Methods and compositions for the production of heterologous pancreatic islet cells and treatment of insulin resistance or deficiency using the same.

Abstract

This invention provides a pharmaceutical composition for the treatment of diabetic patients with inadequate glucose control. [Solution] A pharmaceutical composition comprising a therapeutically effective dose of isolated, genetically engineered transgenic porcine pancreatic islet cells for treating insulin resistance or deficiency in a person in need thereof, wherein the transgenic porcine pancreatic islet cells (a) comprise inactivation of the genes GGTA, B4GALNT2, and CMAH, and (b) express 12 human polypeptides, where the 12 human polypeptides are CD46, CD55, CD59, thrombomodulin (THBD), tissue factor pathway inhibitor (TFPI), CD39, beta-2 microglobulin (B2M), human leukocyte antigen-E (HLAE), TNFα-inducible protein 3 (A20), PD-L1, heme oxygenase (HO-1), and CD47, wherein human blood glucose levels are reduced after treatment with the pharmaceutical composition.

Description

Technical Field

[0001] Cross - reference to related applications

[0001] This application claims priority to International Application No. PCT / CN Patent Application Publication No. 2020 / 070698, filed on January 7, 2020, which is hereby incorporated by reference in its entirety for all purposes.

Background Art

[0002] Background of the Invention

[0002] Current estimates indicate that the prevalence of type 2 diabetes will reach 4.4% globally across all age groups by 2030. Current pharmacological treatments include insulin replacement for type 1 diabetes and, for type 2 diabetes, insulin supplementation either alone or in combination with metformin, sulfonylureas, glinides, DPP - 4 inhibitors, GLP - 1 receptor agonists, SGLT - 2 inhibitors or pioglitazone. All of these strategies require detailed patient management and medication compliance. Furthermore, many patients are unable to achieve glycemic control despite these interventions. (

[0003]

[0003] There is a need for a treatment strategy to improve glucose control in diabetic patients without the complex administration of anti - diabetic drugs or insulin, or to improve glucose control in diabetic patients with insufficient glucose control despite the administration of anti - diabetic drugs or insulin. Allogeneic islet cell transplantation (e.g., intraportal islet cell transplantation) is used for type 1 diabetes with severe risk factors (e.g., unstable T1DM, hypoglycemia unawareness, severe hypoglycemic episodes, glycemic instability). ​​​​​​​​​​​​ While increased use is seen in patients, there are challenges related to the need for strict immunosuppression, graft survival, and The availability of donor cells is important because improved glucose control reduces the risk of long-term complications. When minimizing, type 1 diabetes patients, type 2 diabetes patients, and type 1 or type 2 diabetes in the early stages of the disease This hinders the widespread use of this technology in patients. [Overview of the project] [Means for solving the problem]

[0004] Summary of the Invention

[0004] In one aspect, the present disclosure provides isolated transgenic porcine pancreatic islet cells Herein, these cells are (a) GGTA, B4GALNT2, or CMAH. (b) Not substantially lacking the enzymatic activity of at least one glycosyltransferase enzyme, At least two polypeptide sequences derived from the porcine mammalian species, CD46, CD55 , CD59, THBD, TFPI, CD39, B2M, HLAE, CD47, A20, P At least two polypeptides, including at least two of D-L1, FASL, or HO-2 (c) Expression of the d sequence, and reduction of toxicity by complement derived from non-porcine mammalian species, non-porcine mammalian Reduced induction of activated protein C coagulation from animal species, and thrombin from non-porcine mammalian species. - Reduction of antithrombin complex induction or reduction of toxicity by non-porcine-derived NK cells. Show one or more.

[0005]

[0005] In some embodiments, this cell contains GGTA, B4GALNT2 and Glycosyltransferase enzymes of at least two or three selected from CMAH It essentially lacks basic enzyme activity.

[0006]

[0006] Any of the isolated transgenic porcine pancreatic islet cells disclosed herein In some embodiments, these cells contain CD46, CD55, CD59, THBD , TFPI, CD39, B2M, HLAE, CD47, A20, PD-L1, FASL or HO-1 has at least 3, at least 4, at least 5, at least 6, and fewer At least 7, at least 8, at least 9, at least 10, at least 11, At least 12 or all of them are expressed.

[0007]

[0007] In another aspect, the present disclosure relates to isolated transgenic porcine pancreatic islet cells. Provided, here, these islet cells are (a) low in GGTA, B4GALNT2, or CMAH The enzymatic activity of at least two glycosyltransferase enzymes, including at least two. (b) substantially none, and a polypeptide sequence derived from a non-porcine mammalian species, CD46, CD55, CD59, THBD, TFPI, CD39, B2M, HLAE, CD47, A 20, expressing a polypeptide sequence which is PD-L1, FASL or HO-1, and (c) Toxicity reduction by complement derived from non-porcine mammalian species, and activation of non-porcine activated protein C coagulation. Reduction in induction of, reduction in induction of thrombin-antithrombin complex of non-porcine origin or non This demonstrates the reduction of toxicity by NK T cells derived from pig species.

[0008]

[0008] In some embodiments, this cell contains GGTA, B4GALNT2 and It substantially lacks CMAH enzymatic activity.

[0009]

[0009] Any of the isolated transgenic porcine pancreatic islet cells disclosed herein In some embodiments, this cell expresses CD46, CD55, CD59, THBD , TFPI, CD39, B2M, HLAE, CD47, A20, PD-L1, FASL or at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve or all of HO-1.

[0010]

[0010] In some embodiments of any of the isolated transgenic porcine pancreatic islet cells disclosed herein, this cell expresses CD46, CD55, CD59, CD39 , B2M, HLAE and CD47.

[0011]

[0011] In some embodiments of any of the isolated transgenic porcine pancreatic islet cells disclosed herein, this cell substantially lacks the expression of one or more glycosyltransferase enzymes. In some embodiments, this cell contains a frameshift mutation in one or more glycosyltransferase enzymes, resulting in premature termination of translation, thereby eliminating the activity of the glycosyltransferase enzyme.

[0012]

[0012] In some embodiments of any of the isolated transgenic porcine pancreatic islet cells disclosed herein, one or more nucleic acid sequences encoding one or more polypeptide sequences derived from a non-porcine mammalian species are inserted within a non-orthologous locus of a porcine ortholog.

[0013]

[0013] Any of the isolated transgenic porcine pancreatic islet cells disclosed herein In some embodiments, one or more polypeptides derived from non-porcine mammalian species are used. One or more nucleic acid sequences encoding a sequence are non-orthologous promoters of butorsologs. - is operably connected to. In some embodiments, a non-orthologous promoter It is a non-porcine promoter.

[0014]

[0014] Any of the isolated transgenic porcine pancreatic islet cells disclosed herein In some embodiments, these islet cells are obtained by deaggregation of porcine pancreas.

