, at least one regimen of valganciclovir is administered to the subject after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of valganciclovir 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 later, after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, a valganciclovir regimen between about 300 mg and about 1,000 mg is administered to the subject. In some embodiments of any implementation, a valganciclovir regimen is administered at a lower dose. In some embodiments of any implementation, a regimen of about 450 mg valganciclovir is administered to the subject daily after administration of a dose of engineered, low-immunogenic islets. In some embodiments of any implementation, a valganciclovir regimen is administered at a lower dose. In some embodiments of any implementation, a regimen of about 900 mg valganciclovir is administered to the subject daily after about day 12 following administration of a dose of engineered, low-immunogenic islets. In some embodiments of any implementation, a valganciclovir regimen is administered at a lower dose. In some embodiments of any implementation, a regimen of 900 mg valganciclovir is administered to the subject after administration of a dose of engineered, low-immunogenic islets, continuing until about week 14.

[0073] In some embodiments of any implementation, one or more immunosuppressants include a blood rheology agent. In some embodiments of any implementation, the blood rheology agent is pentoxifylline. In some embodiments of any implementation, at least one regimen of pentoxifylline is administered to the subject before, on the same day, concurrently with, and / or after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of pentoxifylline is administered to the subject before administration of a dose of engineered low-immunogenic islets.

[0074] In some embodiments of any implementation scheme, pentoxifylline is administered to the subject approximately 7 days, approximately 6 days, approximately 5 days, approximately 4 days, approximately 3 days, approximately 2 days, approximately 1 day, approximately 12 hours, approximately 10 hours, approximately 8 hours, approximately 6 hours, approximately 4 hours, approximately 2 hours, or approximately 1 hour prior to the administration of a dose of engineered, low-immunogenic islets. In some embodiments of any implementation scheme, pentoxifylline is administered to the subject approximately 2 days prior to the administration of a dose of engineered, low-immunogenic islets.

[0075] In some embodiments of any implementation scheme, pentoxifylline is administered to the subject in at least one manner after administering a dose of engineered, low-immunogenic islets.

[0076] In some embodiments of any implementation scheme, pentoxifylline 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 later, after administration of a dose of engineered, low-immunogenic islets. In some embodiments of any implementation scheme, pentoxifylline is administered to the subject after administration of a dose of engineered, low-immunogenic islets, continuing until approximately day 7.

[0077] In some embodiments of any implementation, a pentoxifylline regimen between about 300 mg and about 500 mg is administered to the subject. In some embodiments of any implementation, a pentoxifylline regimen is administered at a lower dose.

[0078] In some embodiments of any implementation, one or more immunosuppressants comprise one or more anticoagulants. In some embodiments of any implementation, one or more anticoagulants are selected from the group consisting of aspirin, enoxaparin, and heparin. In some embodiments of any implementation, one or more anticoagulants are aspirin. In some embodiments of any implementation, at least one regimen of aspirin is administered to the subject after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, one or more anticoagulants are enoxaparin. In some embodiments of any implementation, at least one regimen of enoxaparin is administered to the subject after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, one or more anticoagulants are heparin. In some embodiments of any implementation, at least one regimen of heparin is administered to the subject after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of enoxaparin is administered to the subject after administration of at least one regimen of heparin.

[0079] In some embodiments of any implementation scheme, one or more immunosuppressants include a DNA synthesis inhibitor. In some embodiments of any implementation scheme, the DNA synthesis inhibitor is fludarabine. In some embodiments of any implementation scheme, at least one regimen of fludarabine is administered to the subject before, concurrently with, and / or after administration of a dose of engineered, low-immunogenic islets. In some embodiments of any implementation scheme, at least one regimen of fludarabine is administered to the subject before administration of a dose of engineered, low-immunogenic islets.

[0080] In some embodiments of any implementation scheme, at least one regimen of fludarabine is administered to the subject approximately 14 days, approximately 10 days, 7 days, approximately 6 days, approximately 5 days, approximately 4 days, approximately 3 days, approximately 2 days, approximately 1 day, approximately 12 hours, approximately 10 hours, approximately 8 hours, approximately 6 hours, approximately 4 hours, approximately 2 hours, or approximately 1 hour prior to administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, a first regimen of fludarabine is administered to the subject approximately 2 days to approximately 14 days prior to administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, a fludarabine regimen is administered to the subject daily for approximately 2 days, approximately 3 days, or approximately 4 days prior to administration of a dose of engineered low-immunogenic islets. In some implementation schemes of any scheme, the fludarabine regimen is administered to the subject on days 5, 4, and 3, prior to administering a dose of engineered, low-immunogenic islets to the subject.

[0081] In some implementation schemes of any scheme, the concentration will be between approximately 10 mg / m². 2 and approximately 40 mg / m 2 The fludarabine regimen was administered to the subject. In some embodiments of any given regimen, the fludarabine regimen was administered at a lower dose. In some embodiments of any given regimen, approximately 30 mg / m² was used. 2 The fludarabine regimen is administered to the subject. In some embodiments of any implementation, the fludarabine regimen is administered at a lower dose. In some embodiments of any implementation, fludarabine is administered intravenously to the subject.

[0082] In some embodiments of any implementation, one or more immunosuppressants include an alkylating agent. In some embodiments of any implementation, the alkylating agent is cyclophosphamide. In some embodiments of any implementation, at least one regimen of cyclophosphamide is administered to the subject before, concurrently with, and / or after administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of cyclophosphamide is administered to the subject before administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation, at least one regimen of cyclophosphamide is administered to the subject approximately 14 days, approximately 10 days, 7 days, approximately 6 days, approximately 5 days, approximately 4 days, approximately 3 days, approximately 2 days, approximately 1 day, approximately 12 hours, approximately 10 hours, approximately 8 hours, approximately 6 hours, approximately 4 hours, approximately 2 hours, or approximately 1 hour before administration of a dose of engineered low-immunogenic islets. In some embodiments of any implementation scheme, cyclophosphamide is administered to the subject as a first regimen approximately 2 days to approximately 14 days prior to administration of a dose of engineered, low-immunogenic islets. In some embodiments of any implementation scheme, cyclophosphamide is administered to the subject daily for approximately 2 days, approximately 3 days, or approximately 4 days prior to administration of a dose of engineered, low-immunogenic islets. In some embodiments of any implementation scheme, cyclophosphamide is administered to the subject on days 5, 4, and 3 prior to administration of a dose of engineered, low-immunogenic islets.

[0083] In some implementation schemes of any scheme, the concentration will be between approximately 400 mg / m². 2 and approximately 600 mg / m 2 The cyclophosphamide regimen was administered to the subject. In some embodiments of any given implementation, the cyclophosphamide regimen was administered at a lower dose. In some embodiments of any given implementation, approximately 500 mg / m² was used. 2 The cyclophosphamide regimen is administered to the subject. In some embodiments of any implementation, the cyclophosphamide regimen is administered at a lower dose. In some embodiments of any implementation, cyclophosphamide is administered intravenously to the subject.

[0084] In some embodiments of any implementation scheme, at least one regimen of fludarabine and at least one regimen of cyclophosphamide are administered to the subject. In some embodiments of any implementation scheme, at least one regimen of fludarabine is administered to the subject prior to administration of at least one regimen of cyclophosphamide. In some embodiments of any implementation scheme, at least one regimen of fludarabine and at least one regimen of cyclophosphamide are administered to the subject prior to administration of a dose of engineered, hypoimmunogenic islets.

[0085] In some embodiments of any implementation scheme, wherein: i) approximately 30 mg / m² daily for approximately 2 to 7 days prior to administration of a dose of engineered, hypoimmunogenic islets to the subject. 2 The regimen of fludarabine and approximately 500 mg / m² 2 The regimen of cyclophosphamide was administered to the subjects for 3 consecutive days; ii) approximately 30 mg / m² daily for approximately 2 to 14 days prior to administration of a dose of engineered, hypoimmunogenic islets to the subjects. 2 The regimen of fludarabine and approximately 500 mg / m² 2 The cyclophosphamide regimen was administered to the subject for two consecutive days; or, iii) prior to administration of a dose of engineered, low-immunogenic islets to the subject, approximately 30 mg / m² was administered on days 5, 4, and 3. 2 The regimen of fludarabine and approximately 500 mg / m² 2 The cyclophosphamide regimen was administered to the subject. In some embodiments of any implementation, the fludarabine regimen and / or cyclophosphamide regimen were administered at a lower dose.

[0086] In some embodiments of any implementation scheme, tapering of the administration of one or more immunosuppressants is also included. In some embodiments of any implementation scheme, tapering includes gradually reducing the amount of one or more immunosuppressants administered to the subject. In some embodiments of any implementation scheme, tapering is completed when at least one of the one or more immunosuppressants has not been administered to the subject.

[0087] In some embodiments of any implementation, one or more molecules that regulate the expression of cell surface proteins of one or more MHC class I molecules are B2M. In some embodiments of any implementation, the modification includes a modification that regulates the expression of cell surface proteins of one or more MHC class I molecules, and the modification inactivates or disrupts one or more alleles of B2M. In some embodiments of any implementation, the modification that inactivates or disrupts one or more alleles of B2M reduces the mRNA expression of the B2M gene. In some embodiments of any implementation, the modification that inactivates or disrupts one or more alleles of B2M reduces the protein expression of B2M. In some embodiments of any implementation, the modification that inactivates or disrupts one or more alleles of B2M includes: inactivation or disruption of one allele of the B2M gene; inactivation or disruption of two alleles of the B2M gene; or inactivation or disruption of all B2M-coding alleles in the cell. In some embodiments of any implementation, inactivation or disruption includes insertions or deletions in the B2M gene. In some embodiments of any implementation, inactivation or disruption includes frameshift mutations or deletions of a continuous segment of genomic DNA in the B2M gene.

[0088] In some embodiments of any implementation, the modification is a modification that regulates the expression of one or more MHC class II molecules, and the modification inactivates or disrupts one or more alleles of CIITA. In some embodiments of any implementation, the modification that inactivates or disrupts one or more alleles of CIITA reduces the protein expression of CIITA. In some embodiments of any implementation, the modification that inactivates or disrupts one or more alleles of CIITA includes: inactivation or disruption of one allele of the CIITA gene; inactivation or disruption of two alleles of the CIITA gene; or inactivation or disruption of all CIITA-coding alleles in the cell. In some embodiments of any implementation, inactivation or disruption includes insertions or deletions in the CIITA gene. In some embodiments of any implementation, inactivation or disruption is a frameshift mutation or a deletion of a continuous segment of genomic DNA in the CIITA gene.

[0089] In some embodiments of any implementation, the expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR is reduced in engineered, low-immunogenic islets.

[0090] In some embodiments of any implementation scheme, 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.

[0091] In some embodiments of any implementation, at least one of the one or more tolerogenic factors is CD47. In some embodiments of any implementation, the one or more tolerogenic factors is 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 strands; wherein the one or more extracellular domains comprise a signal regulatory protein α (SIRPα) interacting motif, and wherein the engineered protein does not comprise one or more full-length CD47 intracellular domains. In some embodiments, the SIRPα interacting motif is or comprises a CD47 extracellular domain or a portion thereof. In some embodiments, the SIRPα interacting motif is or comprises a SIRPα antibody or a portion thereof.

[0092] In some embodiments of any implementation, the modification that increases the expression of one or more tolerogenic factors includes an exogenous polynucleotide encoding one or more tolerogenic factors. In some embodiments of any implementation, the exogenous polynucleotide encoding one or more tolerogenic factors is integrated into the genome of engineered hypoimmunogenic islets. In some embodiments of any implementation, one or more tolerogenic factors include CD47, and the engineered hypoimmunogenic islets express CD47 at a first level greater than or equal to about 5-fold of a second level expressed by control or wild-type islet cells. In some embodiments of any implementation, CD47 is expressed at a first level greater than or equal to about 10-fold, greater than or equal to about 20-fold, greater than or equal to about 30-fold, greater than or equal to about 40-fold, greater than or equal to about 50-fold, greater than or equal to about 60-fold, or greater than or equal to about 70-fold of the second level expressed by control or wild-type islet cells.

[0093] In some embodiments of any implementation, one or more tolerogenic factors include CD47, and CD47 is expressed by engineered, low-immunogenic islets at greater than or equal to about 20,000 molecules / cell. In some embodiments of any implementation, CD47 is expressed by engineered, low-immunogenic islets at greater than or equal to about 30,000 molecules / cell, greater than or equal to about 50,000 molecules / cell, greater than or equal to about 100,000 molecules / cell, greater than or equal to about 200,000 molecules / cell, greater than or equal to about 300,000 molecules / cell, greater than or equal to about 400,000 molecules / cell, greater than or equal to about 500,000 molecules / cell, or greater than or equal to about 600,000 molecules / cell.

[0094] In some embodiments of any implementation, the engineered low-immunogenic islets have the phenotypes B2M insertion-deletion / insertion-deletion, CIITA insertion-deletion / insertion-deletion, and CD47tg. In some embodiments of any implementation, at least 85% of the cells in the cell dose of engineered low-immunogenic islets are modified. In some embodiments of any implementation, at least 90%, at least 92%, at least 95%, or at least 98% of the cells are modified. In some embodiments of any implementation, at least 85% of the cells in the cell dose of engineered low-immunogenic islets have the phenotypes B2M insertion-deletion / insertion-deletion, CIITA insertion-deletion / insertion-deletion, and CD47tg. In some embodiments of any implementation, at least 90%, at least 92%, at least 95%, or at least 98% of the cells have this phenotype.

[0095] In some embodiments of any implementation, the engineered low-immunogenic islets exhibit one or more functions of wild-type or control β-islet cells, optionally wherein 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 vivo in response to glucose injection, and glucose clearance after glucose injection in vivo. In some embodiments of any implementation, the engineered low-immunogenic islets are capable of glucose-stimulated insulin secretion (GSIS), optionally wherein insulin secretion is performed in a perfusion GSIS assay. In some embodiments of any implementation, the GSIS is dynamic GSIS, which includes dynamic insulin secretion in a first phase and a second phase. In some embodiments of any implementation, the GSIS is static GSIS, optionally wherein the static incubation index is greater than or equal to about 1, greater than or equal to about 2, greater than or equal to about 5, greater than or equal to about 10, or greater than or equal to about 20.

[0096] In some embodiments of any implementation, the insulin secretion level of the engineered low-immunogenic islets is at least 20% of the level observed against primary islets (optionally, cadaveric islets). In some embodiments of any implementation, the insulin secretion level of the engineered low-immunogenic islets is at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the level observed against primary islets (optionally, cadaveric islets).

[0097] In some embodiments of any implementation, the total insulin content of the engineered, low-immunogenic islets is greater than or equal to about 500 µIU insulin / 5000 cells, greater than or equal to about 1000 µIU insulin / 5000 cells, greater than or equal to about 2000 µIU insulin / 5000 cells, greater than or equal to about 3000 µIU insulin / 5000 cells, or greater than or equal to about 4000 µIU insulin / 5000 cells. In some embodiments of any implementation, the proinsulin to insulin ratio of the modified SC-β cells is equal to or between about 0.02 and about 0.1, optionally equal to or between about 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and any value between the foregoing.

[0098] In some embodiments of any implementation scheme, the engineered low-immunogenic islets exhibited functionality for more than 2 weeks after transplantation into the subject. In some embodiments of any implementation scheme, the engineered low-immunogenic islets exhibited functionality for more than 3 weeks, more than 4 weeks, more than 8 weeks, more than 3 months, more than 6 months, or more than 12 months after transplantation into the subject.

[0099] In some implementations of any implementation scheme, the functionality is selected from the group consisting of: maintaining fasting blood glucose levels, secreting insulin in response to glucose injection, and clearing glucose after glucose injection.

[0100] In some embodiments of any implementation, the dose is approximately 1 × 10⁻⁶. 7 One cell to approximately 3 × 10 8 Cells. In some embodiments of any implementation, the dose is approximately 1.25 × 10⁻⁶ cells. 5 Cells / kg to approximately 1.2 × 10⁻⁶ 7 Cells / kg

[0101] In some embodiments of any implementation, the dose is from about 6,500 islet equivalents (IEQ) to about 600,000 IEQ. In some embodiments of any implementation, the dose is from about 80 IEQ / kg to about 24,000 IEQ / kg. In some embodiments of any implementation, no immunosuppressive regimen was administered to the subject. Attached Figure Description

[0102] Figures 1A-1B Results of an allogeneic transplantation study were presented, which evaluated the immune response of non-human primate (NHP) recipients to primary allogeneic NHP islet cells. For transplanted B2M... - / - CIITA - / - CD47 tg NHP primary pancreatic islet cells provided quantitative results of BLI luciferase expression. Figure 1A For quantitative results; Figure 1B (For the corresponding BLI image).

[0103] Figures 2A-2D Results of an intramuscular injection (IM) study of allogeneic transplantation in NHP were presented, and the immune response was evaluated. For transplantation with B2M... - / - CIITA - / - CD47 tg NHP in primary pancreatic islet cells provides interferon-γ (IFNg) levels ( Figure 2A For transplantation with B2M - / - CIITA - / - CD47 tg NHP primary pancreatic islet cells provide donor-specific antibody (DSA) IgM levels ( Figure 2B ) and IgG levels ( Figure 2C For transplantation with B2M - / - CIITA - / - CD47 tg NHP-sensitized primary pancreatic islet cells (with elevated pre-transplant IgG levels) also provide DSA IgG levels ( Figure 2D ).

[0104] Figure 3 Provided B2M - / - CIITA - / - CD47 tg The result of in vitro natural killer (NK) cell-mediated cell killing in NHP primary pancreatic islet cells.

[0105] Figures 4A-4D Display B2M - / - CIITA - / -CD47 tg Phenotypic analysis and allogeneic transplantation of primary pancreatic islet cells from rhesus monkeys. Figure 4A Shown in B2M - / - CIITA - / - CD47 tg Immunofluorescence staining of somatostatin, insulin, and glucagon (top image) and CD47, MHC class I, and DAPI (bottom image) before and after editing. Figure 4B Shown in B2M - / - CIITA - / - CD47 tg Before and after editing, MHC class I, MHC class II and CD47 expression in rhesus monkey pancreatic islets. Figure 4C Shown in B2M - / - CIITA - / - CD47 tg Insulin release from rhesus monkey islets in vitro before and after editing. Figure 4D Shown in B2M - / - CIITA - / - CD47 tg The composition of the pancreatic islets of a rhesus monkey before and after editing.

[0106] Figure 5 It showed that an allogeneic B2M was transplanted. - / - CIITA - / - CD47 tg Blood glucose measurements in diabetic nonhuman primates (NHPs) with primary pancreatic islet cells (NHP). Blood was collected in the morning (morning blood glucose) and afternoon (afternoon blood glucose). Diabetes: >127 mg / dL; Impaired fasting glucose: >80-127 mg / dL; Normal: <80 mg / dL; and Hypoglycemia: <30 mg / dL.

[0107] Figure 6 It showed that an allogeneic B2M was transplanted. - / - CIITA - / - CD47 tg Blood glucose measurements in diabetic nonhuman primates (NHPs) with NHP primary pancreatic islet cells, up to day 111 post-STZ. Blood samples were collected in the morning (morning blood glucose) and afternoon (afternoon blood glucose). Hyperglycemia (diabetes): >127 mg / dL; impaired fasting glucose: >80-127 mg / dL; normal: <80 mg / dL; and hypoglycemia: <30 mg / dL.

[0108] Figure 7 It showed that an allogeneic B2M was transplanted. - / - CIITA - / - CD47 tg Blood glucose measurements in diabetic nonhuman primates (NHPs) with NHP primary pancreatic islet cells, continuing up to day 226 post-STZ. Blood samples were collected in the morning (morning blood glucose) and afternoon (afternoon blood glucose). Hyperglycemia (diabetes): >127 mg / dL; impaired fasting glucose: >80-127 mg / dL; normal: <80 mg / dL; and hypoglycemia: <30 mg / dL.

[0109] Figure 8A The daily administration of exogenous insulin (U / day) varies over time. Figure 8B It shows the changes in blood glucose levels (mg / dL) over time in the morning and evening. Figure 8C Serum C-peptide levels (ng / mL) are shown as changing over time. Figure 8D Displays body weight (kg) over time. An asterisk indicates the time point at which C-peptide was measured.

[0110] Figure 9 It showed that an allogeneic B2M was transplanted. - / - CIITA - / - CD47 tg C-peptide measurements of diabetic nonhuman primates (NHP) from primary NHP pancreatic islet cells. Before STZ: C-peptide measurements before intravenous streptozotocin (STZ); d50 after STZ: C-peptide measurements on day 50 (d50) after STZ injection; d0 (d78 after STZ): C-peptide measurements on day 78 (d78) after STZ injection and day 0 after islet cell transplantation; d7 (d85 after STZ): C-peptide measurements on day 85 (d85) after STZ injection and day 7 after islet cell transplantation; d14 (d92 after STZ): C-peptide measurements on day 92 (d92) after STZ injection and day 14 (d14) after islet cell transplantation; d28 (d106 after STZ): C-peptide measurements on day 106 (d106) after STZ injection and day 28 (d28) after islet cell transplantation; d42 (d120 after STZ): C-peptide measurements on day 120 (d120) after STZ injection and day 42 (d42) after islet cell transplantation; d90 (d172 after STZ): C-peptide measurements on day 172 (d172) after STZ injection and day 90 (d90) after islet cell transplantation.