[0015]

[0015] Any of the isolated transgenic porcine pancreatic islet cells disclosed herein In some embodiments, these pancreatic islet cells are alpha cells, beta cells, delta cells Cells, epsilon cells, pancreatic polypeptide (PP) cells, or any combination thereof ru.

[0016]

[0016] Any of the isolated transgenic porcine pancreatic islet cells disclosed herein In some embodiments, the non-porcine mammalian species is a primate species.

[0017]

[0017] Any of the isolated transgenic porcine pancreatic islet cells disclosed herein In some embodiments, when these cells are transplanted into non-porcine mammalian species, they last for 8 days. It demonstrates survival beyond the intermediate stage.

[0018]

[0018] Any of the isolated transgenic porcine pancreatic islet cells disclosed herein In some embodiments, these cells are PBMCs isolated from non-porcine mammalian species. This shows the decrease in IBMIR.

[0019]

[0019] In another embodiment, the present disclosure provides a therapeutically effective amount of the isolated [disclosed herein] The present invention provides a composition comprising any of the transgenic porcine pancreatic islet cells. Several implementations In terms of form, the isotonic buffer further comprises heparin or a TNF-alpha inhibitor.

[0020]

[0020] In some embodiments of any of the compositions disclosed herein, this The composition contains at least about 12% to about 25% beta cells or at least about 15% to about 30 Contains % alpha cells.

[0021]

[0021] In some embodiments of any of the compositions disclosed herein, this The composition is prepared according to one of the methods disclosed herein.

[0022]

[0022] In another embodiment, the present disclosure describes a treatment for insulin resistance or deficiency, A method for performing in non-porcine mammals requiring the use of a therapeutically effective dose, as specified herein. Any of the isolated transgenic porcine pancreatic islet cells shown or disclosed herein The present invention provides a method comprising administering one of the compositions to a mammal.

[0023]

[0023] In some embodiments, this method involves the internal jugular vein or hepatic portal vein of a mammal This includes administering cells to the central nervous system via a method.

[0024]

[0024] In any one of several embodiments of the methods disclosed herein, The state of insulin resistance includes type 1 diabetes.

[0025]

[0025] In any one of the embodiments of the methods disclosed herein, The state of insulin resistance includes type 2 diabetes.

[0026]

[0026] In any one of the embodiments of the methods disclosed herein, non Before administering transgenic porcine islet cells or compositions to porcine mammals, the therapeutic effect is assessed. Doses of antithymocyte globulin, anti-CD40 antibody, anti-CD20 antibody, rapagnostic, calcinu -Phosphoric inhibitors, ganciclovir or its prodrugs, antihistamines and corticosteroids He is undergoing an introductory regimen that includes Lloyd's.

[0027]

[0027] In any one of the embodiments of the methods disclosed herein, This method involves administering transgenic porcine pancreatic islet cells or a composition followed by a therapeutically effective dose of anti-CD. 40 antibodies, rapalogues, calcineurin inhibitors, and ganciclovir or its prodrugs This further includes administering [the drug].

[0028]

[0028] In any one of the embodiments of the methods disclosed herein, This method involves administering transgenic porcine pancreatic islet cells or a composition, followed by an intermediate dose of the therapeutically effective dose. Insulin analogs for short-acting or long-acting insulin, insulin glargine, insulin detemir or This further includes administering NPH insulin.

[0029]

[0029] In any one of the embodiments of the methods disclosed herein, Any of the therapeutically effective doses disclosed in the specification is less than 1 kg of body weight in non-porcine mammals. At most, it's 5,000 IEQ.

[0030]

[0030] In another aspect, the Disclosure relating to the isolated transges disclosed herein Provides isolated porcine islets containing any of Nick's porcine islet cells. Several implementations Morphologically, pancreatic islets substantially do not contain pancreatic exocrine cells.

[0031]

[0031] In another aspect, the Disclosure relating to isolated transges disclosed herein This provides isolated porcine pancreatic organoids containing any of Nick's porcine islet cells. In some embodiments, the organoid substantially does not contain pancreatic exocrine cells. In several embodiments, the pancreatic organoid is (a) a neonatal piglet before the 7th day of neonatal life. (b) to isolate the pancreas from the material, and (b) to subject the pancreas to mechanical or enzymatic digestion, organo To generate an organoid fragment, and optionally, (c) the organoid of step (b) The fragments are prepared by purification using Ficol gradient sedimentation.

[0032]

[0032] In another aspect, the Disclosure relating to isolated transges disclosed herein This provides isolated porcine pancreas containing any of Nick's porcine islet cells.

[0033]

[0033] In another embodiment, the present disclosure relates to the process of transplanting a pig into a non-porcine mammalian recipient. A method for improving the yield of pancreatic islets from a donor, wherein (a) the purification procedure for neonatal piglets (b) To provide pancreatic organoids in the presence of an effective concentration of a caspase inhibitor (c) The organoids are cultured for at least 90 minutes after purification, and (c) Continue culturing for at least 7 days in the presence of an effective concentration of corticosteroids. This provides a method that includes [something].

[0034]

[0034] In some embodiments, the purification procedure is performed by (a) transting the newborn before day 7. (b) Isolating the pancreas from a sgenic neonatal piglet, and (b) mechanically or enzymatically dissecting the pancreas. Subject to digestion to generate organoid fragments, and optionally, (c) This method includes purifying organoid fragments from digested pancreas by colesial gradient sedimentation.

[0035]

[0035] In some embodiments of any one of the methods disclosed herein, a new Live piglets are isolated transgenic porcine pancreatic islet cells as disclosed herein. This is a transgenic pig containing at least one porcine cell from either of the following sources.

[0036]

[0036] In any one of the embodiments of the methods disclosed herein, The spase inhibitor is Z-VAD-FMK.

[0037]

[0037] In any one of the embodiments of the methods disclosed herein, The luticosteroid is methylprednisolone.

[0038]

[0038] In any one of the embodiments of the methods disclosed herein, pancreas Organ organoids are treated with IBMX, phosphodiesterase inhibitors, or adenosine receptor antagonists. It is cultured in the presence of a gonist.

[0039]

[0039] In any one of the embodiments of the methods disclosed herein, pancreas Organoids are cultured in the presence of nicotinamide or its metabolically acceptable analogues. It can be done.

[0040]

[0040] In another embodiment, the present disclosure describes the treatment of insulin resistance or deficiency, A method for performing treatment on non-porcine mammals requiring treatment, as disclosed herein. An organoid composed of any one of the ganoids is defined as an organoid that meets the following criteria: (a) approximately 5 (b) Endotoxin levels below EU / kg, (c) Negative Gram staining, (c) Over 70% survival rate (d) The pancreatic islet concentration of approximately 20,000 IEQ / mL or higher in total sedimentation volume must be met. The present invention provides a method that includes transplantation into a non-porcine mammal in such cases.

[0041] Built-in by reference

[0041] All publications, patents and patent applications referred to herein are each individual publications. It is specifically and individually indicated that a work, patent, or patent application is incorporated by reference. To the same extent, it is incorporated herein by reference.

[0042] Brief explanation of the drawing

[0042] Novel features of the present invention are specifically described in the appended claims. A better understanding of the features and advantages of this invention will be provided by describing exemplary embodiments in which the principles of this invention are utilized. This can be obtained by referring to the following detailed description and its accompanying drawings. [Brief explanation of the drawing]

[0043] [Figure 1]

[0043] The FACS immunostaining results of 4-7 transgenic endothelial vein porcine cells (PUVECs) incubated in human serum are shown. The upper panel is a FACS plot showing IgG or IgM staining of human umbilical vein endothelial cells ("HUVEC"), transgenic 4-7 porcine umbilical vein endothelial cells ("4-7PUVEC"), or normal porcine umbilical vein endothelial cells ("WT PUVEC"). Transgenic 4-7PUVECs, like HUVEC cells, show reduced binding of IgG and IgM antibodies from human serum compared to their normal porcine counterparts. Data are shown as mean ± standard deviation. Error bars indicate the standard deviation, and p-values ​​are derived from independent two-tailed Student's t-tests. * indicates P<0.05, and ** indicates P<0.01. [Figure 2]

[0044] The results of a human complement toxicity assay performed on 4-7 transgenic endothelial umbilical vein porcine cells (PUVECs) are shown. The left panel is a diagram illustrating the assay workflow, and the right panel is a chart showing the death of human umbilical vein endothelial cells ("HUVECs"), transgenic 4-7 porcine umbilical vein endothelial cells ("4-7PUVECs"), or normal porcine endothelial cells ("WT PUVECs") after incubation with various concentrations of human complement ("HC"). The 4-7 cells, like human HUVEC cells, show a significant reduction in death in response to human complement compared to their normal porcine counterparts. [Figure 3]

[0045] This report presents the results of analyses conducted to examine the expression / functionality of CD39 in 4-7 transgenic porcine umbilical vein endothelial cells (PUVECs). Transgenic 4-7 porcine umbilical vein endothelial cells ("4-7PUVECs") exhibit significantly higher ADPase biochemical activity of hCD39, as measured by phosphate production when incubated with ADP, compared to HUVECs and WT PUVECs. Data are presented as mean ± standard deviation. Error bars indicate the standard deviation, and p-values ​​are derived from independent two-tailed Student's t-tests. ** indicates P<0.01. [Figure 4]

[0046] A schematic diagram of a heterologous cell activation protein C assay using human protein C and human thrombin is shown. [Figure 5]

[0047] The results of a thrombin-antithrombin III (TAT) formation assay in 4-7 cells are shown. The left panel is a diagram illustrating the workflow for measuring thrombin-antithrombin III (TAT) complex formation using human blood, and the right panel is a chart showing the results of the corresponding assays using HUVEC, 4-7 PUVEC, or WT PUVEC. 4-7 cells show a reduction in TAT formation comparable to that of HUVEC cells, compared to WT PUVEC cells. [Figure 6]

[0048] The results of platelet lysis assays performed with 4-7 transgenic cells are shown. FACS traces quantifying the number of platelets remaining in human blood (outlined clusters) after incubation with HUVEC, 4-7PUVEC, or WT PUVEC for 45 or 60 minutes are shown. 4-7 cells continue to show an increased percentage of remaining platelets compared to porcine WT PUVEC, which is comparable to the percentage of remaining platelets when incubated with HUVEC cells. [Figure 7]

[0049] The experimental results shown in Figure 6 at the additional time points (5 minutes and 15 minutes) are quantified as residual CD41-positive platelets (MFI indicates the average fluorescence intensity of the CD41 channel by FAC analysis). [Figure 8]

[0050] The results of NK cytotoxicity assays performed on 4-7 transgenic cells are shown. A chart is presented showing the results of NK toxicity assays performed with an effector:target cell ratio of 10 in HUVEC, 4-7PUVEC, or WT PUVEC cells. 4-7PUVECS represents an intermediate cell death level between normal PUVEC and HUVEC cells. [Figure 9]

[0051] This chart illustrates an exemplary workflow for processing porcine islet cells for transplantation. In an exemplary embodiment, a newborn piglet is subjected to pancreatectomy ("procurement"), and the pancreas is subsequently dissected and digested with collagenase ("islet isolation"). The digested islet cells are then transferred to a gas-permeable, water-impermeable bag and kept at 22-24°C until ready for culture ("transport"). The islet cells are then cultured for a period of time (optionally with EGM2 medium and a caspase inhibitor, "culture"), and quality control procedures such as functional islet equivalence assay (IEQ), endotoxin assay, Gram staining, viability assay, and cell purity assay are performed. [Figure 10]

[0052] Figure 9 shows the results of the pancreatic islet isolation procedure performed in transgenic (4-7) or normal (WT) Bana minipigs. [Figure 11]

[0053] Figure 9 shows the results of platelet lysis or TAT complex formation assays performed on isolated islet cells. The left panel shows that islets 4-7, when incubated with human whole blood, exhibit reduced platelet lysis compared to WT islets and experimental controls (NC, saline only). The right panel shows that islets 4-7, when incubated with human whole blood, exhibit reduced TAT complex formation compared to WT islets and experimental controls (NC, saline only). [Figure 12]

[0054] Figure 9 shows the results of an immediate blood-mediated inflammatory response (IBMIR) assay performed with human blood on the 4-7 islets obtained. IHC micrographs at 200x magnification are shown, illustrating the staining of lesions for antibodies (IgG and IgM, left panel) and complement (C3a and C4d, right panel) after incubation of the 4-7 islet sections with human blood. The 4-7 islet cells show reduced staining and lesions related to IgG, IgM, C3a, and C4d, suggesting that the islet cells exhibit reduced IBMIR. [Figure 13]

[0055] Figure 12 shows neutrophil infiltration into islets 4-7, WT islets, and experimental controls (NC, physiological saline only) after incubation with human blood. Islets 4-7 have a higher number of residual neutrophils compared to WT islets and experimental controls. [Figure 14]

[0056] Figure 9 shows islet cells isolated over time under two different culture conditions. A graph is shown illustrating islet equivalence (IEQ) levels over 7 days of culture in either EGM-2 medium or standard medium ("F-10," indicating Ham's F-10). EGM-2 medium was associated with improved islet yield. [Figure 15]

[0057] The isolated islet cells are shown over time under two different culture conditions: F-10 medium and NEO medium, as shown in Figure 9. [Figure 16]

[0058] The cultures of islets isolated in a corticosteroid-free medium (left panel) versus a corticosteroid-containing medium (right panel) are compared as shown in Figure 9. Corticosteroids were associated with improved islet yield. [Figure 17]

[0059] The cell fractions of islets isolated under initial (top panel, F-10 medium) or improved (EGM-2 medium + corticosteroid, bottom panel) culture conditions are compared as shown in Figure 9. FACS traces comparing intact islet cells (left), beta cells (center), or live beta cells (right) between the two conditions are shown. The improved state was associated with an improvement in the number of intact islet cells and an improvement in the number of beta cells. [Figure 18]