[0111] Figure 10 It showed that an allogeneic B2M was transplanted. - / - CIITA - / - CD47 tgGlucose tolerance measurements in diabetic nonhuman primates (NHP) with NHP primary pancreatic islet cells. Pre-STZ: Glucose tolerance measurements prior to intravenous streptozotocin (STZ); d50 (post-STZ): Glucose tolerance measurements on day 50 (d50) after STZ injection; d103 (d25 post-cell transplantation): Glucose tolerance measurements on day 103 (d103) after STZ injection and day 25 (d25) post-islet cell transplantation; Overlay: Pre-STZ, d50, and d103.

[0112] Figures 11A-11L Showing B2M - / - CIITA - / - CD47 tg Cell- and antibody-mediated responses in primary pancreatic islet cells of rhesus monkeys. Figure 11A The results show the ELISpot assays performed using recipient monkey PBMCs collected at predetermined time points. Figures 11B-11E It showed the use of receptor cynomolgus monkey T cells ( Figure 11B ), PBMC ( Figure 11C NK cells Figure 11D ) and macrophages ( Figure 11E The killing effect was measured. The percentage of target cells killed is displayed on the y-axis. Figures 11F-11I It shows Ig levels, including total serum IgM ( Figure 11F IgG ( Figure 11G Donor-specific antibody (DSA) IgM Figure 11H ) and DSA IgG ( Figure 11I ). Figures 11J-11L This demonstrates the use of complement-removed receptor cynomolgus monkey serum and NK cells ( Figure 11J ) or macrophages ( Figure 11K Antibody-dependent cytotoxicity (ADCC) assay, and assay using intact recipient monkey serum ( Figure 11L The CDC assay was performed. The percentage of target cell killing is shown on the y-axis.

[0113] Figure 12A and Figure 12B Rhesus monkey B2M showed a response to anti-CD47 antibody (magrolimab) treatment. - / - CIITA - / - CD47 tg Primary pancreatic islet cells were killed by cynomolgus monkey NK cells or macrophages.

[0114] Figures 13A-13C Immunohistochemical staining was observed at the sites of pancreatic islets and primary muscle islet transplants. Figure 13A This shows the pancreas from a healthy cynomolgus monkey. Figure 13B The pancreas of the recipient cynomolgus monkey is shown. Figure 13C Showing the muscle implantation site in the recipient cynomolgus monkey. Detailed Implementation

[0115] This document provides methods relating to administering engineered islets to a subject, the engineered islets comprising β cells engineered to evade the immune system (also referred herein as modified immune-evading β cells or low-immunogenic (HIP) β cells). In some embodiments, the engineered islets may be engineered primary islets. In some embodiments, the engineered islets may be engineered islet cells differentiated from pluripotent stem cells. In some embodiments, the engineered islet cells (including engineered β cells) exhibit characteristics that allow them to evade immune recognition. In some embodiments, the engineered islet cells (including engineered β cells) are low-immunogenic (also referred to as low-immunogenic or HIP). In some aspects, the engineered islet cells (including engineered β cells) are not subject to innate immune cell rejection. In some aspects, the engineered islet cells (including engineered β cells) provided herein exhibit reduced innate immune cell rejection and / or adaptive immune cell rejection (e.g., low-immunogenic cells). For example, in some embodiments, engineered islet cells (including engineered β cells) exhibit reduced susceptibility to NK cell-mediated lysis and / or macrophage phagocytosis. In some embodiments, engineered islets and cells can be used as a source of universally compatible cells or tissues (e.g., universal donor cells or tissues) transplanted into recipient subjects. Such low-immunogenic cells retain cell-specific properties and characteristics after administration to a subject (e.g., transplantation or engraftment). In some embodiments, when transplanted or engrafted into a subject, engineered islet cells cluster into effective endocrine organoids, referred to as pseudo-islet grafts (p-islets). Thus, in some embodiments, engineered islets are HIP pseudo-islets (HIP p-islets). In some embodiments, effective endocrine organoids provide stable endocrine function via insulin production and secretion, thereby achieving insulin independence in the subject. In some embodiments, stable endocrine function and insulin independence are achieved without immunosuppression. In some implementations, engineered pancreatic islet cells (including engineered β cells) can be used as a cell source for allogeneic therapy, regardless of the subject's genetic composition.

[0116] In some embodiments, the provided method is used to treat a subject with a β-cell-related condition (e.g., diabetes), such as improving the subject's glucose tolerance. In a particular embodiment, the method is used to treat a subject with type 1 diabetes, such as improving the subject's glucose tolerance. In other embodiments, the method improves graft function of the provided islet cells. In some embodiments, the method restores the subject's glucose metabolism.

[0117] Patients with type 1 diabetes (T1DM) or impaired hypoglycemic awareness (IAH) lack basic hypoglycemic-induced defense mechanisms and are therefore at increased risk of severe hypoglycemic events (Hwang et al., J Clin Invest (2018) 128:1485-195; Lin et al., J Diabetes Investig (2020) 11:1388-1402). Current treatments for patients with type 1 diabetes mellitus (T1DM) include intensive insulin therapy. However, these treatments can lead to severe hypoglycemia, which is associated with altered mental status, seizures, arrhythmias, and even death (Bornstein et al., Nat Rev Endocrinol (2022) 18:389-390).

[0118] Pancreatic islet transplantation has been shown to be superior to insulin therapy, with improved patient survival and quality of life (Boughton et al., Diabetes Obes Metab (2021) 23:1389-1396). However, pancreatic islet transplantation in patients with type 1 diabetes mellitus (T1DM) is severely hampered by the requirement for sustained immunosuppression. Systemic immunosuppression used to prevent rejection of allogeneic islet grafts in patients is accompanied by significant morbidity, including chronic kidney injury, infection, and cancer, and transplant survival is only 4.4 to 5.9 years (Hering et al., 2021). 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 immunosuppression, patients with type 1 diabetes mellitus (T1DM) often become sensitized to allogeneic transplantation and develop elevated population-reactive antibodies, complicating any subsequent transplantation. Therefore, improved pancreatic islet transplantation methods are needed, including those for the treatment of diabetes.

[0119] The provided implementation addresses these needs. The provided implementation involves primary islets engineered to be hypoimmune, thereby reducing or eliminating the need for immunosuppression. Specifically, the results of this paper demonstrate that allogeneic transplantation of primary, hypoimmune-engineered β-islet cells into a fully immunologically active nonhuman primate model of diabetes provides stable endocrine function and achieves insulin independence without immunosuppression, without inducing any detectable immune response. Therefore, this disclosure demonstrates that hypoimmune primary β-islet cells provide a novel and curative cell therapy for T1DM, and can do so with reduced or no immunosuppression.

[0120] In some embodiments, the engineered islets (including engineered β cells) described herein are low immunogenicity upon administration (e.g., transplantation or implantation) and, in some embodiments, evade immune rejection. Non-limiting examples of modifications leading to evasion of immune rejection include: reduced expression of major histocompatibility complex (MHC) human leukocyte antigen (HLA) class I and HLA class II antigens, and increased expression of one or more tolerogenic factors (such as CD47). In some embodiments, engineered islets (including engineered β cells) are administered to MHC-mismatched allogeneic subjects.

[0121] In some embodiments, engineered islet cells (including engineered β cells) contain the following modifications relative to control or wild-type β cells: (a) reduced expression of one or more major histocompatibility complex (MHC) class I molecules and / or one or more MHC class II molecules; and (b) increased expression of one or more tolerogenic factors in the engineered islets. In some embodiments, this modification renders the cells hypoimmune, which in some respects allows the cells to evade immune rejection compared to control or wild-type islet cells (such as primary human islet β cells). For the purposes of this document, the term "engineered islets" may be used interchangeably with the term "islets of hypoimmune origin".

[0122] Engineered islets include engineered cells, such as engineered β cells, which utilize the expression of tolerogenic factors, and the expression (e.g., surface expression) of one or more MHC class I molecules and / or one or more MHC class II molecules is also modulated (e.g., reduced or eliminated). In some embodiments, the modification reducing the expression of one or more MHC class I molecules is a modification reducing the expression of β-2 microglobulin (B2M). In some embodiments, the modification reducing the expression of one or more MHC class II molecules is a modification reducing the expression of CIITA. In some embodiments, engineered cells containing the modifications described herein (including reduced or eliminated expression of MHC class I or MHC class II molecules, and increased expression of CD47 or other tolerogenic factors) survive, colonize, persist, and function after administration (e.g., transplantation or colonization). In some embodiments, compared to control or wild-type islets (such as unmodified islet cells without modifications that induce cellular hypoimmunization), cells of engineered islets exhibit enhanced survival and / or enhanced colonization and / or function over a longer period.

[0123] In some implementations, engineered islets are administered via intramuscular injection (e.g., intramuscular injection in the forearm).

[0124] In some embodiments, genome editing technologies using rare-cutting endonucleases (e.g., CRISPR / Cas, TALEN, zinc finger nucleases, large-scale nucleases, and homing endonuclease systems) (e.g., by deleting the genomic DNA of key immune genes) are used to reduce or eliminate the expression of immune genes, such as those involved in regulating the expression of MHC class I or MHC class II molecules, in islet cells used to generate engineered islets. In some embodiments, genome editing technologies or other gene regulation technologies are used to insert tolerance-inducing (tolerance-inducing) factors (e.g., CD47) into target genomic loci in islet cells used to generate engineered islets, thereby producing engineered islets that can evade immune recognition after being implanted into a recipient subject. Therefore, engineered islets exhibit regulated expression (e.g., reduced or eliminated expression) of one or more genes and factors affecting the expression of MHC class I and / or MHC class II molecules, regulated expression (e.g., reduced or eliminated expression) of tolerogenic factors (such as CD47), and regulated expression (e.g., overexpression), providing recognition of a weakened immune system in the recipient subject. In some embodiments, the modified cells may also exhibit regulated expression (e.g., reduced expression) of CD142, which in some aspects can be reduced by genome editing technologies (e.g., CRISPR / Cas, TALEN, zinc finger nucleases, large-scale nucleases, and homing endonuclease systems), thereby reducing or eliminating CD142 expression (e.g., by deleting the genomic DNA of key immune genes). In some embodiments, engineered islets may exhibit regulated expression (e.g., increased expression) of one or more complement inhibitors selected from CD46, CD59, CD55, and CD35, which in some aspects can be increased by genome editing technologies, thereby inserting or integrating exogenous polynucleotides encoding one or more complement inhibitors into genomic loci in the engineered islets.

[0125] In some implementations, beta-cell-related disorders are metabolic disorders. In some implementations, metabolic disorders include familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabby disease, maple syrup urine disease, metachromatic leukodystrophy, mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), Niemann-Pick disease, phenylketonuria (PKU), porphyria, Ty Sachs disease, Wilson's disease, type 1 diabetes, type 2 diabetes, obesity, hypertension, dyslipidemia, or carbohydrate intolerance. In some implementations, beta-cell-related disorders are type 1 diabetes.

[0126] Unless otherwise expressly stated, the practice of particular embodiments will employ conventional methods within the scope of the art, including chemical, biochemical, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology, many of which are described below for illustrative purposes. Such techniques are well explained in the literature.See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., 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, Volumes I and II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes (Academic Press, New York, 1992); Transcription and Translation (edited by B. Hames and S. Higgins). 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Current Protocols in Immunology QE Coligan, AM Kruisbeek, DH Margulies, EM Shevach and W. Strober (eds., 1991); Annual Review of Immunology; and special issues in journals such as Advances in Immunology.

[0127] All publications (including patent documents, scientific literature, and databases) mentioned in this application are incorporated herein by reference in their entirety for all purposes, as if each individual publication were incorporated individually by reference. Where the definitions listed herein contradict or otherwise differ from those listed in the patents, applications, published applications, and other publications incorporated herein by reference, the definitions listed herein shall prevail over those incorporated herein by reference.

[0128] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this disclosure. The following description illustrates this disclosure and should not in any way be construed as limiting the scope of the invention described herein.

[0129] I. Methods and administration of β-cell therapy In some aspects, this document provides a method for treating a subject with β-cell-related conditions, the method comprising administering engineered islets as described to the subject. Engineered islets administered to a subject according to the method provided herein comprise cells modified to evade immune rejection. In some embodiments, the engineered islets are administered as islet clusters. In a particular embodiment, the engineered islets comprise engineered β-cells. In some embodiments, the engineered β-cells are in a composition comprising additional islet cells. In some embodiments, the islets (such as islet clusters) further comprise α-cells and / or δ-cells. In some embodiments, the islets (such as islet clusters) further comprise ε-cells and / or PP-cells. In some embodiments, the cells of the engineered islets comprise the same low-immunogenic modification. In a particular embodiment, the cells of the engineered islets comprise β-cells modified with a low-immunogenic modification. Exemplary features of engineered islets (including engineered or engineered islets) used in the provided method are described in Section II.

[0130] The engineered cells described herein can be administered to subjects to treat β-cell-related diseases or conditions. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0131] In some embodiments, β-cell-related disorders are metabolic disorders. Metabolic disorders can occur when abnormal chemical reactions in a subject's body interfere with metabolic processes (e.g., processes related to energy metabolism or the breakdown of sugars and acids or the storage of said energy). In some embodiments, metabolic disorders affect the breakdown of amino acids, carbohydrates, or lipids in a subject's body. In some embodiments, metabolic disorders affect a subject's mitochondria (e.g., mitochondrial disease). In some embodiments, metabolic disorders occur when a subject's organs (such as the liver or pancreas) are diseased and / or unable to function properly. Exemplary metabolic disorders described herein may include, but are not limited to, any disease or condition characterized by increased blood pressure, high blood sugar, excessive body fat around the waist, and abnormal levels of cholesterol or triglycerides. In some implementations, metabolic disorders are familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabby disease, maple syrup urine disease, metachromatic leukodystrophy, mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), Niemann-Pick disease, phenylketonuria (PKU), porphyria, Ty Sachs disease, Wilson's disease, type 1 diabetes, type 2 diabetes, obesity, hypertension, dyslipidemia, or carbohydrate intolerance. In some implementations, the metabolic disorder is type 2 diabetes. In some implementations, the metabolic disorder is type 1 diabetes. In some implementations, the metabolic disorder is type 1 diabetes mellitus.

[0132] In some implementations, the β-cell disorder is a metabolic disorder. In some implementations, the metabolic disorder is selected from the group consisting of: familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabby disease, maple syrup urine disease, metachromatic leukodystrophy, mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), Niemann-Pick disease, phenylketonuria (PKU), porphyria, Ty-Sachs disease, Wilson's disease, type 1 diabetes, type 2 diabetes, obesity, hypertension, dyslipidemia, and carbohydrate intolerance. In some implementations, the disorder is diabetes. In some implementations, the disorder is type 1 diabetes.

[0133] A. Pancreatic islet cells In some embodiments, engineered islets (including engineered β cells) possess the ability to evade the immune system. In some embodiments, engineered islets (including engineered β 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 islets relative to control or wild-type islet cells; and (b) increase the expression of one or more tolerogenic factors in the engineered cells relative to control or wild-type islet cells, such as relative to control or wild-type β cells. In some embodiments, engineered islets (including engineered β cells) include modifications that reduce B2M expression in the engineered cells relative to control or wild-type islet cells (such as control or wild-type β cells). In some embodiments, engineered islet cells include modifications that reduce CIITA expression in the modified islet cells relative to control or wild-type islet cells, such as relative to control or wild-type β cells. In some embodiments, engineered islet cells include modifications that increase CD47 expression in the engineered islet cells relative to control or wild-type islet cells, such as relative to control or wild-type β cells. In some implementations, engineered islet cells (such as engineered β cells) include the following modifications: (a) reduced B2M expression relative to control or wild-type islet cells; (b) reduced CIITA expression relative to control or wild-type islet cells; and (c) increased CD47 expression in engineered islet cells relative to control or wild-type islet cells.

[0134] In some embodiments, the islets are primary islets engineered with the low-immunogenicity modifications described. In some embodiments, the primary islets are human. In some embodiments, the islet cells (including β cells) are cells differentiated from stem cells and engineered with the low-immunogenicity modifications 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 pluripotent 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 in a suspension.

[0135] In some embodiments, the islet cells are primary islet cells (also known as pancreatic islet cells). In specific embodiments, primary islet cells include primary β islet cells (pancreatic β islet cells). In some embodiments, primary islets are isolated or obtained from one or more individual donor subjects, such as one or more healthy individual donors (e.g., subjects without known or suspected diseases or infections, such as subjects not exhibiting clinical signs of disease or infection). In some embodiments, the donor is a cadaver. As those skilled in the art will understand, methods for isolating or obtaining islets from an individual can be implemented using known techniques.

[0136] In some embodiments, islet cells are obtained from a subject or individual (e.g., harvested, extracted, removed, or collected). In some embodiments, primary islet cells are generated from an islet cell bank, 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 primary islet cell bank 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 subject is different from a patient (e.g., a recipient subject to be administered therapeutic cells). In some embodiments, the islet cell bank does not include cells from a patient. In some embodiments, one or more of the donor subjects from whom the islet cell bank is derived are different from a patient.

[0137] Further description of pancreatic islet cells (including their use in the present art) can be found in WO2020 / 018615, the disclosure of which is incorporated herein by reference in its entirety.

[0138] In some embodiments, prior to application, engineered primary islet cell populations (including primary β-islet cells) isolated from one or more individual donors (e.g., healthy donors) are maintained in a culture system and, in some cases, expanded. In some embodiments, the engineered islet cell populations are cryopreserved prior to application.

[0139] Exemplary pancreatic islet cell types include, but are not limited to, pancreatic islet progenitor cells, immature pancreatic islet cells, and mature pancreatic islet cells. In some embodiments, the pancreatic cells described herein are administered to a subject to treat diabetes.

[0140] In some embodiments, the pancreatic islet cells disclosed herein, such as primary β-islet cells isolated from one or more individual donors (e.g., healthy donors), secrete insulin. In some embodiments, the pancreatic islet cells exhibit at least two characteristics of endogenous pancreatic islet cells, such as, but not limited to, insulin secretion in response to glucose and expression of β-islet cell markers.

[0141] Exemplary β-islet cell markers or β-islet progenitor cell markers include, but are not limited to, C-peptide, Pdxl, glucose transporter 2 (Glut2), HNF6, VEGF, glucosamine kinase (GCK), prohormone converting enzyme (PC 1 / 3), Cdcpl, NeuroD, Ngn3, Nkx2.2, Nkx6.1, Nkx6.2, Pax4, Pax6, Ptfla, Is11, Sox9, Sox17, and FoxA2.

[0142] In some embodiments, primary pancreatic islet cells can be isolated from primary pancreatic islets, derived from primary pancreatic islet cells within primary pancreatic islets, or as a component of primary pancreatic islets. For example, primary pancreatic β islet cells can be edited into single β islet cells, β islet cell populations, or as a component of primary pancreatic islets (e.g., primary pancreatic β islet cells coexisting with other cell types within primary pancreatic islets). As another example, primary pancreatic β islet cells can be administered to a patient as single β islet cells, β islet cell populations, or as a component of primary pancreatic islets (e.g., primary pancreatic β islet cells coexisting with other cell types within primary pancreatic islets). In embodiments where pancreatic β islet cells coexist with other cell types within pancreatic islets, the other cell types can also be edited using the methods described herein.

[0143] In some implementations, primary pancreatic islet cells are dissociated from primary islets before or after engineering (such as genetic engineering). These dissociated islet cells can cluster before being administered to a patient, and the clusters may include β islet cells as well as other cell types (including, but not limited to, those derived from primary islets). The number of islet cells in a cluster can vary, such as approximately 50, approximately 100, approximately 250, approximately 500, approximately 750, approximately 1000, approximately 1250, approximately 1500, approximately 1750, approximately 2000, approximately 2250, approximately 2500, approximately 2750, approximately 3000, approximately 3500, approximately 4000, approximately 4500, or approximately 5000 cells. 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 clusters can be administered to the patient.