[0060] This image shows the verification of protein expression of gene 4-7 in frozen kidney sections using immunofluorescence staining. Scale bar (white), 75 μm. [Figure 19]

[0060] Blood glucose levels over 60 days in NCG mice transplanted with islets from WT neonatal piglets after STZ, showing that blood glucose levels normalize after approximately 40 days. 4-7 Immunofluorescence staining verification of three knockouts and nine transgenes in kidney frozen sections. Antibody scale bar (white), 75 μm. [Figure 20]

[0061] This shows blood glucose levels over 126 days in NCG mice that underwent islet transplantation using WT porcine islet cells ("WT Tx"), 4-7 islet cells ("4-7Tx"), or sham surgery ("Sham Tx") after STZ. Different islet doses, including 4,000 IEQ, 2,000 IEQ, and 1,000 IEQ, were applied to both the "4-7Tx" and "WT Tx" experimental groups. [Figure 21]

[0062] This specification describes typical induction, immunosuppression, transplantation, and management protocols for NHP transplanted with islets according to the method described herein. [Figure 22]

[0063] This specification presents examples of NHP's glucose tolerance tests regarding blood glucose, insulin, and C-peptide before and after STZ induction in animals with diabetes, demonstrating that the protocol successfully induces diabetes in animals. [Figure 23]

[0064] Table 2 shows the WBC and lymphocyte counts (Figure 23) of the animals listed in the table up to 70 days after islet transplantation. [Figure 24]

[0064] The CD4+ cell type / CD8+ cell type / B cell / NK cell count of the animals listed in Table 2 up to 70 days after islet transplantation (Figure 24) is shown. [Figure 25]

[0064] The rapamycin levels of the animals listed in Table 2 up to 70 days after islet transplantation (Figure 25) are shown. [Figure 26]

[0065] Liver biopsies of animals MA-1 and MA-2 at 12 hours and 1 month post-transplantation are shown, stained with hematoxylin / eosin and anti-chromogranin A, indicating the presence of pancreatic islets in the liver tissue. [Figure 27]

[0066] Immunofluorescence staining analysis of liver biopsies of MB-11 animals 24 hours post-transplantation is shown, indicating the presence of pancreatic islets in liver tissue (clearly indicated by positive signals for insulin and glucagon staining). Monkey IgG, CD41 (platelet marker), fibrinogen (coagulation indicator marker), and CD68 (macrophage marker) were also detected around insulin-positive WT porcine pancreatic islets, indicating the development of an immediate blood-mediated inflammatory response (IBMIR) 24 hours post-transplantation of MB-11. These results also suggest that genetic modification is important for enhancing the survival of porcine pancreatic islets in vivo. [Figure 28]

[0067] This study shows serum concentrations of porcine c-peptide, monkey c-peptide, fasting blood glucose, and exogenous insulin intake in monkey recipients at different time points after transplantation using WT porcine islets. Porcine c-peptide can be reliably detected from animal serum within 55 days after porcine islet transplantation. [Modes for carrying out the invention]

[0044] Detailed description of the invention overview

[0068] This disclosure provides xenopancreatic islet cells for transplantation, thereby relating to transplantation. Addressing challenges related to immunosuppression, graft survival, and donor cell availability. Disclosed xenografts Cells (e.g., genetically modified heterologous cells) exhibit reduced immunogenicity and increased survival. Therefore, it is necessary to reduce the use of immunosuppressants in islet cell transplantation recipients. Suitable for. Methods, compositions and systems for inducing such cells, and the like. Treatment methods, including the use of eel cells, are described further herein.

[0045] definition

[0069] The terms "pig," "swine," and "porcine" are... These terms are used interchangeably in this specification and apply to various types of domestic pigs and wild boars (Sus scrofa). This refers to things related to species.

[0046]

[0070] Terms such as "treatment," "to treat," and "alleviation" are used in relation to disease, injury, or disability. When used in conjunction with other substances, the aim is generally to obtain the desired pharmacological and / or physiological effects. Used herein to enhance flavor and to improve the severity of one or more symptoms of a condition during treatment. It is used to refer to alleviating and / or reducing a disease, condition, or its symptoms. It may be preventive in that it completely or partially delays the onset or recurrence of the disease or / or or partial or complete healing of the condition and / or adverse effects resulting from the disease or condition It may be therapeutic in a specific way. As used herein, “treatment” refers to mammals, especially humans. (a) There may be a predisposition to the disease or condition, but This prevents the development of disease or condition in individuals who have not yet been diagnosed. (b) inhibiting a disease or condition (e.g., preventing its onset), or (c) To alleviate a disease or condition (for example, to cause regression of a disease or condition, and to alleviate one or more symptoms) (including providing an improvement in the condition.)

[0047]

[0071] The term is used to refer to a fragment or derivative of a protein or polypeptide. The term "biologically active" in this context means that the fragment or derivative is a reference full-length protein or a compound. The lipeptide retains at least one measurable and / or detectable biological activity. This means, for example, a biologically active fragment of the CRISPR / Cas9 protein or The derivative binds to gRNA (also referred to herein as single guide RNA (sgRNA)). When combined with the guide RNA, it binds to the target DNA sequence and / or one of the following: It may have the ability to cleave the above DNA strands. For example, biologically active fragments of cell receptors. Alternatively, the derivative has the ability to bind to a native ligand that signals through the receptor. To obtain, or to obtain, an intracellular signal that is generally transmitted by the receptor in response to a ligand. It may have the ability to communicate.

[0048]

[0072] As used herein, the term "indel" means dye This refers to the insertion or deletion of nucleotide bases in the target DNA sequence of a chromosome or episome. Such insertions or deletions may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or It can be a base greater than that. In certain embodiments, the indels are even larger and fewer. It can be at least approximately 20, 30, 40, 50, 60, 70p, 80, 90, or 100 bases. When an indel is introduced into the open reading frame (ORF) of a gene, The process involves creating frameshift mutations that are encoded by ORFs. Indels can interfere with the wild-type expression of the protein. Indels are double-strand breaks in the genome sequence (e.g., at a site). (by specific or programmable nucleases), followed by non-homologous end binding (NHE) This may be the result of cell repair using J).

[0049]

[0073] As used herein, the term "Type 1 diabetes mellitus" (T1DM) refers to pancreatic The inability to produce insulin due to the destruction of beta cells (e.g., autoimmune destruction) This refers to a characteristic condition. In some embodiments, type 1 diabetes is defined as 126 mg / dL Fasting plasma glucose (FPG) level of 7.0 mmol / L or higher, 200 mg / d During the 2-hour period of a 75g oral glucose tolerance test (OGTT) with a glucose level of L (11.1 mmol / L) or higher. Hyperglycemia or high blood glucose levels of 200 mg / dL (11.1 mmol / L) or higher Random plasma glucose or 6.5% or less in patients with typical symptoms of acute onset of blood glucose. Specific clinical criteria including at least one of the above hemoglobin A1c (HbA1c) levels Defined by ("Stage 3 T1DM"). In some embodiments, "1 Type 1 diabetes mellitus (T1DM) is characterized by stages 1, 2, or 3. This is a standard stage. Stage 1 is characterized by the presence of multiple autoantibodies against beta cells. Stage 2 may be asymptomatic, but involves blood glucose abnormalities (IFG and / or IGT). Intermediate FPG levels such as 100-125 mg / dL (5.6-6.9 mmol / L), 1 75g oral glucose negative test with a concentration of 40-199 mg / dL (7.8-11.0 mmol / L) or higher. Intermediate 2-hour plasma glucose level during an oral glucose tolerance test (OGTT) or 5.7-6.4% (39- Possible associated with intermediate hemoglobin A1c (HbA1c) levels of 47 mmol / mol. Yes, it exists. Type 1 diabetes (T1DM) can occur in children, young people, adolescents, or adults. Typically, this manifests as polyuria, polydipsia, polyphagia, diabetic ketoacidosis, or a body with an unknown cause. It is diagnosed after a severe decrease in activity.

[0050]

[0074] As used herein, the term "Type 2 diabetes" (T2DM) is used in This refers to a condition characterized by progressive loss of β-cell insulin secretion, frequently occurring against a background of insulin resistance. In some embodiments, type 1 diabetes is 126 mg / dL (7.0 mmol / L Fasting plasma glucose (FPG) levels above 200 mg / dL (11.1 mmol) 2-hour plasma glucose level during a 75g oral glucose tolerance test (OGTT) of 1 / L or higher , hyperglycemia of 200 mg / dL (11.1 mmol / L) or higher, or typical acute onset of hyperglycemia Random plasma glucose or hemoglobin A1 of 6.5% or higher in symptomatic patients Defined by specific clinical criteria, including at least one level of c(HbA1c).

[0051]

[0075] In some embodiments, the proteins or genes referred to herein are , as shown in the table below.

[0052] [Table 1]

[0053] [Table 2]

[0054] Cells, tissues, methods for generating cells and tissues, and methods for treating with the same.

[0076] This disclosure relates to cells, tissues, and organs having multiple modified genes, and their generation. A method is provided. In some embodiments, cells, tissues, or organs are obtained from animals. In some embodiments, the animal is a mammal. Mammals are non-human mammals, such as horses, primates, pigs, cows, sheep, goats, and It is either a nu or a cat. In some embodiments, the mammal is a pig.

[0055]

[0077] In some embodiments, one or more cells are pig cells. Non-exclusive examples of breeds from which this occurs or originates include the following pig breed: American Landrace , American Yorkshire, Aksai Black Pied, Angeln saddleback, Appalachian English, Arapawa Island, Auckland Island, Australian Yorkshire, Babi Kampung, Ba Xuyen, B antu, Basque, Bazna, Beijing Black, Belarus Black Pied, Belgian Landrace, Bengal i Brown Shannaj, Bentheim Black Pied, Berkshire, Bisaro, Bangur, Black Slavonian , Black Canarian, Breitovo, British Landrace, British Lop, British Saddleback, B ulgarian White, Cambrough, Cantonese, Celtic, Chato Murciano, Chester White, Chi angmai Blackpig, Choctaw Hog, Creole, Czech Improved White, Danish Landrace, Dan ish Protest, Dermantsi Pied, Li Yan, Duroc, Dutch Landrace, East Landrace, East Balkan, Essex, Estonian Bacon, Fengjing, Finnish Landrace, Forest Mountain, Fren ch Landrace, Gascon, German Landrace, Gloucestershire Old Spots, Gottingen minip ig、Grice、Guinea Hog、Hampshire、Hante、Hereford、Hezuo、Hogan Hog、Huntington Black Hog、Iberian、Italian Landrace、Japanese Landrace、Jeju Black、Jinhua、Kak hetian、Kele、Kemerovo、Korean Native、Krskopolje、Kunekune、Lamcombe、Large Bla ck、Large Black-White、Large White、Latvian White、Leicoma、Lithuanian Native、L ithuanian White、Lincolnshire Curly-Coated、Livny、Malhado de Alcobaca、Mangalit sa、Meishan、Middle White、Minzhu、Minokawa Buta、Mong Cai、Mora Romagnola、Mour a、Mukota、Mulefoot、Murom、Myrhorod、Nero dei Nebrodi、Neijiang、New Zealand、N ingxiang、North Caucasian、North Siberian、Norwegian Landrace、Norwegian Yorkshi re、Ossabaw Island、Oxford Sandy and Black、Pakchong 5、Philippine Native、Pietr ain、Poland China、Red Wattle、Saddleback、Semirechensk、Siberian Black Pied、Sm all Black、Small White、Spots、Surabaya Babi、Swabian-Hall、Swedish Landrace、Sw allow Belied Mangalitza, Taihu pig, Tamworth, Thuoc Nhieu, Tibetan, Tokyo-X, Tsi vilsk, Turopolje, Ukrainian Spotted Steppe, Ukrainian White Steppe, Urzhum, Viet namese Potbelly, Welsh, Wessex Saddleback, West French White, Windsnyer, Wuzhish It includes one of the following: anm, Yanan, Yorkshire, and Yorkshire Blue and White. In this embodiment, the pig cells are Yorkshire and Yucatan pig cells.

[0056]

[0078] In some embodiments, the cells of this disclosure are pancreatic islet cells or subsets thereof. Pancreatic islet cells are beta cells, alpha cells, delta cells, epsilon cells, or PP cells. It may include cells (also known as gamma cells or F cells). In some embodiments of this disclosure The cells are pancreatic islets. In some embodiments, the cells of this disclosure are contained in an intact pancreas. In some embodiments, the cells of this disclosure are contained in a pancreatic fragment. In some embodiments, the cells of this disclosure are contained in pancreatic organoids. In this disclosure, the cells are included in a collection of cells. In some embodiments, The cells in the disclosure are divided into a culture medium (e.g., solid, semi-solid, gel, liquid, or a combination thereof). These are dispersed cells. In some embodiments, the cells of this disclosure are in a cell cluster. Included. In some embodiments, the islet cells or organoids of the present disclosure are extrapancreatic. It contains virtually no secretory cells.

[0057]

[0079] In some embodiments, the cells, tissues, organs, or animals of the present disclosure are one or more The above gene is part of the gene, or part of the gene with altered sequence, which is either added, deleted, inactivated, or disrupted. The genes have been modified so that they can be altered by excision.

[0058]

[0080] In some embodiments, the cells, tissues, or organs of the present disclosure are one or more genes. It includes one or more mutations that inactivate the gene. In some embodiments, cells, groups A tissue, organ, or animal exhibits reduced or eliminated expression of one or more genes having one or more mutations. This includes one or more mutations or epigenetic changes that result. In some embodiments, one or more Genes are inactivated by genetically modifying nucleic acids present in cells, tissues, organs, or animals. It is sexualized. In some embodiments, inactivation of one or more genes is used in the assay. This is thus confirmed. In some embodiments, the assay is performed using reverse transcriptase PCR assay. (i) RNA-seq, real-time PCR, or junction PCR mapping assay In some embodiments, the assay is used to determine the function of the gene protein. Immunoassays on proteins transcribed from genes or gene fragments It is an assay.