[0144] In some embodiments, primary pancreatic islet cells isolated from one or more individual donors (e.g., healthy donors) produce insulin in response to increased glucose. In some embodiments, the pancreatic islet cells are β islet cells. In some embodiments, β islet cells are monitored to assess glucose control capacity. Assays for monitoring glucose control may include, but are not limited to: continuous blood glucose level monitoring; monitoring blood glucose levels after a period of fasting; glucose tolerance (e.g., glucose loading) tests; glucose utilization and oxidation; insulin secretion, such as by U-PLEX® Meso Scale Discovery (MSD) assay and / or glucose-stimulated insulin secretion (GSIS) assay; 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 flux, such as glucose-induced Ca2+ elevation and Ca2+-activated exocytosis. Various methods for measuring glucose control are known in the art, such as those described in the following publications: 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, all of which (including figures, illustrations and method descriptions) are incorporated herein by reference in their entirety. In some embodiments, β-islet cells (e.g., modified β-islet cells) may exhibit GSIS. In some embodiments, GSIS is measured in a perfusion GSIS assay. In some embodiments, GSIS is dynamic GSIS, which includes dynamic insulin secretion in a first phase and a second phase. In some embodiments, GSIS is static GSIS. For example, the static incubation index may be greater than or equal to about 1, greater than or equal to about 2, greater than or equal to about 5, greater than or equal to about 10, or greater than or equal to about 20. In various embodiments, pancreatic islet cells secrete insulin in response to increased glucose. In some embodiments, the cells have a distinctive morphology (such as cobblestone cell morphology) and / or a diameter of about 17 pm to about 25 pm.

[0145] In some embodiments, the cells used to generate engineered islet cells are differentiable 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 reprogrammed into an embryonic pluripotent state. In some embodiments, induced pluripotent stem cells are derived from fibroblasts. In some embodiments, the modified cells as provided herein are pluripotent stem cells, or cells differentiated from pluripotent stem cells. The cells may be vertebrate cells, such as mammalian cells, such as human cells or mouse cells. The cells may also be vertebrate stem cells, such as mammalian stem cells, such as human stem cells or mouse stem cells. In embodiments, the cells or stem cells are adapted for modification. The cells or stem cells, or cells derived from such stem cells, may have therapeutic value, such that the cells or stem cells, or cells derived from such stem cells or differentiated from such stem cells, can be used to treat the aforementioned problems of a subject requiring treatment for a disease, condition, defect, or injury.

[0146] In some embodiments, the modified or engineered pancreatic islet cells (including β cells) provided herein are modified pluripotent stem cells (e.g., modified iPSCs). The generation of mammalian (e.g., mouse and human) pluripotent stem cells (commonly referred to as iPSCs; miPSCs for mouse cells or hiPSCs for human cells) is generally known in the art. As those skilled in the art will understand, there are various different methods available for generating iPSCs. Initial induction is performed using a virus to introduce four transcription factors, Oct3 / 4, Sox2, c-Myc, and Klf4, into mouse embryonic or adult fibroblasts; see Takahashi and Yamanaka Cell 126:663-676 (2006), the full text of which, and in particular the techniques outlined therein, are hereby incorporated by reference. Since then, various methods have been developed; see the review by Seki et al., World J. StemCells 7(1): 116-125 (2015), and the full text of both papers, particularly the methods for generating hiPSCs (see, for example, Chapter 3 of the latter reference), which are hereby explicitly incorporated by reference.

[0147] Generally, iPSCs are generated through the transient expression of one or more “reprogramming factors” in host cells typically introduced using an appendage vector. Under these conditions, a small number of cells are induced to become iPSCs (this step is generally inefficient because selection markers are not used). Without being bound by theory, it is believed that once cells are “reprogrammed” and become pluripotent, they lose the appendage vector and use endogenous genes to produce factors.

[0148] Those skilled in the art will also understand that the number of reprogramming factors that can be used or employed can vary. Typically, when fewer reprogramming factors are used, the efficiency of cells in converting to a pluripotent state and the "pluripotency" decrease; for example, fewer reprogramming factors may result in cells that are not fully pluripotent and may only be able to differentiate into fewer cell types.

[0149] 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, SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L T antigens may be used. Generally, these reprogramming factor genes are provided on an add-on vector (such as those known in the art and commercially available).

[0150] In some embodiments, the host cells used for transfection with one or more reprogramming factors are non-pluripotent stem cells. Generally, as known in the art, iPSCs are prepared from non-pluripotent cells such as, but not limited to, blood cells, fibroblasts, etc., by transiently expressing reprogramming factors as described herein. In some embodiments, non-pluripotent cells (such as fibroblasts) are obtained or isolated from one or more individual subjects or donors prior to reprogramming the cells. In some embodiments, iPSCs are prepared from a bank 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., 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), pooled together, reprogrammed into iPSCs, and modified according to the provided methods.

[0151] In some implementations, iPSCs are derived, for example, by transiently transfecting cells from a library of non-pluripotent cells (e.g., fibroblasts) with one or more reprogramming factors, which are derived from one or more donor subjects different from the recipient subject (e.g., the patient to whom the cells are administered). The non-pluripotent cells (e.g., fibroblasts) to be induced into iPSCs may be obtained and aggregated from one, two, three, four, five, six, seven, eight, nine, ten, twenty, fifty, one hundred, or more donor subjects. The non-pluripotent cells (e.g., fibroblasts) may be obtained and aggregated from one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twenty or more, fifty or more, or one hundred or more donor subjects. In some embodiments, non-pluripotent cells (e.g., fibroblasts) are harvested from one or more individuals, and in some cases, a library 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, the library of non-pluripotent cells (e.g., fibroblasts) is modified according to the methods provided herein. In some embodiments, the modified iPSCs or the modified iPSC library are subsequently subjected to a differentiation process to differentiate into any cell type of organism and tissue.

[0152] PSCs can differentiate into β cells of organisms and tissues. In one aspect, this document provides modified cells, which are differentiated from iPSCs into β cells, for subsequent administration to a recipient subject. As is known in the art, differentiation can generally be determined by evaluating the presence of cell-specific markers. As those skilled in the art will understand, differentiated modified (e.g., low immunogenicity) pluripotent cell derivatives can be transplanted using techniques known in the art, depending on the cell type and the end use of these cells. Exemplary types of differentiated cells and methods for generating them are described below. In some embodiments, iPSCs can differentiate into β cells. In some embodiments, iPSCs differentiate into β pancreatic islet cells. In some embodiments, host cells, such as non-pluripotent cells (e.g., fibroblasts) from an individual donor or individual donor bank, are isolated or obtained to generate iPSCs, wherein the iPSCs are subsequently modified to contain the modifications described herein (e.g., genetic modifications) and subsequently differentiated into the desired cell type.

[0153] In some embodiments, the cells are β-islet cells derived from modified iPSCs containing the modifications described herein (e.g., genetic modifications) and differentiated into β-islet cells. As those skilled in the art will understand, the method of differentiation depends on the desired cell type using known techniques. In some embodiments, cells differentiated into various β-islet cells can be used for subsequent transplantation or colonization into a subject (e.g., a recipient). In some embodiments, pancreatic islet cells are derived from modified pluripotent cells described herein. Available methods for differentiating pluripotent stem cells into β pancreatic islet cells are described in, for example, the following references: 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 these references are incorporated herein by reference in their entirety. In some implementations, the modified pluripotent cells described herein differentiate into β-cell-like cells or pancreatic islet organoids for transplantation to address type 1 diabetes mellitus (T1DM). Cellular systems are a promising approach to address T1DM; see, for example, Ellis et al., Nat Rev Gastroenterol Hepatol. 2017 Oct;14(10):612-628, which is incorporated herein by reference. Additionally, Pagliuca et al. (Cell, 2014, 159(2):428-39) reported successful differentiation of hiPSCs into β-cells; the full text of that paper, and particularly the methods and reagents outlined therein for the large-scale production of functional human β-cells from human pluripotent stem cells, is incorporated herein by reference. In addition, Vegas et al. demonstrated the generation of human β 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 full text of which, and in particular the methods and reagents for large-scale production of functional human β cells from human pluripotent stem cells outlined therein, are incorporated herein by reference.

[0154] In some embodiments, a method for generating a modified pancreatic islet cell population from a modified pluripotent cell population via in vitro differentiation comprises: (a) culturing the modified iPSC population 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 inhibitors, ALK inhibitors, BMP type 1 receptor inhibitors, and retinoic acid to generate an immature pancreatic islet cell population; and (b) culturing the immature pancreatic islet cell population in a second culture medium different from the first culture medium to generate a modified pancreatic islet cell population. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration range of the GSK inhibitor is from about 2 mM to about 10 mM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some cases, the concentration range of the ALK inhibitor is from about 1 pM to about 10 pM. In some implementations, the first and / or second culture media do not contain animal serum.

[0155] As is known in the art, differentiation is typically assessed by evaluating the presence of β-cell-related or specific biomarkers, including but not limited to insulin. Differentiation can also be measured functionally, such as by measuring glucose metabolism, as commonly seen in Muraro et al., Cell Syst. 2016 Oct 26; 3(4): 385–394.e3, the full text of which, and in particular the biomarkers outlined therein, are hereby incorporated by reference. Once β-cells are generated, they can be transplanted (as cell suspensions, cell clusters, or in permeable or semi-permeable devices or gel matrices as discussed herein) into the portal vein / liver, omentum, gastrointestinal mucosa, bone marrow, muscle, or subcutaneous sacs.

[0156] In some embodiments, pancreatic islet cells, such as β-islet cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), produce insulin in response to increased glucose. In various embodiments, pancreatic islet cells secrete insulin in response to increased glucose. In some embodiments, the cells have a distinctive morphology (such as cobblestone cell morphology) and / or a diameter of approximately 17 μm to approximately 25 μm.

[0157] Once engineered islets have been generated, their low immunogenicity and / or retention of pluripotency can be determined as described in WO2016183041 and WO2018132783. In some embodiments, low immunogenicity is determined using a variety of techniques illustrated in Figures 13 and 15 of WO2018132783. These techniques include transplantation into an allogeneic host and monitoring the growth of low immunogenic pluripotent cells that escape the host immune system (e.g., teratomas). In some cases, low immunogenic pluripotent cell derivatives are transduced to express luciferase and can subsequently be tracked using bioluminescent imaging. Similarly, the host animal's T-cell and / or B-cell responses to such cells are tested to confirm that the cells do not elicit an immune response in the host animal. T-cell responses can be assessed using Elispot, ELISA, FACS, PCR, or flow cytometry (CYTOF). B-cell responses or antibody responses are assessed using FACS or Luminex. Alternatively, the ability of cells to avoid innate immune responses (e.g., NK cell killing) can be measured, as typically shown in Figures 14 and 15 of WO2018132783.

[0158] In some embodiments, T-cell immunoassays (such as T-cell proliferation assays, T-cell activation assays, and T-cell killing assays) recognized by those skilled in the art are used to evaluate the immunogenicity of the cells. In some cases, the T-cell proliferation assay involves pretreating cells with interferon-γ and co-culturing the cells with labeled T cells, and determining the presence of a T-cell population (or a proliferating T-cell population) after a preselected time period. In some cases, the T-cell activation assay involves co-culturing T cells with cells outlined herein and determining the expression levels of T-cell activation markers in the T cells.

[0159] In vivo assays can be performed to assess the immunogenicity of the cells outlined herein. In some embodiments, allogeneic humanized immunodeficient mouse models are used to determine the survival and immunogenicity of modified iPSCs. In some cases, modified iPSCs are transplanted into allogeneic humanized NSG-SGM3 mice, and cell rejection, cell survival, and teratoma formation are measured. In some cases, the implanted modified iPSCs or their differentiated cells exhibit long-term survival in mouse models.

[0160] Further techniques for determining the immunogenicity (including low immunogenicity) of cells are described, for example, in Deuse et al., Nature Biotechnology, 2019, 37, 252-258 and Han et al., Proc Natl Acad Sci USA, 2019, 116(21), 10441-10446, which (including figures, illustrations and method descriptions) are incorporated herein by reference in their entirety.

[0161] Similarly, pluripotency preservation can be tested in a variety of ways. In one implementation, pluripotency is determined by the expression of certain pluripotency-specific factors, as generally described herein and illustrated in Figure 29 of WO2018132783. Alternatively, differentiation of pluripotent cells into one or more cell types can be used as an indicator of pluripotency.

[0162] Once modified pluripotent stem cells (modified iPSCs) have been generated, they can be maintained in an undifferentiated state, as is known for maintaining iPSCs. For example, cells can be cultured on Matrigel using a medium that prevents differentiation and maintains pluripotency. Alternatively, cells can be kept in a medium under conditions that maintain pluripotency.

[0163] B. Compositions and Formulations In some embodiments, engineered β-islets are provided as pharmaceutical compositions for administration to a subject. In some embodiments, the pharmaceutical composition comprises engineered islets and a pharmaceutically acceptable carrier.

[0164] Acceptable carriers, excipients, or stabilizers are non-toxic to the receptor at the doses and concentrations used and include: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) peptides; proteins, such as serum albumin, etc. Gels 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 includes a pharmaceutically acceptable buffer (e.g., neutral buffered saline or phosphate buffered saline). In some embodiments, the pharmaceutical composition may contain one or more excipients for altering, maintaining, or preserving, for example, the composition's pH, osmotic pressure, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation. In some respects, those skilled in the art understand that pharmaceutical compositions containing cells can differ from pharmaceutical compositions containing proteins.

[0165] In some embodiments, the pharmaceutical composition contains an amount (such as a therapeutically effective amount or a preventatively effective amount) of engineered islets as described herein that are effective in treating or preventing β-cell-related diseases or conditions. In some embodiments, the therapeutic or preventative efficacy is monitored by periodic evaluation of the treated subject. For repeated administration over several days or longer, treatment is repeated as needed until the desired suppression of disease symptoms is achieved. However, other dosing regimens may be useful and can be determined. The desired dose can be delivered by a single bolus injection of the composition, by multiple bolus injections of the composition, or by continuous infusion of the composition.

[0166] In some embodiments, engineered islets are administered using standard application techniques, formulations, and / or devices. In some embodiments, engineered islets or compositions or groups thereof as described herein are administered using standard application techniques, formulations, and / or devices. Formulations and devices, such as syringes and vials, are provided for storing and administering the compositions. Engineered islets can be administered via local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When a therapeutic composition (such as one containing engineered islets) is administered, it is typically formulated as a unit-dose injectable form (solution, suspension, emulsion).

[0167] The formulations include those intended for intravenous, intraperitoneal, or subcutaneous administration. In some embodiments, one or more immunosuppressants are administered parenterally. As used herein, the term "parenterally" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, one or more immunosuppressants are administered to the subject using peripheral systemic delivery, via intravenous, intraperitoneal, or subcutaneous injection.

[0168] In some embodiments, the composition is provided as a sterile liquid formulation, such as an isotonic aqueous solution, suspension, emulsion, or dispersion, which may be buffered to a selected pH in some respects. Liquid compositions are more convenient to administer, especially 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 injectable solutions can be prepared by incorporating one or more immunosuppressants into a solvent, such as by mixing with a suitable carrier, diluent, or excipient (e.g., sterile water, physiological saline, glucose, dextrose).

[0169] In some embodiments, the pharmaceutical composition may be formulated for administration via any route known to those skilled in the art, including intramuscular, intravenous, intradermal, intralesional, intraperitoneal, subcutaneous, intratumoral, epidural, nasal, oral, vaginal, rectal, external, local, ear, inhalation, buccal (e.g., sublingual), and transdermal administration, or any other route. Other administration modalities are also considered in some embodiments. In some embodiments, administration is by bolus infusion, by injection, such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intra-anterior chamber injection, subconjunctival injection, subcapsular injection, retroocular injection, periocular injection, or posterior proximal scleral delivery. In some embodiments, administration is by parenteral, intrapulmonary, and intranasal administration, and, if necessary for local treatment, by intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some implementations, administration is via the portal vein. In other implementations, administration is by injection into the intermuscular space of the subject's forearm.

[0170] In some embodiments, the composition may also be administered sequentially, intermittently, or as part of the same composition with other bioactive agents. In some embodiments, administration may also include a controlled-release system, including controlled-release formulations and devices for controlled release, such as via a pump. In some embodiments, administration is oral. In some embodiments, administration is intravenous.

[0171] In some embodiments, pharmaceutically acceptable carriers may include all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delayers compatible with 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, dextran solution, and 5% human serum albumin. Liposomes and non-aqueous media, such as fixative oils, may also be used. Complementary active compounds may also be incorporated into the composition. The drug carrier should be a carrier suitable for one or more immunosuppressants, such as saline solution, dextran solution, or a solution containing human serum albumin. In some embodiments, a pharmaceutically acceptable carrier or mediator for such compositions is any non-toxic aqueous solution in which engineered islets can maintain or retain activity for a time sufficient to allow administration of live cells. For example, a pharmaceutically acceptable carrier or mediator may be an aqueous saline solution or a buffered saline solution.

[0172] This document also provides compositions suitable for cryopreservation of engineered islets. In some embodiments, engineered islets are cryopreserved in a cryopreservation medium. In some embodiments, the cryopreservation medium is a serum-free cryopreservation medium. In some embodiments, compositions comprising engineered islets or groups thereof contain a cryoprotectant. In some embodiments, the cryoprotectant is or comprises DMSO and / or glycerol. In some embodiments, the cryopreservation medium is equal to or between about 5% and about 10% DMSO (v / v). In some embodiments, the cryopreservation medium is equal to or between about 5% and about 6% DMSO (v / v). In some embodiments, the cryopreservation medium is equal to or between about 7% and about 7.5% DMSO (v / v). In some embodiments, the cryopreservation medium is equal to or between about 8% and about 9% DMSO (v / v). In some embodiments, the cryopreservation medium is equal to 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 ultra-low temperatures), for example, at a temperature range of -40°C to -150°C (such as or about 80°C ± 6.0°C).

[0173] In some embodiments, cryopreserved engineered islets are prepared for administration upon thawing. In some cases, engineered islets can be administered to a subject immediately after thawing. In such embodiments, the composition containing engineered islets is ready for use without any further processing. In other cases, the engineered islets undergo further processing after thawing, such as by resuspending with a pharmaceutically acceptable carrier, incubating with an activator or stimulant, or activating and washing and resuspending in a pharmaceutically acceptable buffer prior to administration. In some embodiments, the composition (including pharmaceutical compositions) is sterile.

[0174] In some embodiments, the pharmaceutical composition comprises engineered islets and a pharmaceutically acceptable carrier comprising 31.25% (v / v) Plasma-Lyte A, 31.25% (v / v) 5% dextran / 0.45% sodium chloride, 10% dextran 40 (LMD) / 5% dextran, 20% (v / v) 25% human serum albumin (HSA), and 7.5% (v / v) dimethyl sulfoxide (DMSO).

[0175] C. Administration and application In some embodiments, engineered islets can be administered via any route known to those skilled in the art, including intramuscular, intravenous, intradermal, intralesional, intraperitoneal, subcutaneous, renal sac, intratumoral, epidural, nasal, oral, vaginal, rectal, topical, local, ear, inhalation, buccal (e.g., sublingual), and transdermal administration, or any other route. Other administration modalities are also considered in some embodiments. In some embodiments, administration is by bolus infusion, by injection, such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intra-anterior chamber injection, subconjunctival injection, subcapsular injection, retroocular injection, periocular injection, or posterior proximal scleral delivery. In some embodiments, administration is by parenteral, intrapulmonary, and intranasal administration, and, if necessary for local treatment, by intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, administration is via the portal vein. In some implementations, administration is performed by injection into the intermuscular space of the subject's forearm. In some implementations, administration is performed via the renal sac.

[0176] In some embodiments, engineered islets can be administered at any suitable location within the subject's body. For example, in some embodiments, engineered islets are administered to the subject's kidneys, forearm, mouth, anus, nose, upper arm, hip, thigh, buttocks, liver, spleen, muscle, subcutaneous tissue, or white adipose tissue. In some embodiments, engineered cells are administered to the subject's liver, muscle, or white adipose tissue. In some embodiments, the white adipose tissue is the omentum.

[0177] In certain embodiments, engineered islets are administered via intramuscular injection. In some embodiments, engineered islets are applied to the subject's forearm. In some embodiments, engineered islets are applied to the intermuscular space of the subject's forearm.

[0178] In some implementations, injection into the muscle avoids this risk compared to early islet loss due to the immediate blood-mediated inflammatory response (IBMIR) known to occur after portal vein injection. (Bennet et al., Diabetes(1999) 48:1907-1914). Muscles have a rich vascular system, and the transplantation of pancreatic islets into striated muscles has been clinically successful (Christoffersson et al., ). Diabetes (2010) 59:2569-2578; Rafael et al., Am J Transplant (2008) 8:458-462).

[0179] In some aspects, the method of administration involves implanting engineered islet cells into a subject. In some aspects, the engineered islets may be implanted as dispersed cells or formed into clusters. In some embodiments, the engineered islets are administered as a suspension of islet cell populations. In some embodiments, the engineered islets are engineered tissue grafts comprising engineered islet cell populations and a matrix. In some embodiments, the engineered islet cells are in a composition administered as a suspension of engineered islet cell populations.

[0180] The specific dosage / regimen of engineered islets will vary depending on factors such as the subject's weight, sex, age, and health status; the formulation, biochemical properties, biological activity, bioavailability, and side effects of the engineered islets; and the number and type of engineered cells. The administered dose may depend on a variety of factors, including the patient's condition and response to therapy, and can be determined by someone skilled in the art.