[0059]

[0081] The cells, tissues, or organs described herein may be genetically modified by any appropriate method. This is possible. Knockout (KO) and knock-in (KI) as disclosed and described herein. Non-limiting examples of methods suitable for and / or genome replacement strategies include Cas9, Cas12a( Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas12g Cas12h, Cas12i or other CRISPR endonucleases, Argon Endoendonucleases, transcription activator-like (TAL) effectors and nucleases ( TALEN), zinc finger nuclease (ZFN), expression vector, transposo Use a transposase system (e.g., PiggyBac transposase) or any combination thereof. This includes CRISPR-mediated gene modifications in some embodiments.

[0060]

[0082] In some embodiments, a cell, tissue, or organ has at least one glycerinary cell. The enzyme has substantially no enzymatic activity of the cosyltransferase enzyme, and the glycosyltransferase The enzymes involved are GGTA, B4GALNT2, or CMAH. (Cells, tissues, or organs) This is a combination of at least two glycosides selected from GGTA, B4GALNT2, and CMAH. The enzyme may substantially lack the enzymatic activity of the transferase enzyme. Cells, tissues or The organ has at least three glycans selected from GGTA, B4GALNT2 and CMAH. It may be that the cosyltransferase enzyme has substantially no enzymatic activity. In this case, at least one Glico selected from GGTA, B4GALNT2 and CMAH Cells that substantially lack the enzymatic activity of the siltransferase enzyme are detectable at a level It substantially does not contain a full-length copy of the glycosyltransferase enzyme protein. In that case, at least one of the following will be selected from GGTA, B4GALNT2 and CMAH. Cells that substantially lack the enzymatic activity of the lycosyltransferase enzyme are at a detectable level. A functional polypeptide fragment of the glycosyltransferase enzyme protein is substantially Not included. In some cases, a small selection is made from GGTA, B4GALNT2 and CMAH. Cells that substantially lack the enzymatic activity of at least one glycosyltransferase enzyme are It substantially contains a transcript of mRNA encoding a full-length glycosyltransferase enzyme. No. In some cases, fewer options are available from GGTA, B4GALNT2 and CMAH. Cells that substantially lack the enzymatic activity of even one glycosyltransferase enzyme are, Substantially, the transcript of mRNA encoding a functional fragment of the lycosyltransferase enzyme Not included. In some cases, a small selection is made from GGTA, B4GALNT2 and CMAH. Cells that substantially lack the enzymatic activity of at least one glycosyltransferase enzyme are , open reading frame of at least one glycosyltransferase enzyme It contains indels. The indels can be produced using site-specific nucleases. Rundel is open reading of at least one glycosyltransferase enzyme. Frames (ORFs) (or, in the case of a gene that has multiple copies within the genome, all ORFs) ) is destroyed, and as a result, when the glycosyltransferase gene is transcribed, the full length or This inhibits the production of functional fragment mRNA or protein.

[0061]

[0083] In some embodiments, the cells, tissues, or organs are derived from non-porcine mammalian species. at least two polypeptide sequences (for example, at least two heterologous polypeptide sequences) ) expresses, and the at least two polypeptide sequences are CD46, CD55, CD59 , THBD, TFPI, CD39, B2M, HLAE, CD47, A20, PD-L1, Contains at least two of FASL or HO-1. Cells, tissues, or organs contain CD46, CD 55, CD59, THBD, TFPI, CD39, B2M, HLAE, CD47, A20 , PD-L1, FASL or HO-1, at least three, at least four, at least five , at least 6, at least 7, at least 8, at least 9, at least 1 0, at least 11, at least 12, or all may be expressed. In some cases, non At least two polypeptide sequences derived from the porcine mammalian species are CD46, CD55, and CD 59, THBD, TFPI, CD39, B2M, HLAE, CD47, A20, PD-L 1. Includes a full-length sequence of FASL or HO-1 or a combination thereof. In addition, at least two polypeptide sequences derived from non-porcine mammalian species are CD46, CD55 , CD59, THBD, TFPI, CD39, B2M, HLAE, CD47, A20, P Includes functional fragments of D-L1, FASL, or HO-1, or combinations thereof. In Tsuka's case, cells expressing at least two polypeptide sequences derived from non-porcine mammalian species. , tissues or organs, CD46, CD55, CD59, THBD, TFPI, CD39, B 2M, HLAE, CD47, A20, PD-L1, FASL or HO-1 or similar It expresses mRNA encoding the full-length sequence of the combination. In some cases, non-porcine milk Cells, tissues, or organs expressing at least two polypeptide sequences derived from an animal species are classified as CD. 46, CD55, CD59, THBD, TFPI, CD39, B2M, HLAE, CD4 7. Functional breakdown of A20, PD-L1, FASL, or HO-1 or a combination thereof It expresses mRNA that codes for one of the components.

[0062]

[0084] In some embodiments, the heterologous polypeptide sequences disclosed herein One of them is a polypeptide sequence encoded by the target human gene or a fragment thereof. And at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 7 8%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 8 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 They are 8%, 99%, or 100% identical. In some embodiments, as disclosed herein A polynucleotide sequence encoding any one of the heterologous polypeptide sequences is the target Human genes or fragments thereof, and at least about 70%, 71%, 72%, 73%, 74%, 75% %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 They are identical by %, 96%, 97%, 98%, 99%, or 100%. In some examples, The human genes of interest disclosed herein are CD46, CD55, CD59, THBD, TFPI, CD39, B2M, HLAE, CD47, A20, PD-L1, FASL and It may include one or more members selected from HO-1 (for example, two or more members). .

[0063]

[0085] In some embodiments, genetically modified cells disclosed herein, Either a tissue or organ is a genetically modified cell used to treat a target of a different species. It can be used for the purpose of. In some embodiments, the present disclosure is used for the genetic modification described herein. The present invention provides a method for transplanting altered cells, tissues, or organs into a target that requires them. In some embodiments, the subject is a human. In some embodiments, The subjects are non-human primates.

[0064]

[0086] Non-porcine mammalian species may be primate species. In some embodiments, non Pigs are a mammalian species that is not a human primate.

[0065]

[0087] In some embodiments, non-porcine mammalian species are found in humans (Homo sapiens). be.