[0181] In some implementations, the engineered islets are administered in doses equal to or greater than about 1000 islet equivalent units (IEQ) to about 1 × 10⁶ IEQ, such as equal to or greater than about 1000 IEQ to about 500,000 IEQ, equal to or greater than about 1000 IEQ to about 250,000 IEQ, equal to or greater than about 1000 IEQ to about 100,000 IEQ, equal to or greater than about 1000 IEQ to about 50,000 IEQ, equal to or greater than about 1000 IEQ to about 25,000 IEQ. 000 IEQ, equal to or approximately 1000 IEQ to equal to or approximately 10000 IEQ, equal to or approximately 1000 IEQ to equal to or approximately 5000 IEQ, equal to or approximately 5000 IEQ to equal to or approximately 1×10⁶ IEQ, equal to or approximately 5000 IEQ to equal to or approximately 500,000 IEQ, equal to or approximately 5000 IEQ to equal to or approximately 250,000 IEQ, equal to or approximately 5000 IEQ to equal to or approximately 100,000 IEQ, equal to or approximately 50,000 IEQ, equal to or approximately 5000 IEQ to equal to or approximately 250000 IEQ, equal to or approximately 5000 IEQ to equal to or approximately 10000 IEQ, equal to or approximately 10000 IEQ to equal to or approximately 1×10⁶ IEQ, equal to or approximately 10000 IEQ to equal to or approximately 500000 IEQ, equal to or approximately 10000 IEQ to equal to or approximately 250000 IEQ, equal to or approximately 10000 IEQ to equal to or approximately 100,000 IEQs are equivalent to approximately 10,000 IEQs to approximately 50,000 IEQs; approximately 10,000 IEQs to approximately 250,000 IEQs; approximately 25,000 IEQs to approximately 1 × 10⁶ IEQs; approximately 25,000 IEQs to approximately 500,000 IEQs; approximately 25,000 IEQs to approximately 250,000 IEQs; approximately 25,000 IEQs to... This is equivalent to approximately 100,000 IEQs, or approximately 25,000 IEQs to approximately 50,000 IEQs, or approximately 1 × 10⁶ IEQs, or approximately 50,000 IEQs to approximately 500,000 IEQs, or approximately 150,000 IEQs, or approximately 100,000 IEQs, or approximately 100,000 IEQs. One IEQ is equal to or equal to approximately 1 × 10⁶ IEQs, equal to or equal to approximately 100,000 IEQs is equal to or equal to approximately 500,000 IEQs, equal to or equal to approximately 100,000 IEQs is equal to or equal to approximately 250,000 IEQs, equal to or equal to approximately 250,000 IEQs is equal to or equal to approximately 1 × 10⁶ IEQs, equal to or equal to approximately 250,000 IEQs is equal to or equal to approximately 500,000 IEQs, or equal to or equal to approximately 500,000 IEQs is equal to or equal to approximately 1 × 10⁶ IEQs. In some implementations, modified SB-β cells are administered in amounts equal to or greater than about 50,000 IEQ, about 100,000 IEQ, about 200,000 IEQ, about 300,000 IEQ, about 400,000 IEQ, or about 500,000 IEQ, or any value between the foregoing. IEQ provides a standardized estimate of islet volume, where one IEQ corresponds to the volume of an ideally spherical islet with a diameter of 150 μm (Ricordi et al., Acta Diabetol. Lat. 27, 185-195 (1990)).

[0182] In some embodiments, the engineered islets administered to the subject are given in doses equal to or greater than about 500 IEQ / kg body weight to or greater than about 10,000 IEQ / kg; equal to or greater than about 500 IEQ / kg to or greater than about 5,000 IEQ / kg; equal to or greater than about 500 IEQ / kg to or greater than about 2,500 IEQ / kg; equal to or greater than about 500 IEQ / kg to or greater than about 1,000 IEQ / kg; equal to or greater than about 1,000 IEQ / kg to or greater than about 1,000 IEQ / kg. 0 IEQ / kg, equal to or approximately 1000 IEQ / kg to or approximately 5000 IEQ / kg, equal to or approximately 1000 IEQ / kg to or approximately 2500 IEQ / kg, equal to or approximately 2500 IEQ / kg to or approximately 10000 IEQ / kg, equal to or approximately 2500 IEQ / kg to or approximately 5000 IEQ / kg, or equal to or approximately 5000 IEQ / kg to or approximately 10000 IEQ / kg.

[0183] Any therapeutically effective amount of the cells described herein may be included in the pharmaceutical composition, depending on the indication being treated. Non-limiting examples of cells include primary islet cells as described (e.g., engineered low-immunogenic islet cells). In some embodiments, the pharmaceutical composition comprises at least about 1 × 10⁻⁶ cells. 7 1, 2×10 7 1, 3×10 7 1, 4×10 7 5×10 7 6×10 7 7×10 7 8×10 7 9×10 7 1×10 8 1, 2×10 8 1, 3×10 8 Cells. In some embodiments, the pharmaceutical composition comprises up to about 1 × 10⁶ cells. 7 1, 2×10 7 1, 3×10 7 1, 4×10 7 5×10 7 6×10 7 7×10 7 8×10 7 9×10 7 1×10 8 1, 2×10 8 1, 3×108 Cells. In some embodiments, the pharmaceutical composition comprises up to about 1 × 10⁶ cells. 7 Cells. In some embodiments, the pharmaceutical composition comprises up to about 3 × 10⁶ cells. 8 Cells. In some embodiments, the pharmaceutical composition comprises at least about 1 × 10⁶ cells. 7 3×10 7 1, 2×10 7 4×10 7 1, 3×10 7 5×10 7 1, 4×10 7 6×10 7 5×10 7 7×10 7 6×10 7 8×10 7 7×10 7 9×10 7 8×10 7 1×10 8 9×10 7 2×10 8 One or 1×10 8 3×10 8 Cells. In an exemplary embodiment, the pharmaceutical composition comprises about 1 × 102 cells. 7 Approximately 3 × 10 8 Cells. In some embodiments, the pharmaceutical composition comprises at least about 25 × 10⁶ cells. 6 Each - at least approximately 25 x 10 7 Cells. In some embodiments, the pharmaceutical composition comprises at least about 80 × 10⁸ cells. 6 Each - at least approximately 80 x 10 7 10 cells. In another exemplary embodiment, the pharmaceutical composition comprises about 25 × 10 cells. 6 Approximately 80 × 10 6 Cells. In some embodiments, the pharmaceutical composition comprises about 25 × 10⁶ cells. 6 Approximately 80 × 10 7 Each cell.

[0184] In some embodiments, the pharmaceutical composition is prepared at a concentration of approximately 1.25 × 10⁻⁶ kg body weight. 5 Approximately 1.2 × 10⁻⁶ 7 A single dose of engineered, low-immunogenic pancreatic islet cells is administered. In some embodiments, the pharmaceutical composition is administered at approximately 1.25 × 10⁻⁶ per kg of body weight. 5 Approximately 1.25 × 10⁻⁶ 6 One, approximately 1.5 × 10 5Approximately 1.5 × 10⁻⁶ 6 One, approximately 2.0 × 10 5 Approximately 2.0 × 10⁻⁶ 6 Each, approximately 2.5 × 10 5 Approximately 2.5 × 10⁻⁶ 6 Each, approximately 3.0 × 10 5 Approximately 3.0 × 10⁻⁶ 6 Each, approximately 3.5 × 10 5 Approximately 3.5 × 10⁻⁶ 6 One, approximately 4.0 × 10 5 Approximately 4.0 × 10⁻⁶ 6 Each, approximately 4.5 × 10 5 Approximately 4.5 × 10⁻⁶ 6 Each, approximately 5.0 × 10 5 Approximately 5.0 × 10⁻⁶ 6 Each, approximately 5.5 × 10 5 Approximately 5.5 × 10⁻⁶ 6 Each, approximately 6.0 × 10 5 Approximately 6.0 × 10⁻⁶ 6 Each, approximately 6.5 × 10 5 Approximately 6.5 × 10⁻⁶ 6 Each, approximately 7.0 × 10 5 Approximately 7.0 × 10⁻⁶ 6 7.5 × 10 5 Approximately 7.5 × 10⁻⁶ 6 One, approximately 8.0 × 10 5 Approximately 8.0 × 10⁻⁶ 6 8.5 × 10 5 Approximately 8.5 × 10⁻⁶ 6 1, approximately 9.0 × 10 5 Approximately 9.0 × 10⁻⁶ 6 One, approximately 1.0 × 10 6 Approximately 1.0 × 10⁻⁶ 7 Or approximately 1.2 × 10 6 Approximately 1.2 × 10⁻⁶ 7 Single-dose administration per cell. In several embodiments, the dose is below approximately 1.25 × 10⁻⁶ cells per kg of body weight. 5 Approximately 1.2 × 10⁻⁶ 7 Within the range of individual cells. In several embodiments, the dosage is above approximately 1.25 × 10⁻⁶ cells per kg of body weight. 5 Approximately 1.2 × 10⁻⁶ 7 Within the range of individual cells. In some implementations, the dose is administered intravenously.

[0185] In some embodiments, the pharmaceutical composition comprises an islet equivalent (IEQ). In some embodiments, the pharmaceutical composition comprises 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 comprises 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 comprises up to about 6,500 IEQ. In some embodiments, the pharmaceutical composition comprises up to about 600,000 IEQ. In some embodiments, the pharmaceutical composition comprises at least about 6,500 IEQs, 50,000 IEQs, 100,500 IEQs, 200,000 IEQs, 300,000 IEQs, 400,000 IEQs, 500,000 IEQs, or 600,000 IEQs. In an exemplary embodiment, the pharmaceutical composition comprises about 6,500 to about 600,000 IEQs.

[0186] In some embodiments, the pharmaceutical composition is administered in a single dose of about 80 IEQ / kg to about 24,000 IEQ / kg. In some embodiments, the pharmaceutical composition is administered in a single dose of about 80 IEQ / kg to about 800 IEQ / kg, about 100 IEQ / kg to about 1,000 IEQ / kg, about 200 IEQ / kg to about 2,000 IEQ / kg, about 300 IEQ / kg to about 3,000 IEQ / kg, about 400 IEQ / kg to about 4,000 IEQ / kg, about 500 IEQ / kg to about 5,000 IEQ / kg, about 1,000 IEQ / kg to about 10,000 IEQ / kg, about 5,000 IEQ / kg to about 15,000 IEQ / kg, about 10,000 IEQ / kg to about 20,000 IEQ / kg, or about 14,000 IEQ / kg to about 24,000 IEQ / kg. In several embodiments, the dose is in the range of less than about 80 IEQ / kg to about 24,000 IEQ / kg. In several embodiments, the dose is in the range of more than about 80 IEQ / kg to about 24,000 IEQ / kg. In some embodiments, the dose is administered intravenously.

[0187] In some embodiments, the pharmaceutical composition is administered as a single dose of about 500 to about 1500 islets per cluster. In some embodiments, the pharmaceutical composition is administered as a single dose of about 500, 1000, or 1500 islets per cluster.

[0188] D. Subjects 1. β-cell-related diseases The modified cells provided herein can be administered to any suitable subject (e.g., a patient), including candidates for cell therapies, such as those for treating β-cell-related diseases or conditions. Candidates for cell therapy include any subject suffering from a β-cell-related disease or condition who may potentially benefit from the therapeutic effects of the modified β-cells and one or more immunosuppressants provided herein. In some embodiments, the subject is an allogeneic recipient of the administered modified β-cells. In some embodiments, the provided modified β-cells and one or more immunosuppressants are effectively used for allogeneic cell therapy. Subjects who benefit from the therapeutic effects of the modified β-cells and one or more immunosuppressants provided herein exhibit elimination, reduction, or improvement of their β-cell-related disease or condition. In some aspects, the subject has a β-cell-related condition or an increased risk of developing a β-cell-related condition.

[0189] In some embodiments, β-cell-related disorders are metabolic disorders. Metabolic disorders can occur when abnormal chemical reactions in a subject's body interfere with metabolic processes (e.g., processes related to energy metabolism or the breakdown of sugars and acids or the storage of said energy). In some embodiments, metabolic disorders affect the breakdown of amino acids, carbohydrates, or lipids in a subject's body. In some embodiments, metabolic disorders affect a subject's mitochondria (e.g., mitochondrial disease). In some embodiments, metabolic disorders occur when a subject's organs (such as the liver or pancreas) are diseased and / or unable to function properly. Exemplary metabolic disorders described herein may include, but are not limited to, any disease or condition characterized by increased blood pressure, high blood sugar, excessive body fat around the waist, and abnormal levels of cholesterol or triglycerides. In some implementations, metabolic disorders are familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabby disease, maple syrup urine disease, metachromatic leukodystrophy, mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), Niemann-Pick disease, phenylketonuria (PKU), porphyria, Ty Sachs disease, Wilson's disease, type 1 diabetes, type 2 diabetes, obesity, hypertension, dyslipidemia, or carbohydrate intolerance. In some implementations, the metabolic disorder is type 2 diabetes. In some implementations, the metabolic disorder is type 1 diabetes. In some implementations, the metabolic disorder is type 1 diabetes mellitus.

[0190] In some embodiments, the subject has been diagnosed with a β-cell-related disease or condition (e.g., type 1 diabetes) prior to administration of one or more immunosuppressants and / or modified β-cells or compositions (such as any of the immunosuppressants and / or compositions containing modified β-cells described herein). In some embodiments, the subject has been diagnosed with a β-cell-related disease or condition between approximately 1 year and approximately 5 years prior to administration of one or more immunosuppressants and / or modified β-cells or compositions. In some embodiments, the subject has been diagnosed with a β-cell-related disease or condition at least approximately 1 year prior to administration of one or more immunosuppressants and / or modified β-cells or compositions, such as at least approximately 2, 3, 4, 5, or earlier prior to administration of one or more immunosuppressants and / or modified β-cells or compositions. In some embodiments, the subject has been diagnosed with a β-cell-related disease or condition less than approximately 5 years prior to administration of one or more immunosuppressants and / or modified β-cells or compositions, such as less than approximately 4, 3, 2, 1, or less prior to administration of one or more immunosuppressants and / or modified β-cells or compositions. In some embodiments, the subject has been diagnosed with type 1 diabetes for at least about one year prior to the administration of one or more immunosuppressants and / or modified β-cells or compositions, such as for at least about two, three, four, five, or earlier years prior to the administration of one or more immunosuppressants and / or modified β-cells or compositions. In some embodiments, the subject has been diagnosed with type 1 diabetes for less than about five years prior to the administration of one or more immunosuppressants and / or modified β-cells or compositions, such as for less than about four, three, two, one, or less years prior to the administration of one or more immunosuppressants and / or modified β-cells or compositions.

[0191] 2. Inclusion criteria In some implementations, subjects exhibit one or more inclusion criteria prior to administration of a dose of engineered, low-immunogenic islets. As used herein, the term "inclusion criteria" refers to the clinical phenotype of a subject that qualifies them for the methods and uses provided herein.

[0192] In some implementations, the subjects are teenagers, adolescents, middle-aged, or elderly. In some implementations, the subjects are teenagers. In some implementations, the subjects are between about 1 month and about 18 years old, such as between about 1 month and about 1 year old, between about 6 months and about 5 years old, between about 2 years and about 10 years old, or between about 8 years and about 15 years old. In some implementations, the subjects are older than about 1 month, such as older than about 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. In some implementations, the subjects are less than about 18 years of age, such as less than about 17, 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 less. In some implementations, the subjects are between about 18 and about 90 years of age, such as between about 18 and about 40 years of age, between about 20 and about 60 years of age, between about 50 and about 80 years of age, or between about 60 and about 90 years of age. In some implementations, the subjects are older than approximately 18 years of age, such as being older than approximately 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or any other age. In some implementations, the subjects are younger than approximately 90 years of age, such as being younger than approximately 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 18, or any other age.

[0193] In some implementations, the subjects to be treated are characterized by one or more of the following: diagnosed before the age of 18; receiving intensive diabetes management; aged between 18 and 45 years; and weighing <80 kg. In some implementations, the subjects to be treated are diagnosed before the age of 18. In some implementations, the subjects to be treated receive intensive diabetes management. In some implementations, intensive diabetes management includes self-monitoring of subcutaneous blood glucose levels via continuous glucose monitoring or intermittent scanning glucose monitoring at least three times per day on average per week. In some implementations, intensive diabetes management includes administering insulin injections or insulin pump therapy three or more times per day. In some implementations, intensive diabetes management includes self-monitoring of subcutaneous blood glucose levels via continuous glucose monitoring or intermittent scanning glucose monitoring at least three times per day on average per week, and administering insulin injections or insulin pump therapy three or more times per day. In some implementations, the subjects to be treated are aged between 18 and 45 years. In some implementations, the subjects to be treated weigh <80 kg.

[0194] 3. Exclusion Criteria In some implementations, the subject does not exhibit any of the exclusion criteria prior to administration of a dose of engineered, low-immunogenic islets. As used herein, the term "exclusion criterion" refers to the clinical phenotype of the subject that disqualifies the subject from using the methods and uses provided herein.

[0195] In some implementation schemes, subjects are characterized by the absence of: any prior organ transplantation; any history of malignancy; use of any investigational drug within 4 weeks of administration of this dose of engineered low-immunogenic islets; use of any antidiabetic medication other than insulin within 4 weeks of administration of this 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 corresponding reference range; serological evidence of HTLVI or HTLVII infection; pregnancy, lactation, or intention to become pregnant; grade 3 or higher chronic kidney disease (e.g., GFR <60 ml / min estimated by creatine measurement); history of heart disease or symptoms consistent with heart disease at screening; HLA immunity, MICA / B immunity; known autoimmune diseases other than type 1 diabetes (e.g., Hashimoto's disease); administration of a live attenuated vaccine <6 months prior to administration of this dose of engineered low-immunogenic islets; islet antibody GADA >2000 IE / mL or IA2A. >4000 IE / mL or ZnT8 autoantibodies; untreated proliferative diabetic retinopathy; persistent mental illness; persistent substance abuse (drugs or alcohol) or treatment non-compliance; and known hypersensitivity to ciprofloxacin, gentamicin, or amphotericin B.

[0196] In some implementations, the subject has no prior organ transplantation. In some implementations, the subject has no history of malignancy. In some implementations, the subject has not used any investigational drugs within 4 weeks of receiving this dose of engineered low-immunogenic islets. In some implementations, the subject has not used any antidiabetic medications other than insulin within 4 weeks of receiving this dose of engineered low-immunogenic islets. In some implementations, the subject has no active infection, including tuberculosis, HIV, HBV, and HCV. In some implementations, the subject's liver function test values ​​for AST, ALT, GGT, or ALP are within the corresponding reference ranges. In some implementations, the subject has no serological evidence of HTLVI or HTLVII infection. In some implementations, the subject is not pregnant, lactating, or intends to become pregnant. In some implementations, the subject does not have grade 3 or higher chronic kidney disease (e.g., GFR <60 ml / min estimated by creatine measurement). In some implementations, the subject has no history of heart disease or symptoms consistent with heart disease at screening. In some implementations, the subject has no history of HLA or MIC A / B immunization. In some implementations, the subject has no known autoimmune diseases other than type 1 diabetes (e.g., Hashimoto's disease). In some implementations, the subject has not received administration of a live attenuated vaccine within 6 months prior to receiving this dose of engineered, low-immunogenic islets. In some implementations, the subject does not have islet antibodies GADA >2000 IE / mL, IA2A >4000 IE / mL, or ZnT8 autoantibodies. In some implementations, the subject does not have untreated proliferative diabetic retinopathy. In some implementations, the subject does not have persistent mental illness. In some implementations, the subject does not have persistent substance abuse (drug or alcohol) or treatment non-compliance. In some implementations, the subject is known not to be allergic to ciprofloxacin, gentamicin, or amphotericin B.

[0197] E. Results of the method This article provides methods relating to administering engineered islets (typically comprising engineered β-islet cells) to a subject. In some embodiments, the provided methods can be used to treat a subject with β-cell-related conditions (e.g., type 1 diabetes), promote the colonization or survival of β-cells in the subject, and / or restore the subject's glucose metabolism.

[0198] In some embodiments, the provided method improves glucose tolerance in a subject. Glucose tolerance can be measured by any suitable method, such as those described herein (e.g., insulin secretion assay). In some embodiments, engineered islets exhibit glucose-stimulated insulin secretion (GSIS). Therefore, in some embodiments, improved glucose tolerance is measured in a GSIS perfusion assay. Glucose intolerance is associated with insulin resistance and can lead to diabetes (e.g., type 1 and type 2 diabetes). Therefore, in some embodiments, a method for treating a β-cell-related condition (e.g., diabetes) is provided, comprising administering the provided engineered islets to a subject. In some embodiments, the subject is a diabetic patient. In some embodiments, the subject has type 1 diabetes. In some embodiments, the subject has type 2 diabetes. Specifically, in some embodiments, a method for improving glucose tolerance in a subject is provided, comprising administering the subject an engineered islet as described herein. In some embodiments, glucose tolerance is improved relative to the subject's glucose tolerance before administration of the engineered islet. In some embodiments, the engineered islet reduces the subject's exogenous insulin use. In some embodiments, glucose tolerance is improved, as measured by HbA1c levels. In some implementations, the subject is in a fasting state. In some implementations, engineered islets improve insulin secretion in the subject. In some implementations, insulin secretion is improved relative to the subject's insulin secretion before administration of engineered islets.

[0199] In some embodiments, the methods disclosed herein also include monitoring the patient's insulin independence. In some embodiments, achieving "insulin independence" or "insulin-independent" means that the subject (e.g., islet cell receptor) is able to gradually discontinue insulin therapy for at least one week and meet one or more (e.g., all) of the following criteria: (i) fasting capillary blood glucose levels exceeding 140 mg / dL (7.8 mmol / L) no more than three times within one week (based on at least seven capillary blood glucose measurements over seven days); (ii) 2-hour postprandial capillary blood glucose levels exceeding 180 mg / dL (10.0 mmol / L) no more than three times within one week (based on at least 21 capillary blood glucose measurements over seven days); and (iii) evidence of endogenous insulin production, defined as fasting or post-stimulation C-peptide levels >0.5 ng / mL (0.16 pmol / L). In some embodiments, the subject is characterized by one of (i)-(iii). In some embodiments, the subject is characterized by two of (i)-(iii). In some implementations, the subject is characterized by each of (i)-(iii).

[0200] In some embodiments, subjects are monitored approximately 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 after administration of any of the cells provided herein (e.g., a dose of engineered low-immunogenic islet cells). In some embodiments, the methods disclosed herein include monitoring subjects' insulin independence for up to one year after administration of any of the cells provided herein (e.g., a dose of engineered low-immunogenic islet cells).

[0201] In some embodiments, subjects exhibit reduced insulin dependence (e.g., a 10% or greater reduction in the dose of exogenous insulin, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) compared to the amount of exogenous insulin required for a subject receiving non-hypoimmunogenic islets to treat β-cell disease or for an untreated subject with β-cell disease. In some embodiments, this reduction in insulin dependence is achieved 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 hypoimmunogenic islet cells).

[0202] In some implementations, the subject is insulin-independent. In some implementations, insulin independence is achieved for 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 islet cells).

[0203] In some implementations, the methods disclosed herein also include monitoring the patient once or multiple times or continuously throughout the transplant survival period. In some implementations, the transplant survival period may be about 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years or longer (e.g., about 1 year or longer, about 2 years or longer, about 5 years or longer, about 7 years or longer, or about 10 years or longer).

[0204] In some embodiments, the methods disclosed herein further include administering one or more additional doses of engineered islets to subjects who are not insulin-insulin-independent or insulin-dependent at the end of the monitoring period. In some embodiments, a subject is "insulin-dependent" if the subject (e.g., islet cell receptors) does not meet the insulin-independence criteria described above. In some embodiments, the methods disclosed herein further include administering one or more additional doses of cells to subjects whose C-peptide levels in serum samples are below about 0.2 ng / ml, 0.3 ng / ml, 0.4 ng / ml, 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 serum samples are below about 0.2 ng / ml, 0.3 ng / ml, 0.4 ng / ml, or 0.5 ng / ml (e.g., about 0.3 ng / ml) are not insulin-insulin-independent.

[0205] In some embodiments, administration of the provided engineered islet 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 can be assessed by measuring the IFNg cytokine secretion levels of CD8+ T cells. In some embodiments, the IFNg levels generated after administration of the engineered islets are lower than those of wild-type primary islet cells, or reduced to, for example, about 1 / 400, 1 / 300, 1 / 200, 1 / 100, 1 / 50, 1 / 25, or 1 / 10 compared to SC-derived islet cells derived from unmodified pluripotent stem cells. 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 levels of donor-specific antibody (DSA) IgG or IgM. In some embodiments, the engineered islets exhibit lower DSA levels compared to wild-type primary islet cells, such as DSA levels reduced to about 1 / 2, 2 / 3, or the same as those of control or wild-type β cells.

[0206] In some embodiments, engineered islet cells are less immunogenic and exhibit a reduced or diminished immune response compared to unmodified engineered islet cells. In some embodiments, the immune response against engineered cells is reduced or diminished 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 generated by the administration of immunogenic cells (e.g., a cell population having the same or similar cell type or phenotype but without modifications such as genetic modification). In some embodiments, the administered engineered islets do not elicit an immune response against the modified cells in a subject.

[0207] In some embodiments, the applied engineered islets induce a reduced or decreased level of systemic TH1 activation in the subject. In some cases, the level of systemic TH1 activation induced by the cells is reduced 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 systemic TH1 activation induced by the application of immunogenic cells (e.g., cell populations with the same or similar cell types or phenotypes but without modifications such as genetic modifications). In some embodiments, the applied engineered islets do not induce systemic TH1 activation in the subject.

[0208] In some embodiments, the applied engineered islets induce a reduced or decreased level of immune activation in peripheral blood mononuclear cells (PBMCs) in the subject. In some cases, the level of immune activation in PBMCs induced by these cells is reduced 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 activation in PBMCs induced by the application of immunogenic cells (e.g., a population of cells having the same or similar cell type or phenotype but without modifications such as genetic modifications).

[0209] In some embodiments, the applied engineered islets induce a reduced or decreased level of donor-specific IgG antibodies in the subject. In some cases, the level of donor-specific IgG antibodies induced by the cells is reduced 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 donor-specific IgG antibodies induced by the application of immunogenic cells (e.g., cell populations with the same or similar cell type or phenotype but without modifications, such as genetic modifications). In some embodiments, the applied modified cell population fails to induce donor-specific IgG antibodies in the subject.

[0210] In some embodiments, the applied engineered islets induce reduced or decreased levels of IgM and IgG antibody production in the subject. In some cases, the levels of IgM and IgG antibodies induced by immunogenic cells (e.g., cell populations having the same or similar cell type or phenotype but without modifications such as genetic modifications) are reduced 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 levels induced by the application of immunogenic cells (e.g., cell populations having the same or similar cell type or phenotype but without modifications such as genetic modifications). In some embodiments, the applied engineered islets do not induce IgM and IgG antibody production in the subject.

[0211] In some embodiments, the applied engineered islets induce a reduced or decreased level of cytotoxic T-cell killing in the subject. In some cases, the level of cytotoxic T-cell killing induced by immunogenic cells (e.g., cell populations having the same or similar cell type or phenotype but without modifications such as genetic modifications) is reduced 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 cytotoxic T-cell killing induced by the application of immunogenic cells (e.g., cell populations having the same or similar cell type or phenotype but without modifications such as genetic modifications).

[0212] Following administration of the engineered islets described herein, subjects exhibited no systemic immune response or a reduced level of systemic immune response compared to a response to non-hypoimmunogenic cells. In some embodiments, subjects exhibited no adaptive immune response or a reduced level of adaptive immune response compared to a response to non-hypoimmunogenic cells. In some embodiments, subjects exhibited no innate immune response or a reduced level of innate immune response compared to a response to non-hypoimmunogenic cells. In some embodiments, subjects exhibited no T-cell response or a reduced level of T-cell response compared to a response to non-hypoimmunogenic cells. In some embodiments, subjects exhibited no B-cell response or a reduced level of B-cell response compared to a response to non-hypoimmunogenic cells.

[0213] In some implementations, the subject did not experience any adverse events after administration of engineered islets as described herein. In some implementations, the subject experienced fewer adverse events compared to a subject who did not receive one or more immunosuppressants. In some implementations, adverse events were assessed using the Common Terminology Criteria for Adverse Events (CTCAE) v5.0. Adverse events may include, but are not limited to: hypoglycemia and hyperglycemia limits related to glucose risk, muscle pain during administration of engineered islets, local bleeding during administration of engineered islets and / or one or more immunosuppressants, and / or cytokine release syndrome.

[0214] In some implementations, as assessed by PBMC and serum, the administered engineered islets evade the subject's immune system. In some implementations, the engineered islets evade the subject's immune system at weeks 0, 2, 4, 8, 12, 18, 26, and 52 following administration of the engineered islets. In some implementations, as assessed by MRI, the administered engineered islets survive in the subject. In some implementations, the engineered islets survive within 48 hours following administration. In some implementations, the engineered islets survive at weeks 2, 4, 6, 8, 12, 26, and 52 following administration. In some implementations, the subject exhibits a C-peptide peak >0.01 nmol / L in response to a mixed meal tolerance test (MMTT) after administration of the engineered islets. In some implementations, at weeks 4, 8, 12, 18, 26, and 52 following administration of engineered islets, the peak C-peptide concentration in response to the MMTT is >0.01 nmol / L. In some implementations, the peak C-peptide concentration is measured using the area under the curve (AUC). In some implementations, subjects exhibit non-fasting C-peptide concentrations >0.01 nmol / L following administration of engineered islets. In some implementations, non-fasting C-peptide concentrations are >0.01 nmol / L at weeks 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 52 following administration of engineered islets. In some implementations, subjects exhibit a reduction in insulin requirements per kilogram of body weight (BW) following administration of engineered islets. In some implementations, insulin requirements per kilogram of blood weight (BW) are reduced at 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 52 weeks after administration of engineered islets. In some implementations, subjects exhibit a decrease in HbA1c after administration of engineered islets. In some implementations, HbA1c is reduced at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 26, and 52 weeks after administration of engineered islets. In some implementations, subjects exhibit reduced glycemic variability after administration of engineered islets. In some implementations, glycemic variability is reduced at 4, 8, 12, 18, 26, and 52 weeks after administration of engineered islets. In some implementations, subjects exhibit reduced hypoglycemia after administration of engineered islets. In some implementations, hypoglycemia was reduced at 4, 8, 12, 18, 26, and 52 weeks after administration of engineered islets to subjects. In some implementations, subjects exhibited reduced hyperglycemia after administration of engineered islets.In some implementations, hyperglycemia was reduced at 4, 8, 12, 18, 26, and 52 weeks after administration of engineered islets to subjects.

[0215] II. Methods and administration of combining β-cell therapy with immunosuppressants This article provides methods and uses for combination therapies comprising engineered islets (such as a dose of engineered hypoimmunogenic islets) and one or more immunosuppressants.

[0216] A. Immunosuppressants and their regimens In some aspects 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 colonization and / or survival of modified β cells or compositions (e.g., compositions comprising modified β cells) in the subject while minimizing drug toxicity, infection, and malignancy in the subject. In some embodiments, one or more immunosuppressants are administered in combination with a composition comprising modified β cells to a subject for the treatment of β cell-related conditions, including diabetes (e.g., type 1 diabetes). In some embodiments, the provided methods of administering one or more immunosuppressants and a composition comprising modified β cells can be used to restore or provide glucose metabolism to a subject in need.

[0217] 1. Application In some implementations, the provided method involves administering to a subject one or more immunosuppressants and a composition comprising modified β cells.

[0218] In some embodiments, the provided method involves administering at least one regimen of one or more immunosuppressants before, after, during, throughout, concurrently with, sequentially and / or intermittently, the administration of modified β cells or a composition. In some embodiments, the provided method involves administering a first dose of one or more immunosuppressants before, subsequently (after), during, throughout, concurrently with, sequentially or intermittently, the administration of modified β cells or a composition. In some embodiments, "simultaneously" means that the administration of one or more immunosuppressants and the administration of modified β cells or a composition overlap with each other, at least one regimen of one or more immunosuppressants overlaps with the administration of a composition containing modified β, and / or the administration of one or more immunosuppressants and the administration of modified β cells or a composition occur at the same time (e.g., on the same day and / or simultaneously).

[0219] In some embodiments, the method includes administering one or more immunosuppressants (e.g., one or more regimens of one or more immunosuppressants) before, concurrently with, and / or after administering modified β cells or a composition to the subject.

[0220] In some embodiments, administering one or more immunosuppressants includes at least one regimen of administering one or more immunosuppressants prior to administering the modified β-cells or composition to the subject. In some embodiments, one or more immunosuppressants are administered to the subject only prior to the first and / or second regimen of administering the modified β-cells or composition. In some embodiments, one or more immunosuppressants are administered between approximately 30 seconds and approximately 10 weeks prior to administering the modified β-cells or composition to the subject, such as between approximately 30 seconds and approximately 1 hour, between approximately 30 minutes and approximately 12 hours, between approximately 6 hours and approximately 1 day, between approximately 10 hours and approximately 5 days, between approximately 2 days and approximately 7 days, between approximately 5 days and approximately 14 days, between approximately 7 days and approximately 4 weeks, or between approximately 2 weeks and approximately 10 weeks. In some implementations, one or more immunosuppressants are administered at least about 30 seconds before the administration of the modified β 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 earlier. In some implementations, one or more immunosuppressants are administered to the subject less than about 10 weeks prior to administration of the modified β cells or composition, such as less than about 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 prior to administration of the modified β cells or composition. 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 prior to administration of the modified β cells or composition. In some embodiments, one or more immunosuppressants are administered to the subject prior to administration of the modified β cells or composition and are continued throughout the subject's lifespan. In some embodiments, one or more immunosuppressants are administered to the subject prior to each round of administration of the modified β cells or composition.In some implementations, one or more immunosuppressants are administered to the subject prior to each round of administration of modified β cells or the composition, and are continued to be administered throughout the subject's lifespan.

[0221] In some embodiments, administering one or more immunosuppressants includes administering one or more immunosuppressants on the same day as administering modified β cells or a composition to the subject. In some embodiments, administering one or more immunosuppressants includes administering one or more immunosuppressants concurrently with administering modified β cells or a composition to the subject. In some embodiments, administering one or more immunosuppressants includes administering one or more immunosuppressants on the same day as administering modified β cells or a composition to the subject, and continuing administration throughout the subject's lifespan. In some embodiments, administering one or more immunosuppressants concurrently with administering modified β cells or a composition to the subject, and continuing administration throughout the subject's lifespan. In some embodiments, a first regimen of administering one or more immunosuppressants to the subject concurrently with administering modified β cells or a composition to the subject. In some embodiments, a second regimen of administering one or more immunosuppressants to the subject concurrently with administering modified β cells or a composition to the subject. In some embodiments, one or more immunosuppressants are administered to the subject on the same day as each round of administration of modified β cells or a composition. In some embodiments, one or more immunosuppressants are administered to the subject on the same day as each round of administration of modified β cells or a composition, and continuing administration throughout the subject's lifespan. In some embodiments, one or more immunosuppressants are administered to the subject concurrently with each round of administration of the modified β-cells or composition. In some embodiments, one or more immunosuppressants are administered to the subject concurrently with each round of administration of the modified β-cells or composition, and are continued to be administered throughout the subject's lifespan.

[0222] In some embodiments, administering one or more immunosuppressants includes a regimen (e.g., at least one regimen) of administering one or more immunosuppressants after administering modified β-cells or a composition to the subject. In some embodiments, one or more immunosuppressants are administered to the subject only after a first and / or second regimen of administering modified β-cells or a composition. In some embodiments, one or more immunosuppressants are administered between approximately 30 seconds and approximately 10 weeks after administering modified β-cells or a composition to the subject, such as between approximately 30 seconds and approximately 1 hour, between approximately 30 minutes and approximately 12 hours, between approximately 6 hours and approximately 1 day, between approximately 10 hours and approximately 5 days, between approximately 2 days and approximately 7 days, between approximately 5 days and approximately 14 days, between approximately 7 days and approximately 4 weeks, or between approximately 2 weeks and approximately 10 weeks. In some implementations, one or more immunosuppressants are administered at least about 30 seconds after administration of the modified β 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 later after administration of the modified β cells or composition to the subject. In some implementations, one or more immunosuppressants are administered to the subject less than about 10 weeks after administration of the modified β cells or composition, such as less than about 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 prior to administration of the modified β cells or composition. 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 β cells or composition. In some embodiments, one or more immunosuppressants are administered to the subject after administration of the modified β cells or composition and are continued throughout the subject's lifespan. In some embodiments, one or more immunosuppressants are administered to the subject after each round of administration of the modified β cells or composition.In some implementations, one or more immunosuppressants are administered to the subject after each round of administration of modified β cells or a composition, and are continued to be administered throughout the subject's lifespan.

[0223] In some implementations, one or more immunosuppressants are administered to the subject at a lower dose than the dose of one or more immunosuppressants administered to the subject, in order to reduce immune rejection of modified immunogenic cells that do not contain modified β cells.

[0224] In some embodiments, one or more immunosuppressants are administered to the subject in a single regimen (e.g., a dose). In some embodiments, one or more immunosuppressants are administered to the 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 daily. In some embodiments, the total daily dose of one or more immunosuppressants is provided as a single daily regimen. In some embodiments, one or more immunosuppressants are administered as a divided dose regimen.

[0225] In some embodiments, the total daily dose of one or more immunosuppressants is divided into two, three, or four daily regimens. In some embodiments, the total daily dose of one or more immunosuppressants is divided into two daily regimens. In some embodiments, 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 daily regimens. In some embodiments, the total daily dose of one or more immunosuppressants is divided into four daily regimens.

[0226] In some embodiments, following administration of the modified β-cells or composition to the subject, one or more immunosuppressants are administered (e.g., daily) for approximately 3 months, approximately 6 months, approximately 12 months, approximately 24 months, approximately 36 months, approximately 48 months, approximately 60 months, or longer. In some embodiments, one or more immunosuppressants are administered for approximately 3 months following administration of the modified β-cells or composition to the subject. In some embodiments, one or more immunosuppressants are administered for approximately 6 months following administration of the modified β-cells or composition to the subject. In some embodiments, one or more immunosuppressants are administered for approximately 9 months following administration of the modified β-cells or composition to the subject. In some embodiments, one or more immunosuppressants are administered for approximately 12 months following administration of the modified β-cells or composition to the subject. In some embodiments, one or more immunosuppressants are administered for approximately 24 months following administration of the modified β-cells or composition to the subject. In some embodiments, one or more immunosuppressants are administered for approximately 48 months following administration of the modified β-cells or composition to the subject. In some embodiments, one or more immunosuppressants are administered for approximately 60 months following administration of the modified β-cells or composition to the subject.

[0227] In some embodiments, one or more immunosuppressants are administered (e.g., daily) while the modified β cells or composition are alive in the subject. In some embodiments, one or more immunosuppressants are administered (e.g., daily) throughout the subject's life. In some embodiments, one or more immunosuppressants may be formulated for administration via any route known to those skilled in the art, including intramuscular, intravenous, intradermal, intralesional, intraperitoneal, subcutaneous, intratumoral, epidural, nasal, oral, vaginal, rectal, topical, local, ocular, inhalation, buccal (e.g., sublingual), and transdermal administration, or any other route. Other administration modalities are also considered in some embodiments. In some embodiments, administration is by bolus infusion, by injection, e.g., intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intra-anterior chamber injection, subconjunctival injection, subcapsular injection, retroocular injection, periocular injection, or posterior proximal scleral delivery. In some implementations, administration is via parenteral, intrapulmonary, and intranasal routes, and, if necessary, intralesional administration if needed for local treatment. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some implementations, administration is via the portal vein. In some implementations, administration is by injection into the intermuscular space of the subject's forearm.

[0228] In some embodiments, one or more immunosuppressants may be administered at any suitable site within the subject's body. For example, in some embodiments, one or more immunosuppressants may be administered to the subject's kidney, forearm, mouth, anus, nose, upper arm, hip, thigh, buttock, liver, spleen, muscle, subcutaneous tissue, or white adipose tissue. In some embodiments, one or more immunosuppressants may be administered to the subject's forearm. In some embodiments, one or more immunosuppressants may be administered to the intermuscular space of the subject's forearm. In some embodiments, one or more immunosuppressants may be administered to the subject's liver, muscle, or white adipose tissue. In some embodiments, the white adipose tissue is the omentum.

[0229] 2. Exemplary immunosuppressants Most clinically used immunosuppressive regimens consist of a combination of one or more immunosuppressants used according to a selected protocol. Immunosuppressive regimens can be categorized as induction, maintenance, or anti-rejection. Induction regimens provide strong early postoperative immunosuppression (e.g., before, concurrently with, and / or after administration of modified β-cells or a composition to the subject); while maintenance regimens are used throughout the subject's life to prevent acute and chronic rejection of the modified β-cells or composition. In some exemplary embodiments, the immunosuppressive regimens provided herein employ the highest intensity of immunosuppression immediately before, concurrently with, and / or after administration of the modified β-cells or composition to the subject (e.g., induction immunosuppression), and gradually reduce the intensity over approximately one year after administration of the modified β-cells or composition (e.g., maintenance immunosuppression), because immune reactivity and the probability of rejection are highest early after administration of the modified β-cells or composition and decrease over time. In such embodiments, a minimum maintenance level of immunosuppression compatible with preventing rejection and minimizing drug toxicity is reached over time. According to some embodiments, maintenance immunosuppression may also be tapered and, in some cases, withdrawn entirely.

[0230] This document 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 based on the specific subject and / or the state of β-cell-related diseases or conditions. Various immunosuppressive regimens are known in the art and are each suitable for the methods and uses provided herein.See, for example, 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].