[0066]

[0088] In some cases, at least two polypeptide sequences derived from non-porcine mammalian species. Cells, tissues, or organs that express CD46, CD55, CD59, THBD, TFPI CD39, B2M, HLAE, CD47, A20, PD-L1, FASL or HO -1, including genome sequences encoding combinations or fusions thereof. In this case, the genome sequences are CD46, CD55, CD59, THBD, TFPI, CD39 , B2M, HLAE, CD47, A20, PD-L1, FASL or HO-1, Open reading frames that encode a full-length copy of a combination or fusion thereof. It includes the genome. In some cases, the genome sequence is CD46, CD55, CD59, THBD , TFPI, CD39, B2M, HLAE, CD47, A20, PD-L1, FASL Young Or HO-1, an O that codes for functional fragments of a combination of them or a fusion thereof. Includes open reading frame. In some embodiments, open reading The frame is operably connected to the promoter. In some embodiments, the The motor is a ubiquitous promoter. In some embodiments, the promoter - is a human promoter. In some embodiments, the promoter is non-porcine. It is a promoter. In some embodiments, the promoter is a virus promoter It is a promoter. In some embodiments, the promoter is a porcine promoter. In some embodiments, the promoter is a ubiquitous promoter. In one embodiment, the promoter is a natural human promoter of a gene derived from a non-porcine mammalian species. Motors or their functional components, e.g., CD46, CD55, CD59, THBD, TFP I, CD39, B2M, HLAE, CD47, A20, PD-L1, FASL or HO- It is a natural promoter. In some embodiments, the promoter is a non-porcine promoter. Porcine promoters or functional fragments thereof of butorsologs of genes derived from dairy species, for example CD46, CD55, CD59, THBD, TFPI, CD39, B2M, HLAE, C It is the promoter for D47, A20, PD-L1, FASL, or HO-1.

[0067]

[0089] A genome sequence encoding at least two polypeptide sequences is found in cells, tissues, or It can be located at any appropriate position within the organ's genome. In some embodiments, less The genome sequences encoding at least two polypeptide sequences are AAVS1, CEP112, "SafeHerbal" is a pig genome such as ROSA26, Pifts302, or Pifts501. — Located at the gene locus. In some embodiments, at least two polypeptides The genome sequence encoding the sequence is a site-specific or programmable nuclease (e.g., By forming indels using the above-mentioned GGTA, B4GALNT2, CMAH) It is located at or near the locus of another gene that has been "knocked out". Several implementations In this state, the genome sequence encoding at least two polypeptide sequences is non-porcine milk The corresponding orthologous pig locus for polypeptides derived from animal species, e.g., CD46, CD 55, CD59, THBD, TFPI, CD39, B2M, HLAE, CD47, A20 It is located at the locus of the orthologs of PD-L1, FASL, or HO-1. In the application form, the genome sequences encoding at least two polypeptide sequences correspond to each other. Orthologous porcine polypeptides, for example, CD46, CD55, CD59, THBD, T FPI, CD39, B2M, HLAE, CD47, A20, PD-L1, FASL or H It occupies a position in place of the O-1 orthologue.

[0068]

[0090] In some embodiments, at least two polyps derived from non-porcine mammalian species The peptide sequences are CD46, CD55, CD59, THBD, TFPI, CD39, B2 Includes a subset of M, HLAE, CD47, A20, PD-L1, FASL, or HO-1. The subsets are CD46, CD55, CD59, CD39, B2M, HLAE and C It could be D47. The subsets are inflammatory response transgenes, immune response transgenes, and immunomodulation. From drug-introducing genes, coagulation response-introducing genes, complement response-introducing genes, and combinations thereof It may be at least two types of transgenes selected from the group. Inflammatory response transgenes The offspring contains TNFα-inducible protein 3 (A20), heme oxygenase (HO-1), and differentiation It may include raster 47 (CD47) or a combination thereof. The immune response transgene is Human leukocyte antigen-E (HLA-E), beta-2 microglobulin (B2M), or related substances. This may include combinations of immunomodulatory agents. The immunomodulatory agent transgene is programmed death ligand 1 (PD-L 1) May include Fas ligand (FasL) or a combination thereof. Coagulation response induction residue The genes include differentiation cluster 39 (CD39), thrombomodulin (THBD), and tissue factor. This may include pathway inhibitors (TFPIs) and combinations thereof. Complement response transgenes are membrane Cofactor protein (hCD46), complement disintegration promoter (hCD55), MAC inhibitor ( This may include hCD59) or a combination thereof.

[0069]

[0091] In some embodiments, at least two polyps derived from non-porcine mammalian species The peptide sequence is incorporated as a tandem sequence (for example, by homologous recombination). It can be provided (as a single structure).

[0070]

[0092] In some embodiments, the cells, tissues, or organs described herein are non-b When transplanted into mammalian species, the incubation period is approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days. More than 1 day, More than 8 days, More than 9 days, More than 10 days, More than 11 days, More than 12 days, More than 13 days, More than 14 days, More than 15 days , more than 16 days, more than 20 days, more than 24 days, more than 36 days, more than 48 days, more than 60 days, more than 72 days, more than 84 days Or it may demonstrate survival beyond that.

[0071]

[0093] In some embodiments, the cells, tissues, or organs described herein are as described herein. When transplanted into non-porcine mammalian species as described in this document, it may exhibit an altered immune response. The cells, tissues, or organs described in this book are reduced in number compared to PBMCs isolated from non-porcine mammalian species. A slight IBMIR may be observed. An immediate blood-mediated immune response (IBMIR) involves coagulation and complement A cascade, a powerful innate immune response involving leukocytes and platelet populations, residing in donor pancreatic islets Immediately after transplantation into Piento, an assay is used to monitor complement activation, for example. It can be measured (JD Lambris et al. (eds.), Immune Responses to Biosurf aces (2015), Advances in Experimental Medicine and Biology, Springer Internationa Kourtzelis et al., Chapter 11 “Regulation of Instant Blood Mediated Inf lammatory Reaction (IBMIR) in Pancreatic Islet Xeno-Transplantation:Points for T "Herapeutic Interventions" IBMIR states that the ratio is at least approximately 0.25 times, approximately 0.5 times, Approximately 0.75 times, approximately 1 times, approximately 2 times, approximately 3 times, approximately 4 times, approximately 5 times, approximately 6 times, approximately 7 times, approximately 8 times, approximately The reduction may be 9 times, approximately 10 times, or more. Cells, tissues, or organs as described herein. The government may demonstrate a reduction in toxicity due to complement derived from non-porcine species. Toxicity from complement derived from non-porcine species This is a radioactive assay (for example, 51 Cr assay), live cell staining (e.g., flow cell (by measurement), activity of released intracellular enzymes such as LDH or GAPDH, or dead cells It can be measured using staining. An exemplary method is described in Yamamoto et al. Scientific Reports. 10): 9771 (2020) describes the toxicity to complement derived from non-porcine species, at least approximately 0.25x, approx. 0.5x, approx. 0.75x, approx. 1x, approx. 2x, approx. 3x, approx. 4x, approx. 5x, approx. It may decrease by 6 times, approximately 7 times, approximately 8 times, approximately 9 times, or approximately 10 times, or more. The cells, tissues, or organs described herein induce activated protein C coagulation derived from non-porcine mammalian species. This may show a reduction. Induction of activated protein C coagulation from non-porcine mammalian species is at least Approximately 0.25 times, approximately 0.5 times, approximately 0.75 times, approximately 1 time, approximately 2 times, approximately 3 times, approximately 4 times, approximately 5 times It may decrease by approximately 6 times, 7 times, 8 times, 9 times, or 10 times, or more. The cells, tissues, or organs described in the document are thrombin-antithrombin complexes of non-porcine origin. It may show a reduction in the induction of body formation. Non-porcine thrombin-antithrombin complex form The induction of growth was at least about 0.25 times, about 0.5 times, about 0.75 times, about 1 time, about 2 times, about 3 times, approximately 4 times, approximately 5 times, approximately 6 times, approximately 7 times, approximately 8 times, approximately 9 times, or approximately 10 times or more The number of cells, tissues, or organs described herein may decrease. This may show a reduction in toxicity. Toxicity from NK cells derived from non-porcine species can be measured using radioactive assays (for example) Ba, 51 Cr assay), live cell staining (e.g., by flow cytometry), LDH Alternatively, evaluate the activity of released intracellular enzymes such as GAPDH, dead cell staining, or cytotoxicity. Toxicity by non-porcine-derived NK cells can be measured using other techniques. This is at least about 0.25 times, about 0.5 times, about 0.75 times, about 1 time, about 2 times, about 3 times, about A decrease of 4 times, approximately 5 times, approximately 6 times, approximately 7 times, approximately 8 times, approximately 9 times, or approximately 10 times, or more. It is possible.