[0231] In some embodiments, one or more immunosuppressants are small molecules. In some embodiments, the small molecule is a chemical compound. In some embodiments, the small molecule is a nucleic acid. In some embodiments, one or more immunosuppressants are biological products. In some embodiments, the biological product is a protein. In some embodiments, the biological product is an antibody. In some embodiments, one or more immunosuppressants are pharmaceutical salts and / or their precursor forms.

[0232] 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, small molecules are chemical compounds. In some embodiments, small molecules are nucleic acids. In some embodiments, one or more immunomodulators are biological products. In some embodiments, biological products are proteins. In some embodiments, biological products are antibodies. In some embodiments, one or more immunomodulators are pharmaceutical salts and / or their precursor forms.

[0233] Non-limiting examples of immunosuppressants include calcineurin inhibitors, steroids, alkylating agents, antibiotics, analgesics, anti-inflammatory agents, antihistamines, antiviral agents, antifungal agents, anticoagulants, DNA synthesis inhibitors, blood rheology modifiers, inosine monophosphate dehydrogenase (IMDH) inhibitors, Janus kinase inhibitors, mTOR inhibitors, TNF inhibitors, and antiCD25 inhibitors. In some implementations, one or more immunosuppressants include, but are not limited to: anti-thymocyte globulin (ATG), corticosteroids, prednisone, cortisone, prednisolone methylprednisolone, dexamethasone, betamethasone, hydrocortisone, methotrexate, acetaminophen, diphenhydramine, sirolimus (rapamycin), one or more immunosuppressants (FK-506), mycophenolic acid (MPA), mycophenolic acid ethyl ester (MMF), mycophenolic acid sodium, cyclosporine, etanercept (TNFR-Fc), azathioprine, gold salts, sulfasalazine, antimalarial drugs, buquina, leflunomide, imidazolidin, 15-deoxyguanidin, 6-mercaptopurine, cyclophosphamide, OKT3, anti-thymocyte globulin, thymopentin (thymosin-α), fludarabine, cyclophosphamide, and immunosuppressive antibodies. Any suitable combination of any immunosuppressants, regimens, and dosages described herein may be used in conjunction with compositions comprising modified β-cells in the methods and uses provided. Anti-thymocyte globulin (ATG) In some aspects, one or more immunosuppressants include anti-thymocyte globulin (ATG). In some embodiments, ATG is administered to the subject (e.g., one or more ATG regimens are administered to the subject). In some embodiments, ATG is administered to the subject in the form of 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 administration of modified β-cells or a composition. In some embodiments, at least one ATG regimen is administered to the subject before, concurrently with, and / or after administration of modified β-cells or a composition.

[0234] In some embodiments, ATG is administered to the subject prior to administration of the modified β-cells or composition. In some embodiments, ATG is administered to the subject only prior to administration of the modified β-cells or composition. In some embodiments, at least one regimen of ATG is administered to the subject prior to administration of the modified β-cells or composition. In some embodiments, more than one regimen of ATG is administered to the subject prior to administration of the modified β-cells or composition. In some embodiments, ATG (e.g., at least one regimen of ATG) is administered between approximately 30 seconds and approximately 10 weeks prior to administration of the modified β-cells or composition, such as between approximately 30 seconds and approximately 1 hour, between approximately 30 minutes and approximately 12 hours, between approximately 6 hours and approximately 1 day, between approximately 10 hours and approximately 5 days, between approximately 2 days and approximately 7 days, between approximately 5 days and approximately 14 days, between approximately 7 days and approximately 4 weeks, or between approximately 2 weeks and approximately 10 weeks. In some implementations, ATG is administered at least about 30 seconds before the subject is given the modified β cells or composition, 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 earlier. In some implementations, ATG is administered to the subject less than about 10 weeks prior to administration of the modified β cells or composition, such as less than about 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 prior to administration of the modified β cells or composition. In some embodiments, ATG 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 prior to administration of the modified β cells or composition. In some embodiments, a first regimen of ATG is administered to the subject approximately 2 days prior to administration of the modified β cells or composition. In some embodiments, a first regimen of ATG is administered to the subject approximately 1 day prior to administration of the modified β cells or composition.In some implementations, ATG is administered to the subject in a first regimen approximately 2 days prior to administration of the modified β-cells or composition, and ATG is administered to the subject in a second regimen approximately 1 day prior to administration of the modified β-cells or composition.

[0235] In some embodiments, ATG is administered to the subject prior to the administration of the modified β-cells or composition, and is continued throughout the subject's lifespan. In some embodiments, ATG is administered to the subject prior to each round of administration of the modified β-cells or composition. In some embodiments, ATG is administered to the subject prior to each round of administration of the modified β-cells or composition, and is continued throughout the subject's lifespan.

[0236] In some embodiments, ATG (e.g., an ATG regimen) is administered to the subject on the same day as the administration of modified β-cells or the composition. In some embodiments, at least one ATG regimen is administered to the subject on the same day as the administration of modified β-cells or the composition. In some embodiments, ATG is administered to the subject concurrently with the administration of modified β-cells or the composition. In some embodiments, at least one ATG regimen is administered to the subject concurrently with the administration of modified β-cells or the composition. In some embodiments, ATG is administered to the subject concurrently with the administration of modified β-cells or the composition and is continued throughout the subject's lifespan. In some embodiments, ATG is administered to the subject concurrently with the administration of modified β-cells or the composition and is continued throughout the subject's lifespan. In some embodiments, a first ATG regimen is administered to the subject concurrently with the administration of modified β-cells or the composition. In some embodiments, a second ATG regimen is administered to the subject concurrently with the administration of modified β-cells or the composition. In some embodiments, ATG is administered to the subject on the same day as each round of administration of the modified β-cells or composition. In some embodiments, ATG is administered to the subject on the same day as each round of administration of the modified β-cells or composition, and is continued throughout the subject's lifespan. In some embodiments, ATG is administered to the subject concurrently with each round of administration of the modified β-cells or composition. In some embodiments, ATG is administered to the subject concurrently with each round of administration of the modified β-cells or composition, and is continued throughout the subject's lifespan.

[0237] In some embodiments, ATG (e.g., an ATG regimen) is administered to the subject after administration of modified β-cells or the composition. In some embodiments, at least one ATG regimen is administered to the subject after administration of modified β-cells or the composition. In some embodiments, more than one ATG regimen is administered to the subject after administration of modified β-cells or the composition. In some embodiments, ATG is administered to the subject only after administration of the first and / or second regimen of modified β-cells or the composition. In some embodiments, ATG is administered between approximately 30 seconds and approximately 10 weeks after administration of modified β-cells or the composition, such as between approximately 30 seconds and approximately 1 hour, between approximately 30 minutes and approximately 12 hours, between approximately 6 hours and approximately 1 day, between approximately 10 hours and approximately 5 days, between approximately 2 days and approximately 7 days, between approximately 5 days and approximately 14 days, between approximately 7 days and approximately 4 weeks, or between approximately 2 weeks and approximately 10 weeks. In some implementations, ATG is administered at least about 30 seconds after the administration of the modified β-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 later. In some implementations, ATG is administered to a subject less than about 10 weeks after administration of the modified β-cells or composition, such as less than about 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 prior to administration of the modified β-cells or composition. In some embodiments, ATG is administered to the subject at 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 β cells or composition. In some embodiments, ATG is administered to the subject 48 hours after administration of the modified β cells or composition.In some embodiments, ATG is administered to the subject after administration of the modified β-cells or composition, and is continued throughout the subject's lifespan. In some embodiments, ATG is administered to the subject after each round of administration of the modified β-cells or composition. In some embodiments, ATG is administered to the subject after each round of administration of the modified β-cells or composition, and is continued throughout the subject's lifespan.

[0238] In some embodiments, ATG is administered to the subject before and after administration of the modified β-cells or composition. In some embodiments, ATG is administered to the subject before, on the same day, and after administration of the modified β-cells or composition. In some embodiments, ATG is administered to the subject before, concurrently with, and after administration of the modified β-cells or composition. In some embodiments, ATG is administered to the subject approximately 2 days before administration of the composition containing modified β-cells: i) approximately 1 day before; ii) approximately 1 day before; iii) on the same day; iv) approximately 1 day after; and / or v) approximately 2 days after.

[0239] In some embodiments, an ATG regimen and / or total daily dose between about 0.05 mg / kg and about 4.0 mg / kg, such as between about 0.05 mg / kg and about 1.0 mg / kg, between about 0.1 mg / kg and about 2.0 mg / kg, between about 1.0 mg / kg and about 3.0 mg / kg, or between about 2.0 mg / kg and about 4.0 mg / kg, is administered to the subject. In some embodiments, an ATG regimen between about 0.1 mg / kg and about 2.0 mg / kg is administered to the subject. In some embodiments, a regimen of more than about 0.05 mg / kg of ATG, such as a regimen of more than any of about 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 more of ATG, is administered to the subject. In some embodiments, a regimen of less than about 4.0 mg / kg of ATG, such as a regimen of less than any of about 3.5 mg / kg, 3.0 mg / kg, 2.5 mg / kg, 2.0 mg / kg, 1.5 mg / kg, 1.0 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, 0.1 mg / kg, 0.05 mg / kg or less of ATG, is administered to the subject. In some embodiments, a regimen of approximately 0.5 mg / kg ATG is administered to the subject. In some embodiments, a regimen of approximately 1.0 mg / kg ATG is administered to the subject. In some embodiments, a regimen of approximately 1.5 mg / kg ATG is administered to the subject.

[0240] In some embodiments, a regimen of about 0.5 mg / kg ATG is administered to the subject about 2 days prior to administration of the modified β-cells or composition. In some embodiments, a regimen of about 1.0 mg / kg ATG is administered to the subject about 1 day prior to administration of the modified β-cells or composition. In some embodiments, a regimen of about 1.5 mg / kg ATG is administered to the subject on the same day as administration of the modified β-cells or composition. In some embodiments, a regimen of about 1.5 mg / kg ATG is administered to the subject 1 day after administration of the modified β-cells or composition. In some embodiments, a regimen of about 1.5 mg / kg ATG is administered to the subject about 2 days after administration of the modified β-cells or composition. In some embodiments, i) a regimen in which approximately 0.5 mg / kg of ATG is administered to the subject approximately 2 days prior to administration of the modified β-cells or composition; ii) a regimen in which approximately 1.0 mg / kg of ATG is administered to the subject approximately 1 day prior to administration of the modified β-cells or composition; and iii) a regimen in which approximately 1.5 mg / kg of ATG is administered to the subject on the same day as administration of the modified β-cells or composition, approximately 1 day after administration of the modified β-cells or composition, and approximately 2 days after administration of the modified β-cells or composition. In some embodiments, ATG is administered to the subject at a lower dose.

[0241] a. Steroids In some aspects, one or more immunosuppressants include steroids (e.g., one or more steroids). Steroids can be used to reduce inflammation in a subject. In some embodiments, the steroid is a corticosteroid. In some aspects, one or more immunosuppressants include prednisone, cortisone, prednisolone methylprednisolone, dexamethasone, betamethasone, and / or hydrocortisone. In some embodiments, the steroid is administered to the 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 the subject in the form of one or more compositions (e.g., a pharmaceutical composition containing one or more steroids). In some embodiments, one or more steroids are administered to the subject before, concurrently with, and / or after administration of modified β-cells or a composition (e.g., a composition containing modified β-cells). In some embodiments, at least one regimen of steroids is administered to the subject before, concurrently with, and / or after administration of modified β-cells or a composition.

[0242] In some embodiments, one or more immunosuppressants do not include steroids. In some embodiments, subjects who have previously received or are currently receiving steroid treatment are not suitable for treatment with any of the methods or uses described herein.

[0243] In some embodiments, one or more immunosuppressants include methylprednisolone. In some embodiments, methylprednisolone (e.g., a methylprednisolone regimen) is administered to the subject before, concurrently with, and / or after administration of the modified β-cells or composition. In some embodiments, at least one regimen of methylprednisolone is administered to the subject before, concurrently with, and / or after administration of the modified β-cells or composition.

[0244] In some embodiments, methylprednisolone (e.g., a methylprednisolone regimen) is administered to the subject before administration of the modified β-cells or composition. In some embodiments, methylprednisolone is administered to the subject only before administration of the modified β-cells or composition. In some embodiments, at least one regimen of methylprednisolone is administered to the subject before administration of the modified β-cells or composition. In some embodiments, more than one regimen of methylprednisolone is administered to the subject before administration of the modified β-cells or composition. In some embodiments, methylprednisolone (e.g., at least one regimen of methylprednisolone) is administered between approximately 30 seconds and approximately 10 weeks prior to administration of the modified β-cells or composition to the subject, such as between approximately 30 seconds and approximately 1 hour, between approximately 30 minutes and approximately 12 hours, between approximately 6 hours and approximately 1 day, between approximately 10 hours and approximately 5 days, between approximately 2 days and approximately 7 days, between approximately 5 days and approximately 14 days, between approximately 7 days and approximately 4 weeks, and between approximately 2 weeks and approximately 10 weeks prior to administration of the modified β-cells or composition to the subject. In some implementations, methylprednisolone is administered at least about 30 seconds before the administration of the modified β-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 earlier. In some implementations, methylprednisolone is administered to the subject less than about 10 weeks prior to administration of the modified β-cells or composition, such as less than about 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 prior to administration of the modified β-cells or composition. 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 prior to administration of the modified β-cells or composition. In some embodiments, the methylprednisolone regimen is administered to the subject approximately 2 days prior to administration of the modified β-cells or composition.

[0245] In some embodiments, the methylprednisolone regimen is administered to the subject before the ATG regimen is administered. In some embodiments, the methylprednisolone regimen is administered to the subject only before the ATG regimen is administered. In some embodiments, the methylprednisolone regimen is administered to the subject before administering the first regimen of ATG. In some embodiments, the methylprednisolone regimen is administered to the subject only before administering the first regimen of ATG. In some embodiments, the methylprednisolone regimen is administered to the subject between approximately 30 minutes and approximately 24 hours before administering the ATG regimen (e.g., the first regimen), such as between approximately 30 minutes and approximately 5 hours, between approximately 1 hour and approximately 3 hours, between approximately 4 hours and approximately 10 hours, or between approximately 8 hours and approximately 24 hours before administering the ATG regimen. In some embodiments, the methylprednisolone regimen is administered to the subject approximately 1 hour before administering the ATG regimen. In some embodiments, the methylprednisolone regimen is administered to the subject approximately 1 hour before administering the first regimen of ATG. In some embodiments, the methylprednisolone regimen is administered to the subject concurrently with the administration of the ATG regimen. In some embodiments, the methylprednisolone regimen is administered to the subject concurrently with the administration of the first ATG regimen. 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). In some embodiments, the methylprednisolone regimen is administered to the subject before the administration of the ATG, and before the administration of the modified β-cells or composition, and before the administration of the methylprednisolone regimen. In some embodiments, both the methylprednisolone regimen and the ATG regimen are administered to the subject before the administration of the modified β-cells or composition.

[0246] In some embodiments, a methylprednisolone regimen and / or total daily dose between about 0.05 mg / kg and about 4.0 mg / kg, such as between about 0.05 mg / kg and about 1.0 mg / kg, between about 0.1 mg / kg and about 2.0 mg / kg, between about 1.0 mg / kg and about 3.0 mg / kg, or between about 2.0 mg / kg and about 4.0 mg / kg, is administered to the subject. In some embodiments, a methylprednisolone regimen between about 0.1 mg / kg and about 2.0 mg / kg is administered to the subject. In some implementations, a regimen of more than about 0.05 mg / kg of methylprednisolone, such as a regimen of more than about 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 more of methylprednisolone, is administered to the subject. In some embodiments, a regimen of less than about 4.0 mg / kg of methylprednisolone, such as less than about 3.5 mg / kg, 3.0 mg / kg, 2.5 mg / kg, 2.0 mg / kg, 1.5 mg / kg, 1.0 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or less, is administered to the subject. In some embodiments, a regimen of about 1.0 mg / kg of methylprednisolone is administered to the subject. In some embodiments, methylprednisolone is administered intravenously to the subject.

[0247] In some embodiments, a regimen of approximately 1.0 mg / kg of methylprednisolone is administered to the subject approximately 1 hour prior to administration of the first regimen of ATG. In some embodiments, a regimen of approximately 1.0 mg / kg of methylprednisolone is administered to the subject approximately midway through administration of the first regimen of ATG. In some embodiments, a first regimen of approximately 0.5 mg / kg of ATG is administered to the subject approximately 2 days prior to administration of the modified β-cells or composition. In some embodiments, a regimen of approximately 1.0 mg / kg of ATG is administered to the subject approximately 1 day prior to administration of the modified β-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 β-cells or composition. In some embodiments, a regimen of approximately 1.5 mg / kg of ATG is administered to the subject 1 day after administration of the modified β-cells or composition. In some implementations, approximately 1.5 mg / kg of ATG is administered to the subject about 2 days after administration of the modified β cells or composition. In some embodiments, i) a regimen of approximately 1.0 mg / kg of methylprednisolone is administered to the subject approximately 1 hour before administration of the first regimen of ATG; ii) a regimen of approximately 1.0 mg / kg of methylprednisolone is administered to the subject approximately midway through administration of the first regimen of ATG; iii) a regimen of approximately 0.5 mg / kg of ATG is administered to the subject approximately 2 days before administration of the modified β-cells or composition; iv) a regimen of approximately 1.0 mg / kg of ATG is administered to the subject approximately 1 day before administration of the modified β-cells or composition; 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 β-cells or composition, approximately 1 day after administration of the composition containing modified β-cells, and approximately 2 days after administration of the modified β-cells or composition. In some embodiments, the methylprednisolone regimen is administered at a lower dose. In some embodiments, the ATG regimen is administered at a lower dose.

[0248] b. Analgesics In some aspects, one or more immunosuppressants include analgesics (e.g., one or more analgesics). Analgesics are medicines that can be used to relieve pain. In some embodiments, the analgesic is acetaminophen, an opioid, or a nonsteroidal anti-inflammatory drug (NSAID). In some embodiments, the analgesic is administered to a subject (e.g., one or more regimens of the analgesic are administered to the subject). In some embodiments, the analgesic is administered to the subject in the form of one or more compositions (e.g., a pharmaceutical composition containing the analgesic). In some embodiments, the analgesic is administered to the subject before, concurrently with, and / or after administration of modified β-cells or a composition (e.g., a composition containing modified β-cells). In some embodiments, at least one regimen of the analgesic is administered to the subject before, concurrently with, and / or after administration of modified β-cells or a composition.

[0249] In some embodiments, one or more immunosuppressants include acetaminophen. In some embodiments, acetaminophen is administered to the subject before, concurrently with, and / or after administration of the modified β-cells or composition. In some embodiments, at least one regimen of acetaminophen is administered to the subject before, concurrently with, and / or after administration of the modified β-cells or composition.

[0250] In some embodiments, acetaminophen is administered to the subject prior to administration of the modified β-cells or composition. In some embodiments, acetaminophen is administered to the subject only prior to administration of the modified β-cells or composition. In some embodiments, at least one regimen of acetaminophen is administered to the subject prior to administration of the modified β-cells or composition. In some embodiments, more than one regimen of acetaminophen is administered to the subject prior to administration of the modified β-cells or composition. In some embodiments, acetaminophen (e.g., at least one regimen of acetaminophen) is administered between approximately 30 seconds and approximately 10 weeks prior to administration of the modified β-cells or composition, such as between approximately 30 seconds and approximately 1 hour, between approximately 30 minutes and approximately 12 hours, between approximately 6 hours and approximately 1 day, between approximately 10 hours and approximately 5 days, between approximately 2 days and approximately 7 days, between approximately 5 days and approximately 14 days, between approximately 7 days and approximately 4 weeks, or between approximately 2 weeks and approximately 10 weeks. In some implementations, acetaminophen is administered to the subject at least about 30 seconds prior to administration of the modified β-cells or composition, 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 earlier. In some implementations, acetaminophen is administered to the subject less than about 10 weeks prior to administration of the modified β-cells or composition, such as less than about 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 prior to administration of the modified β-cells or composition. In some embodiments, acetaminophen 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 prior to administration of the modified β-cells or composition. In some embodiments, the acetaminophen regimen is administered to the subject approximately 2 days prior to administration of the modified β-cells or composition.

[0251] In some embodiments, the acetaminophen regimen is administered to the subject before the ATG regimen is administered. In some embodiments, the acetaminophen regimen is administered to the subject only before the ATG regimen is administered. In some embodiments, the acetaminophen regimen is administered to the subject before administering the first ATG regimen. In some embodiments, the acetaminophen regimen is administered to the subject only before administering the first ATG regimen. In some embodiments, the acetaminophen regimen is administered to the subject between approximately 30 minutes and approximately 24 hours before administering the ATG regimen (e.g., the first regimen), such as between approximately 30 minutes and approximately 5 hours, approximately 1 hour and approximately 3 hours, approximately 4 hours and approximately 10 hours, or approximately 8 hours and approximately 24 hours before administering the ATG regimen. In some embodiments, the acetaminophen regimen is administered to the subject approximately 30 minutes before administering the ATG regimen. In some embodiments, the acetaminophen regimen is administered to the subject approximately 30 minutes before administering the first ATG regimen. In some embodiments, the acetaminophen regimen is administered to the subject concurrently with the administration of the ATG regimen. In some embodiments, the acetaminophen regimen is administered to the subject concurrently with the administration of the first ATG regimen. 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). In some embodiments, the acetaminophen regimen is administered to the subject before the administration of ATG, and before the administration of the modified β-cells or composition, and before the administration of the acetaminophen regimen. In some embodiments, both the acetaminophen regimen and the ATG regimen are administered to the subject before the administration of the modified β-cells or composition.

[0252] In some embodiments, a regimen of acetaminophen between about 10 mg and about 5,000 mg and / or a total daily dose, such as between about 10 mg and about 100 mg, between about 100 mg and about 1,000 mg, or between about 500 mg and about 5,000 mg, is administered to the subject. In some embodiments, a regimen of acetaminophen between about 100 mg and about 10,000 mg is administered to the subject. In some embodiments, a regimen of more than about 10 mg of acetaminophen, such as a regimen of more than about 20 mg, 30 mg, 40 mg, 50 mg, 100 mg, 500 mg, 1,000 mg, 2,000 mg, 3,000 mg, 4,000 mg, 5,000 mg, or more of acetaminophen, is administered to the subject. In some embodiments, a regimen of less than about 5,000 mg of acetaminophen, such as less than about 4,000 mg, 3,000 mg, 2,000 mg, 1,000 mg, 500 mg, 100 mg, 50 mg, 40 mg, 30 mg, 20 mg, 10 mg, or less of acetaminophen, is administered to the subject. In some embodiments, a regimen of about 650 mg of acetaminophen is administered to the subject. In some embodiments, acetaminophen is administered to the subject orally or rectally.

[0253] In some embodiments, approximately 650 mg of acetaminophen is administered to the subject about 30 minutes before administration of the first regimen of ATG. In some embodiments, approximately 650 mg of acetaminophen is administered to the subject approximately midway through administration of the first regimen of ATG. In some embodiments, approximately 0.5 mg / kg of ATG is administered to the subject about 2 days before administration of the modified β-cells or composition. In some embodiments, approximately 1.0 mg / kg of ATG is administered to the subject about 1 day before administration of the modified β-cells or composition. In some embodiments, approximately 1.5 mg / kg of ATG is administered to the subject on the same day as administration of the modified β-cells or composition. In some embodiments, approximately 1.0 mg / kg of ATG is administered to the subject about 1 day after administration of the modified β-cells or composition. In some embodiments, approximately 2 days after administration of the modified β-cells or composition to the subject, a regimen of approximately 1.0 mg / kg of ATG is administered. In some embodiments, i) approximately 30 minutes before administration of the first regimen of ATG to the subject, a regimen of approximately 650 mg of acetaminophen is administered; ii) approximately midway through administration of the first regimen of ATG to the subject, a regimen of approximately 650 mg of acetaminophen is administered; iii) approximately 2 days before administration of the modified β-cells or composition to the subject, a regimen of approximately 0.5 mg / kg of ATG is administered; iv) approximately 1 day before administration of the modified β-cells or composition to the subject, a regimen of approximately 1.0 mg / kg of ATG is administered; and / or, v) approximately 1.5 mg / kg of ATG is administered to the subject on the same day as administration of the modified β-cells or composition, approximately 1 day after administration of the modified β-cells or composition to the subject, and approximately 2 days after administration of the modified β-cells or composition to the subject. In some embodiments, acetaminophen is administered at a lower dose. In some embodiments, ATG is administered at a lower dose.

[0254] c. Antihistamines In some aspects, one or more immunosuppressants include antihistamines (e.g., one or more antihistamines). Antihistamines are medications that can be used to relieve allergy symptoms such as runny nose, sneezing, and nasal congestion. In some embodiments, the antihistamine is an H1-antihistamine, H2-antihistamine, H3-antihistamine, H4-antihistamine, histidine decarboxylase inhibitor, or mast cell inhibitor. In some embodiments, the antihistamine may be, but is not limited to, diphenhydramine, doxylamine, hydroxyzine, promethazine, bensulfanilamine, olphenadine, tropineamine, cimetidine, clofenac, thiamethoxam, sodium cromoglycate, or catechin. In some embodiments, the antihistamine is administered to a subject (e.g., one or more regimens of the antihistamine are administered to the subject). In some embodiments, the antihistamine is administered to the subject in the form of one or more compositions (e.g., pharmaceutical compositions containing an antihistamine). In some embodiments, an antihistamine is administered to the subject before, concurrently with, and / or after administration of the modified β-cells or composition. In some embodiments, at least one regimen of the antihistamine is administered to the subject before, concurrently with, and / or after administration of the modified β-cells or composition.

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

[0256] In some embodiments, diphenhydramine (e.g., a diphenhydramine regimen) is administered to the subject before administration of the modified β-cells or composition. In some embodiments, diphenhydramine is administered to the subject only before administration of the modified β-cells or composition. In some embodiments, at least one regimen of diphenhydramine is administered to the subject before administration of the modified β-cells or composition. In some embodiments, more than one regimen of diphenhydramine is administered to the subject before administration of the modified β-cells or composition. In some embodiments, diphenhydramine (e.g., at least one regimen of diphenhydramine) is administered to the subject between approximately 30 seconds and approximately 10 weeks prior to administration of the modified β cells or composition, such as between approximately 30 seconds and approximately 1 hour, between approximately 30 minutes and approximately 12 hours, between approximately 6 hours and approximately 1 day, between approximately 10 hours and approximately 5 days, between approximately 2 days and approximately 7 days, between approximately 5 days and approximately 14 days, between approximately 7 days and approximately 4 weeks, and between approximately 2 weeks and approximately 10 weeks prior to administration of the modified β cells or composition. In some implementations, diphenhydramine is administered at least about 30 seconds before the administration of the modified β-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 earlier. In some implementations, diphenhydramine is administered to the subject less than about 10 weeks prior to administration of the modified β-cells or composition, such as less than about 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 prior to administration of the modified β-cells or composition. 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 prior to administration of the modified β-cells or composition. In some embodiments, the diphenhydramine regimen is administered to the subject approximately 2 days prior to administration of the modified β-cells or composition.

[0257] In some embodiments, the diphenhydramine regimen is administered to the subject before the ATG regimen is administered. In some embodiments, the diphenhydramine regimen is administered to the subject only before the ATG regimen is administered. In some embodiments, the diphenhydramine regimen is administered to the subject before administering the first regimen of ATG. In some embodiments, the diphenhydramine regimen is administered to the subject only before administering the first regimen of ATG. In some embodiments, the diphenhydramine regimen is administered to the subject between approximately 30 minutes and approximately 24 hours before administering the ATG regimen (e.g., the first regimen), such as between approximately 30 minutes and approximately 5 hours, approximately 1 hour and approximately 3 hours, approximately 4 hours and approximately 10 hours, or approximately 8 hours and approximately 24 hours before administering the ATG regimen. In some embodiments, the diphenhydramine regimen is administered to the subject approximately 30 minutes before administering the ATG regimen. In some embodiments, the diphenhydramine regimen is administered to the subject approximately 30 minutes before administering the first regimen of ATG. In some embodiments, the diphenhydramine regimen is administered to the subject concurrently with the administration of the ATG regimen. In some embodiments, the diphenhydramine regimen is administered to the subject concurrently with the administration of the first ATG regimen. 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). In some embodiments, the diphenhydramine regimen is administered to the subject before the administration of ATG, and before the administration of modified β-cells or the composition, and before the administration of the diphenhydramine regimen. In some embodiments, both the diphenhydramine regimen and the ATG regimen are administered to the subject before the administration of modified β-cells or the composition.

[0258] In some embodiments, a diphenhydramine regimen and / or a total daily dose between about 1 mg and about 1,000 mg, such as between about 1 mg and about 100 mg, about 50 mg and about 500 mg, or about 500 mg and about 1,000 mg, is administered to the subject. In some embodiments, a diphenhydramine regimen between about 10 mg and about 100 mg is administered to the subject. In some embodiments, a diphenhydramine regimen of more than about 1 mg, such as a regimen of more than about 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 more, is administered to the subject. In some embodiments, a regimen of less than about 1,000 mg of diphenhydramine, such as less than about 500 mg, 400 mg, 300 mg, 200 mg, 100 mg, 50 mg, 10 mg, 5 mg, 4 mg, 3 mg, 2 mg, 1 mg, or less, is administered to the subject. In some embodiments, a regimen of about 50 mg of diphenhydramine is administered to the subject. In some embodiments, diphenhydramine is administered to the subject orally or rectally.

[0259] In some embodiments, approximately 50 mg of diphenhydramine is administered to the subject about 30 minutes before administration of the first regimen of ATG. In some embodiments, approximately 50 mg of diphenhydramine is administered to the subject midway through administration of the first regimen of ATG. In some embodiments, approximately 0.5 mg / kg of ATG is administered to the subject about 2 days before administration of the modified β-cells or composition. In some embodiments, approximately 1.0 mg / kg of ATG is administered to the subject about 1 day before administration of the modified β-cells or composition. In some embodiments, approximately 1.5 mg / kg of ATG is administered to the subject on the same day as administration of the modified β-cells or composition. In some embodiments, approximately 1.5 mg / kg of ATG is administered to the subject about 1 day after administration of the modified β-cells or composition. In some embodiments, a regimen of approximately 1.5 mg / kg ATG is administered to the subject 2 days after administration of the modified β-cells or composition. In some embodiments, i) approximately 30 minutes before administration of the first regimen of ATG is administered to the subject; ii) approximately 50 mg of diphenhydramine is administered to the subject midway through administration of the first regimen of ATG; iii) approximately 2 days before administration of the modified β-cells or composition is administered to the subject; iv) approximately 1 day before administration of the modified β-cells or composition is administered to the subject; and / or, v) approximately 1.5 mg / kg ATG is administered to the subject on the same day as administration of the modified β-cells or composition, approximately 1 day after administration of the modified β-cells or composition, and approximately 2 days after administration of the modified β-cells or composition. In some implementations, diphenhydramine is administered at a lower dose. In some implementations, ATG is administered at a lower dose.

[0260] d. Anti-inflammatory agents In some embodiments, one or more immunosuppressants include anti-inflammatory agents (e.g., one or more anti-inflammatory agents). Anti-inflammatory agents are drugs 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. TNF inhibitors may be, but are not limited to, infliximab, adalimumab, etanercept (TNFR-Fc), golimumab, and sertozumab. 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 the anti-inflammatory agent are administered to the subject). In some embodiments, the anti-inflammatory agent is administered to the subject in the form of one or more compositions (e.g., a pharmaceutical composition containing an anti-inflammatory agent). In some embodiments, the anti-inflammatory agent is administered to the subject before, concurrently with, and / or after administration of modified β-cells or a composition (e.g., a composition containing modified β-cells). In some embodiments, at least one regimen of an anti-inflammatory agent is administered to the subject before, concurrently with, and / or after the administration of modified β-cells or a composition.

[0261] In some embodiments, one or more immunosuppressants include dexamethasone. In some embodiments, dexamethasone (e.g., a dexamethasone regimen) is administered to the subject before, concurrently with, and / or after administration of the modified β-cells or composition. In some embodiments, at least one regimen of dexamethasone is administered to the subject before, concurrently with, and / or after administration of the modified β-cells or composition.

[0262] In some embodiments, one or more immunosuppressants include etanercept. In some embodiments, etanercept (e.g., an etanercept regimen) is administered to the subject before, concurrently with, and / or after administration of modified β-cells or a composition. In some embodiments, at least one regimen of etanercept is administered to the subject before, concurrently with, and / or after administration of modified β-cells or a composition.

[0263] In some embodiments, etanercept (e.g., an etanercept regimen) is administered to the subject before administration of the modified β-cells or composition. In some embodiments, etanercept is administered to the subject only before administration of the modified β-cells or composition. In some embodiments, at least one regimen of etanercept is administered to the subject before administration of the modified β-cells or composition. In some embodiments, more than one regimen of etanercept is administered to the subject before administration of the modified β-cells or composition. In some embodiments, etanercept (e.g., at least one regimen of etanercept) is administered to the subject between approximately 30 seconds and approximately 10 weeks prior to administration of the modified β cells or composition, such as between approximately 30 seconds and approximately 1 hour, between approximately 30 minutes and approximately 12 hours, between approximately 6 hours and approximately 1 day, between approximately 10 hours and approximately 5 days, between approximately 2 days and approximately 7 days, between approximately 5 days and approximately 14 days, between approximately 7 days and approximately 4 weeks, and between approximately 2 weeks and approximately 10 weeks prior to administration of the modified β cells or composition. In some implementations, etanercept is administered at least about 30 seconds before the administration of the modified β-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 earlier. In some implementations, etanercept is administered to the subject less than about 10 weeks prior to administration of the modified β-cells or composition, such as less than about 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 prior to administration of the modified β-cells or composition. In some implementations, 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 prior to administration of the modified β cells or composition to the subject.

[0264] In some embodiments, etanercept (e.g., etanercept regimens, such as the first regimen) is administered to the subject on the same day as the administration of modified β-cells or the composition. In some embodiments, at least one regimen of etanercept is administered to the subject on the same day as the administration of modified β-cells or the composition. In some embodiments, etanercept is administered to the subject concurrently with the administration of modified β-cells or the composition. In some embodiments, at least one regimen of etanercept is administered to the subject concurrently with the administration of modified β-cells or the composition. In some embodiments, etanercept is administered to the subject on the same day as the administration of modified β-cells or the composition, and is continued throughout the subject's lifespan. In some embodiments, etanercept is administered to the subject concurrently with the administration of modified β-cells or the composition, and is continued throughout the subject's lifespan. In some embodiments, a first regimen of etanercept is administered to the subject concurrently with the administration of modified β-cells or the composition. In some embodiments, etanercept is administered to the subject concurrently with the administration of the modified β-cells or composition. In some embodiments, etanercept is administered to the subject on the same day as each round of administration of the modified β-cells or composition. In some embodiments, etanercept is administered to the subject on the same day as each round of administration of the modified β-cells or composition, and is continued throughout the subject's lifespan. In some embodiments, etanercept is administered to the subject concurrently with each round of administration of the modified β-cells or composition. In some embodiments, etanercept is administered to the subject concurrently with each round of administration of the modified β-cells or composition, and is continued throughout the subject's lifespan.

[0265] In some embodiments, etanercept (e.g., an etanercept regimen) is administered to the subject after administration of modified β-cells or a composition. In some embodiments, at least one regimen of etanercept is administered to the subject after administration of modified β-cells or a composition. In some embodiments, more than one regimen of etanercept is administered to the subject after administration of modified β-cells or a composition. In some embodiments, etanercept is administered to the subject only after administration of the first and / or second regimen of modified β-cells or a composition. In some embodiments, etanercept is administered to a subject between approximately 30 seconds and approximately 10 weeks after administration of the modified β-cells or composition, such as between approximately 30 seconds and approximately 1 hour, between approximately 30 minutes and approximately 12 hours, between approximately 6 hours and approximately 1 day, between approximately 10 hours and approximately 5 days, between approximately 2 days and approximately 7 days, between approximately 5 days and approximately 14 days, between approximately 7 days and approximately 4 weeks, and between approximately 2 weeks and approximately 10 weeks. In some implementations, etanercept is administered at least about 30 seconds after administration of the modified β-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 later. In some implementations, etanercept is administered to a subject less than about 10 weeks after administration of the modified β-cells or composition, such as less than about 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 prior to administration of the modified β-cells or composition. In some embodiments, etanercept 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 after administration of the modified β cells or composition. In some embodiments, etanercept is administered to the subject approximately 3 days after administration of the modified β cells or composition. In some embodiments, etanercept is administered to the subject approximately 7 days after administration of the modified β cells or composition.In some embodiments, etanercept is administered to the subject approximately 10 days after administration of the modified β-cells or composition. In some embodiments, etanercept is administered to the subject approximately 3 days, approximately 7 days, and approximately 10 days after administration of the modified β-cells or composition. In some embodiments, etanercept is administered to the subject after administration of the modified β-cells or composition and is continued throughout the subject's lifespan. In some embodiments, etanercept is administered to the subject after each round of administration of the modified β-cells or composition. In some embodiments, etanercept is administered to the subject after each round of administration of the modified β-cells or composition and is continued throughout the subject's lifespan.

[0266] In some embodiments, etanercept is administered to the subject on the same day and after administration of the modified β-cells or composition. In some embodiments, etanercept is administered to the subject simultaneously with and after administration of the modified β-cells or composition. In some embodiments, etanercept is administered to the subject on the same day as: i) administration of the composition containing modified β-cells; approximately 3 days after: ii) administration; approximately 7 days after: iii) administration; and / or approximately 10 days after: iv) administration.

[0267] In some embodiments, a regimen of etanercept between about 1 mg and about 1,000 mg and / or a total daily dose, such as between about 1 mg and about 100 mg, about 50 mg and about 500 mg, or about 500 mg and about 1,000 mg, is administered to the subject. In some embodiments, a regimen of etanercept between about 10 mg and about 100 mg is administered to the subject. In some embodiments, a regimen of more than about 1 mg of etanercept, such as a regimen of more than about 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 more of etanercept, is administered to the subject. In some embodiments, a regimen of less than about 1,000 mg of etanercept, such as less than about 500 mg, 400 mg, 300 mg, 200 mg, 100 mg, 50 mg, 10 mg, 5 mg, 4 mg, 3 mg, 2 mg, 1 mg, or less, is administered to the subject. In some embodiments, a regimen of about 50 mg of etanercept is administered to the subject. In some embodiments, a regimen of about 25 mg of etanercept is administered to the subject.

[0268] In some embodiments, a regimen of about 50 mg of etanercept is administered to the subject on the same day as administration of the modified β-cells or composition. In some embodiments, a regimen of about 25 mg of etanercept is administered to the subject approximately 3 days after administration of the modified β-cells or composition. In some embodiments, a regimen of about 25 mg of etanercept is administered to the subject approximately 7 days after administration of the modified β-cells or composition. In some embodiments, a regimen of about 25 mg of etanercept is administered to the subject approximately 10 days after administration of the modified β-cells or composition. In some embodiments, i) a regimen of about 50 mg of etanercept is administered to the subject on the same day as administration of the modified β-cells or composition; and ii) a regimen of about 25 mg of etanercept is administered to the subject approximately 3 days, approximately 7 days, and approximately 10 days after administration of the modified β-cells or composition. In some embodiments, the etanercept regimen is administered at a lower dose.

[0269] In some embodiments, the subject is given both an etanercept regimen and an ATG regimen. In some embodiments, the subject is given at least one etanercept regimen and at least one ATG regimen. In some embodiments, the subject is given at least one ATG regimen before, on the same day, concurrently with, and / or after at least one etanercept regimen. In some embodiments, the subject is given at least one ATG regimen before at least one etanercept regimen. In some embodiments, the subject is given a regimen of approximately 40 mg / kg of ATG daily for four consecutive days. In some embodiments, after the ATG regimen, the subject is given a first regimen of approximately 25 mg of etanercept twice weekly for two consecutive weeks. In some embodiments, after the first etanercept regimen, the subject is given a regimen of approximately 25 mg of etanercept once monthly for approximately four months. In some embodiments, the etanercept regimen is administered at a lower dose. In some embodiments, the ATG regimen is administered at a lower dose.

[0270] In some embodiments, the subject is administered an etanercept regimen and an IL-1 receptor antagonist regimen. In some embodiments, the subject is administered at least one etanercept regimen and at least one IL-1 receptor antagonist regimen. In some embodiments, the subject is administered at least one IL-1 receptor antagonist regimen before, on the same day, concurrently with, and / or after at least one etanercept regimen.

[0271] e. mTOR inhibitors In some aspects, one or more immunosuppressants include mechanistic target (mTOR) inhibitors of rapamycin (e.g., one or more mTOR inhibitors). mTOR inhibitors are drugs that inhibit mTOR (a serine / threonine-specific protein kinase belonging to the phosphatidylinositol-3 kinase (PI3K)-associated kinase (PIKK) family). In some embodiments, the mTOR inhibitor is rapamycin or an analogue thereof, such as, but not limited to, sirolimus, tesimolimus, everolimus, ridaforolimus, umirolimus, or zotacolimus. 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 the mTOR inhibitor are administered to a subject). In some embodiments, the mTOR inhibitor is administered to a subject in the form of one or more compositions (e.g., a pharmaceutical composition containing an mTOR inhibitor). In some embodiments, the mTOR inhibitor is administered to the subject before, concurrently with, and / or after administration of modified β cells or the composition to the subject. In some embodiments, at least one regimen of the mTOR inhibitor is administered to the subject before, concurrently with, and / or after administration of the modified β cells or composition.

[0272] 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 administration of the modified β-cells or composition. In some embodiments, at least one regimen of sirolimus is administered to the subject before, concurrently with, and / or after administration of the modified β-cells or composition.

[0273] In some embodiments, sirolimus (e.g., a sirolimus regimen) is administered to the subject before administration of the modified β-cells or composition. In some embodiments, sirolimus is administered to the subject only before administration of the modified β-cells or composition. In some embodiments, at least one regimen of sirolimus is administered to the subject before administration of the modified β-cells or composition. In some embodiments, more than one regimen of sirolimus is administered to the subject before administration of the modified β-cells or composition. In some embodiments, sirolimus (e.g., at least one regimen of sirolimus) is administered between approximately 30 seconds and approximately 10 weeks prior to administration of the modified β-cells or composition to the subject, such as between approximately 30 seconds and approximately 1 hour, between approximately 30 minutes and approximately 12 hours, between approximately 6 hours and approximately 1 day, between approximately 10 hours and approximately 5 days, between approximately 2 days and approximately 7 days, between approximately 5 days and approximately 14 days, between approximately 7 days and approximately 4 weeks, and between approximately 2 weeks and approximately 10 weeks prior to administration of the modified β-cells or composition to the subject. In some implementations, sirolimus is administered at least about 30 seconds before administration of the modified β-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 earlier. In some implementations, sirolimus is administered to the subject less than about 10 weeks prior to administration of the modified β-cells or composition, such as less than about 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 prior to administration of the modified β-cells or composition. In some implementations, sirolimus 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...

Claims

1. A method for treating or preventing β-cell disease in a subject of need, the method comprising administering to the subject a dose of engineered, low-immunogenic islets. The dose is administered to the subject via intramuscular injection, and The dose mentioned above is from the following: A) Approximately 1×10 7 One cell to approximately 3 × 10 8 One cell; B) Approximately 1.25 × 10 5 Cells / kg to approximately 1.2 × 10⁻⁶ 7 Cells / kg; C) Approximately 6,500 islet equivalents (IEQ) to approximately 600,000 IEQ; or D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg.

2. A method for reducing exogenous insulin dependence in subjects who have or are at risk of developing β-cell disorders, the method comprising administering to the subject a dose of engineered, low-immunogenic islets. The dose is administered via intramuscular injection. The dose mentioned above is from the following: A) Approximately 1×10 7 One cell to approximately 3 × 10 8 One cell; B) Approximately 1.25 × 10 5 Cells / kg to approximately 1.2 × 10⁻⁶ 7 Cells / kg; C) Approximately 6,500 islet equivalents (IEQ) to approximately 600,000 IEQ; or D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg, and The amount of exogenous insulin required is less than that required by subjects treated with non-hypoimmunogenic islets or less than that required by untreated subjects with the aforementioned β-cell disease.

3. A method for stabilizing blood glucose levels in a subject who has or is at risk of developing β-cell disorders, the method comprising administering to the subject a dose of engineered, low-immunogenic islets. The dose is administered via intramuscular injection. The dose mentioned above is from the following: A) Approximately 1×10 7 One cell to approximately 3 × 10 8 One cell; B) Approximately 1.25 × 10 5 Cells / kg to approximately 1.2 × 10⁻⁶ 7 Cells / kg; C) Approximately 6,500 islet equivalents (IEQ) to approximately 600,000 IEQ; or D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg The blood glucose levels were stable compared to subjects receiving alternative insulin therapy or to untreated subjects.

4. A method for stabilizing / increasing C-peptide levels in a subject who has or is at risk of developing β-cell disorders, the method comprising administering to the subject a dose of engineered, low-immunogenic islets. The dose is administered via intramuscular injection, and The dose mentioned above is from the following: A) Approximately 1×10 7 One cell to approximately 3 × 10 8 One cell; B) Approximately 1.25 × 10 5 Cells / kg to approximately 1.2 × 10⁻⁶ 7 Cells / kg; C) Approximately 6,500 islet equivalents (IEQ) to approximately 600,000 IEQ; or D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg The C-peptide levels were stable or increased compared to subjects receiving alternative insulin therapy or to untreated subjects.

5. A method for reducing HbA1c levels in subjects who have or are at risk of having β-cell disorders, the method comprising administering to the subject a dose of engineered, low-immunogenic islets. The dose is administered via intramuscular injection, and The dose mentioned above is from the following: A) Approximately 1×10 7 One cell to approximately 3 × 10 8 One cell; B) Approximately 1.25 × 10 5 Cells / kg to approximately 1.2 × 10⁻⁶ 7 Cells / kg; C) Approximately 6,500 islet equivalents (IEQ) to approximately 600,000 IEQ; or D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg The HbA1c levels were lower in subjects who received alternative insulin therapy or in untreated subjects.

6. A method for reducing adverse side effects associated with islet cell therapy in subjects who have or are at risk of developing β-cell disorders, the method comprising: i) Introducing low-immunogenic modifications into a population of pancreatic islet cells containing β cells to generate engineered low-immunogenic islets, and ii) Administer a dose of the engineered, low-immunogenic islets to subjects who have or are at risk of developing β-cell disease. The dose is administered via intramuscular injection, and The dose mentioned above is from the following: A) Approximately 1×10 7 One cell to approximately 3 × 10 8 One cell; B) Approximately 1.25 × 10 5 Cells / kg to approximately 1.2 × 10⁻⁶ 7 Cells / kg; C) Approximately 6,500 islet equivalents (IEQ) to approximately 600,000 IEQ; or D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg.

7. A method for increasing the time-in-target range (TIR) ​​in subjects who have or are at risk of developing β-cell disorders, the method comprising administering to the subject a dose of engineered, low-immunogenic islets. The dose is administered via intramuscular injection, and The dose mentioned above is from the following: A) Approximately 1×10 7 One cell to approximately 3 × 10 8 One cell; B) Approximately 1.25 × 10 5 Cells / kg to approximately 1.2 × 10⁻⁶ 7 Cells / kg; C) Approximately 6,500 islet equivalents (IEQ) to approximately 600,000 IEQ; or D) Approximately 80 IEQ / kg to approximately 24,000 IEQ / kg The TIR was increased compared to subjects receiving alternative islet therapy or to untreated subjects.

8. The method of any one of claims 1-7, wherein the method results in a reduction in the demand for other drugs used to treat the β-cell disease, optionally wherein the β-cell disease drug is insulin.

9. The method of any one of claims 1-8, wherein the subject exhibits reduced insulin dependence.

10. The method of claim 2 or claim 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 receiving non-hypoimmunogenic islets to treat the β-cell disease or for an untreated subject with the β-cell disease.

11. The method of any one of claims 2, 9, and 10, wherein the method is characterized in that the subject meets one or more of the following criteria: (i) fasting capillary blood glucose level exceeding 140 mg / dL (7.8 mmol / L) no more than three times in one week (based on at least 7 measurements of capillary blood glucose level over seven days); (ii) 2-hour postprandial capillary blood glucose exceeding 180 mg / dL (10.0 mmol / L) no more than three times in one week (based on at least 21 measurements of capillary blood glucose level over seven days); and (iii) evidence of endogenous insulin production, defined as fasting or post-stimulation C-peptide level >0.5 ng / mL (0.16 pmol / L).

12. The method of any one of claims 1-6, wherein the method results in the subject exhibiting insulin independence.

13. The method of claim 11, wherein the subject exhibits insulin independence for a period of time that is longer 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 of any one of claims 1-13, characterized in that the subject satisfies one or more of the following: a) C-peptide peak > 0.20 nmol / L (e.g., as assessed by a mixed-meal tolerance test); b) Non-fasting C-peptide >0.10 nmol / L (e.g., as assessed by mixed meal tolerance test); c) Daily exogenous insulin requirement <0.25 U / kg; d) Daily exogenous insulin requirement = 0 U / kg; e) Reduced exogenous insulin requirements (per kg of body weight); f) HbA1c (per kg of body weight) decreases; g) Reduced (stable) blood glucose variability; h) Shortened duration of hypoglycemia and / or hyperglycemia (improved normal blood glucose levels); i) Blood glucose control HbA1c ≤ 6.5% (48 mmol / mol); and j) Blood glucose control: HbA1c <7.0% (53 mmol / mol).

15. The method of any one of claims 1-14, wherein the engineered low-immunogenic islets comprise the following modifications: (a) Inactivating or destroying one or more alleles of: (i) one or more major histocompatibility complex (MHC) class I molecules or one or more molecules that regulate 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 regulate the expression of said one or more MHC class II molecules; and / or (b) Increase the expression of one or more tolerogenic factors, wherein the increase in expression is relative to control or wild-type islets that do not contain the modification.

16. The method of any one of claims 1-15, wherein the engineered low-immunogenic islets comprise engineered β-islet cells.

17. The method of claim 16, wherein the engineered low-immunogenic islets further comprise additional engineered islet cells, optionally wherein the additional engineered islet cells comprise α cells and / or δ cells.

18. The method of claim 17, wherein the additional engineered islet cells comprise cells containing the same modifications as the engineered β islet cells.

19. The method of any one of claims 1-18, 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 engineered low-immunogenic islets comprise engineered β-islet cells.

20. The method of any one of claims 1-19, wherein the engineered low-immunogenic islets are islet clusters.

21. The method of any one of claims 1-20, wherein the engineered low-immunogenic islets are engineered from primary islets.

22. The method of claim 21, wherein the primary islets are derived from the pancreas.

23. The method of claim 21 or claim 22, wherein the primary islets are derived from human or animal subjects, optionally wherein the primary islets are from pigs, cattle, or sheep.

24. The method of any one of claim 22 or claim 23, wherein the primary islets are derived from a donor subject who is not suspected of having β-cell-related disease.

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

26. The method of any one of claims 1-25, wherein the engineered low-immunogenic islets are ABO blood type O.

27. The method of any one of claims 1-26, wherein the engineered low-immunogenic islets are rhesus factor negative (Rh-).

28. The method of any one of claims 1-27, wherein the engineered low-immunogenic islets are derived from stem cell differentiation.

29. The method of any one of claims 1-28, wherein the β-cell disorder is diabetes.

30. The method of any one of claims 1-29, wherein the β-cell disorder is type 1 diabetes.

31. The method of any one of claims 1-30, 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) C-peptide in response to a mixed meal tolerance test (MMTT), positive antibody against GAD or IA2, and HbA1c. 1c ≥70mmol / mol, and exogenous insulin requirement <1U / kg.

32. The method of any one of claims 1-31, wherein the dose of engineered low-immunogenic islets comprises a pharmaceutically acceptable carrier.

33. The method of any one of claims 1-32, wherein intramuscular administration is via the intermuscular space of the forearm, upper arm, hip, thigh, or buttock.

34. The method of any one of claims 1-33, wherein administration comprises administering one or more additional doses of the said low-immunogenic engineered cells.

35. The method of claim 34, wherein one or more additional doses of the low-immunogenic engineered cells are administered to the subject when the following occurs after the initial dose: (a) The subject did not exhibit a reduced requirement for other medications used to treat the β-cell disease, optionally wherein the β-cell disease medication is insulin; and / or (b) No low-immunogenicity engineered cells were detected by imaging.

36. The method of claim 34, wherein the one or more additional doses of the low-immunogenic engineered cells are administered to the subject if the subject does not meet one or more of the following criteria after the initial dose: (i) a fasting capillary blood glucose level greater than 140 mg / dL (7.8 mmol / L) no more than three times in one week (based on at least 7 measurements of capillary blood glucose levels over seven days); (ii) a 2-hour postprandial capillary blood glucose level greater than 180 mg / dL (10.0 mmol / L) no more than three times in one week (based on at least 21 measurements of capillary blood glucose levels over seven days); and (iii) evidence of endogenous insulin production, defined as a fasting or post-stimulation C-peptide level >0.5 ng / mL (0.16 pmol / L).

37. The method of claim 34, wherein the one or more additional doses of the low-immunogenic engineered cells are administered to the subject when: (a) The subject did not achieve insulin independence for a period of time after the initial dose; and / or (b) The subject did not exhibit a reduced need for other medications used to treat the β-cell disease over a period of time, optionally wherein the β-cell disease medication is insulin. Optionally, the subjects did not achieve insulin non-dependency for a period of more than one week, more than two weeks, more than three weeks, more than one month, more than two months, more than three months, more than four months, more than five months, more than six months, more than seven months, more than eight months, more than nine months, more than ten months, more than eleven months, or more than twelve months; alternatively, the subjects did not achieve insulin non-dependency for a period of two weeks.

38. The method of claim 34, wherein the one or more additional doses of the low-immunogenic engineered cells are administered to the subject if the subject does not meet one or more of the following criteria after the initial dose: a) C-peptide peak > 0.20 nmol / L (e.g., as assessed by a mixed-meal tolerance test); b) Non-fasting C-peptide >0.10 nmol / L (e.g., as assessed by mixed meal tolerance test); c) Daily exogenous insulin requirement <0.25 U / kg; d) Daily exogenous insulin requirement = 0 U / kg; e) Reduced exogenous insulin requirements (per kg of body weight); f) HbA1c (per kg of body weight) decreases; g) Reduced (stable) blood glucose variability; h) Shortened duration of hypoglycemia and / or hyperglycemia (improved normal blood glucose levels); i) Blood glucose control HbA1c ≤ 6.5% (48 mmol / mol); and j) Blood glucose control: HbA1c <7.0% (53 mmol / mol).

39. The method of any one of claims 35-38, wherein the number of engineered hypoimmune islets in the subject from the initial dose is cleared or reduced prior to administration of the one or more additional doses of engineered hypoimmune islets.

40. The method of claim 39, wherein after administration of an exogenously administered agent to guide the targeted death of the engineered hypoimmunogenic islets, the number of engineered hypoimmunogenic islets in the subject is reduced, optionally wherein the exogenously administered agent activates a suicide gene or safety switch in the engineered cells, or recognizes one or more tolerogenic factors on the surface of the engineered hypoimmunogenic islets.

41. The method of any one of claims 1-40, wherein the immunosuppressive regimen is administered to the subject.

42. The method of claim 41, wherein the immunosuppressive regimen is administered to the subject only prior to the administration of the said dose of the engineered hypoimmunogenic islets.

43. The method of claim 41 or 42, wherein the immunosuppressive regimen is administered to the subject only after administration of the said dose of the engineered hypoimmunogenic islets.

44. The method of any one of claims 41-43, wherein the immunosuppression regimen comprises one or more immunosuppressants.

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

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

47. The method of claim 45, wherein the small molecule is a chemical compound or a nucleic acid.

48. The method of any one of claims 44-47, 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, blood rheology modifiers, inosine monophosphate dehydrogenase (IMPDH) inhibitors, Janus kinase inhibitors, mTOR inhibitors, TNF inhibitors, and antiCD25 inhibitors.

49. The method of claim 48, wherein the one or more immunosuppressants are selected from the group consisting of: anti-thymocyte globulin (ATG), corticosteroids, prednisone, cortisone, prednisolone methylprednisolone, dexamethasone, betamethasone, hydrocortisone, methotrexate, acetaminophen, diphenhydramine, sirolimus (rapamycin), tacrolimus (FK-506), mycophenolic acid (MPA), mycophenolic acid ethyl ester (MMF), mycophenolic acid sodium, cyclosporine, etanercept (TNFR-Fc), azathioprine, gold salts, sulfasalazine, antimalarial drugs, buquina, leflunomide, imidazolidin, 15-deoxyguanidin, 6-mercaptopurine, cyclophosphamide, OKT3, anti-thymocyte globulin, thymopentin (thymosin-α), fludarabine, and immunosuppressive antibodies.

50. The method of any one of claims 44-47, wherein the one or more immunosuppressants comprise: Antibodies that bind to MHC, CD2, CD3, CD4, CD7, CD28, B7, CD25, CD40, CD45, CD95, IFN-γ, TNF-α, IL-2Rα, IL-4, IL-5, IL-6R, IL-6, IGF, IGFR1, IL-7, IL-8, IL-10, CD11α, or CD58, and antibodies that bind to any of their ligands; soluble IL-15R, IL-10, B7 molecules (such as B7-1, B7-2), their variants and fragments, ICOS and OX40; and inhibitors of negative T-cell regulators, such as antibodies against CTLA-4, or similar agents.

51. The method of any one of claims 1-50, further comprising tapering the administration of the one or more immunosuppressants.

52. The method of any one of claims 15-51, 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 of any one of claims 15-52, wherein at least one of the one or more tolerogenic factors is CD47.

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

55. The method of claim 54, wherein the engineered CD47 protein comprises: (a) One or more extracellular domains; and (b) One or more membrane chains; The one or more extracellular domains contained signal regulatory protein α (SIRPα) interaction motifs, and The engineered protein described therein does not contain one or more full-length CD47 intracellular domains.

56. The method of claim 55, wherein the SIRPα interaction motif is or comprises a CD47 extracellular domain or a portion thereof.

57. The method of claim 55, wherein the SIRPα interacting motif is or comprises a SIRPα antibody or a portion thereof.

58. The method of any one of claims 1-56, wherein the engineered low-immunogenic islets have the B2M phenotype. 插入缺失 / 插入缺失 CIITA 插入缺失 / 插入缺失 CD47 tg .

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

60. The method of claim 59, wherein the GSIS is a dynamic GSIS comprising dynamic insulin secretion in a first phase and a second phase.

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

62. The method of any one of claims 1-61, wherein the insulin secretion level of the engineered low-immunogenic islets is at least 20% of the level observed in primary islets (optionally, cadaveric islets).

63. The method of any one of claims 1-62, wherein the total insulin content of the engineered low-immunogenic islets is greater than or equal to about 500 µIU insulin / 5000 cells, greater than or equal to about 1000 µIU insulin / 5000 cells, greater than or equal to about 2000 µIU insulin / 5000 cells, greater than or equal to about 3000 µIU insulin / 5000 cells, or greater than or equal to about 4000 µIU insulin / 5000 cells.

64. The method of any one of claims 1-63, wherein the engineered low-immunogenic islets exhibited functionality for more than 2 weeks after administration to a subject.

65. The method of any one of claims 1-64, wherein the dose is selected from: about 1 × 10⁻⁶ 7 One cell to approximately 3 × 10 8 One cell, approximately 25 × 10 6 80 × 10⁶ cells per cell 7 1 cell, approximately 25 × 10 6 1 cell to approximately 25 × 10 7 One cell, approximately 80 × 10 6 80 × 10⁶ cells per cell 7 One cell, approximately 25 × 10 6 80 × 10⁶ cells per cell 6 One cell or approximately 1.25 × 10⁻⁶ 5 One cell to approximately 1.2 × 10⁻⁶ 7 Each cell.

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

67. The method of any one of claims 1-66, wherein the method is characterized in that the subject satisfies one or more of the following: a) Immune evasion by engineered, low-immunogenic islets, as evaluated in systemic PBMCs and serum; b) Peak C-peptide concentration in response to mixed meal tolerance test (MMTT) > 0.01 nmol / L; c) Non-fasting C-peptide concentration > 0.01 nmol / L; d) Survival of engineered, low-immunogenic islets, as evaluated by MRI; e) Insulin requirement / kg BW decreases; f) Decrease in HbA1c; and g) Reduced blood glucose variability, i.e., decreased hypoglycemia and hyperglycemia.

68. The method of claim 67, wherein the engineered low-immunogenic islets exhibit immune evasion at weeks 0, 2, 4, 8, 12, 18, 26, and 52 following administration to the subject.

69. The method of claim 67, wherein, after administration to the subject, the engineered low-immunogenic islets survive for 2, 4, 6, 8, 12, 26, and 52 weeks.

70. The method of 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 engineered low-immunogenic islets to the subject.

71. The method of claim 67, wherein the HbA1c reduction occurs at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 26 and 52 weeks after administration of the engineered low-immunogenic islets to the subject.

72. The method of claim 67, wherein glycemic variability, i.e., hypoglycemia and hyperglycemia, is reduced at 4, 8, 12, 18, 26 and 52 weeks after administration of the engineered hypoimmunogenic islets to the subject.

73. The method of any one of claims 1-72, wherein the subject is characterized by not having: any prior organ transplantation; any history of malignancy; use of any investigational drug within 4 weeks of receiving the said dose of engineered low-immunogenic islets; use of any antidiabetic drug other than insulin within 4 weeks of receiving the said 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 corresponding reference range; serological evidence of HTLVI or HTLVII infection; pregnancy, lactation, or intention to become pregnant; grade 3 or higher chronic kidney disease (e.g., GFR <60 ml / min estimated by creatine measurement); history of heart disease or symptoms consistent with heart disease at screening; HLA immunity, MIC A / B immunity; known autoimmune diseases other than type 1 diabetes (e.g., Hashimoto's disease); administration of a live attenuated vaccine <6 months prior to receiving the said dose of engineered low-immunogenic islets; islet antibody GADA >2000 IE / mL or IA2A. >4000 IE / mL or ZnT8 autoantibodies; untreated proliferative diabetic retinopathy; persistent mental illness; persistent substance abuse (drugs or alcohol) or treatment non-compliance; and known hypersensitivity to ciprofloxacin, gentamicin, or amphotericin B.

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