[0072]

[0094] In some cases, this disclosure refers to the therapeutically effective dose of the embodiments described herein. The present invention provides a composition containing porcine islet cells. Islet cells are naturally occurring cells found in the pancreas. It may contain a proportion of islet cells, or a subset of islet cells or naturally occurring cells in the pancreas. It may contain different proportions of islet cells. Islet cells include beta cells, alpha cells, and delta cells. This may include T cells, epsilon cells, or PP cells (also known as gamma cells or F cells).

[0073]

[0095] In some cases, islet cells make up at least about 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%, Beta in amounts of 27%, 28%, 29%, 30%, 35%, 40%, 50%, or more. It may contain cells. In some cases, islet cells make up up to approximately 50%, 45%, 40%, and 35%. %, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21 %, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less. It may contain beta cells. In some cases, islet cells make up about 10% to 30% of the range. It may contain a circumferential amount of beta cells. In some cases, islet cells make up about 12% to about 25%. It may contain a quantity of beta cells in the range of %.

[0074]

[0096] In some cases, islet cells make up at least about 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%, 35%, 40%, 50% or more of alpha It may contain pancreatic islet cells. In some cases, pancreatic islet cells make up up to approximately 50%, 45%, 40%, and 3%. 5%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 2 1%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than that. It may contain a certain amount of alpha cells. In some cases, islet cells make up about 10% to about 40%. It may contain an amount of alpha cells in the range of [number]. In some cases, islet cells make up about 15%~ It may contain beta cells in amounts ranging from approximately 30%.

[0075]

[0097] In some cases, islet cells make up at least about 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%, Delta in amounts of 27%, 28%, 29%, 30%, 35%, 40%, 50%, or more. It may contain cells. In some cases, islet cells make up up to approximately 50%, 45%, 40%, and 35%. %, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21 %, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less. It may contain delta cells. In some cases, islet cells make up about 10% to about 40% of the range. It may contain a considerable amount of delta cells. In some cases, islet cells make up about 15% to about 30%. It may contain delta cells in amounts ranging from %.

[0076]

[0098] In some cases, islet cells make up at least about 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%, 35%, 40%, 50% or more of ipsi It may contain Rhon cells. In some cases, islet cells make up up to approximately 50%, 45%, and 40%. 35%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less. It may contain an amount of epsilon cells. In some cases, islet cells make up about 10% to about 4%. It may contain epsilon cells in amounts ranging from 0%. In some cases, islet cells are approximately 1 It may contain epsilon cells in amounts ranging from 5% to approximately 30%.

[0077]

[0099] In some cases, islet cells make up at least about 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%, 35%, 40%, 50% or more of pancreatic phosphate It may contain lipeptide (PP) cells. In some cases, pancreatic islet cells make up up to about 50%, 4 5%, 40%, 35%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 2 3%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 1 3%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 It may contain % or less of PP cells. In some cases, islet cells are about 10 It may contain PP cells in the range of % to approximately 40%. In some cases, islet cells are approximately It may contain PP cells in amounts ranging from 15% to approximately 30%.

[0078]

[0100] In some cases, the number of beta cells compared to the number of alpha cells is less than Approximately 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%, 35%, 40% It may be 45%, 50%, or more. In some cases, the number of alpha cells The number of beta cells compared to the others was up to 50%, 45%, 40%, 35%, 30%, and 29%. 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19% , 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, It could be 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less. In the example, the number of beta cells compared to the number of alpha cells ranged from approximately 10% to 40%. It is possible. In some cases, the number of beta cells compared to the number of alpha cells is approximately It could be in the range of 15% to approximately 30%.

[0079]

[0101] The cells are first collected from the culture medium or from the deaggregated pancreas, and then in an effective dose. Wash the cells with a culture medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) and concentrate them. It can be formulated by doing so. A suitable infusion medium can be any isotonic medium, and a normal biological medium. Saline solution, Normosol R (Abbott), Plasma-Lyte A (Baxter), 5% dextrose in water CMRL 1066 (e.g., 10) which does not contain lactated Ringer's solution, phenol red, and heparin. (0 U / kg recipient) etc. can be used. The injection medium is human serum albumin, bovine fetus It can be supplemented with infant serum or other human serum components. In some cases, an anticoagulant (e.g., heparin) may be used. (n) is at least 1 unit (U / kg), 2 U / kg per kilogram of recipient. 3U / kg, 4U / kg, 5U / kg, 10U / kg, 15U / kg, 20U / kg, 3 0U / kg, 40U / kg, 50U / kg, 60U / kg, 70U / kg, 80U / kg , 90U / kg, 100U / kg, 150U / kg, 200U / kg, 250U / kg, 300U / kg, 350U / kg, 400U / kg, 450U / kg, 500U / kg, 600U / kg, 700U / kg, 800U / kg, 900U / kg, 1,000U / k It may be administered in amounts of g or more. The anticoagulant may be used in the same solution as the pancreatic islet cells (e.g., buffered solution). It may be administered in liquid form. In other embodiments, the anticoagulant and pancreatic islet cells are administered separately. Obtain. In some cases, TNF-alpha inhibitors (e.g., etanercept) are used in recipes. Ent contains at least approximately 0.1 milligrams (mg / kg) per kilogram, and 0.2 mg / kg, 0.3mg / kg, 0.4mg / kg, 0.5mg / kg, 1mg / kg, 2mg / kg, 3mg / kg, 4mg / kg, 5mg / kg, 6mg / kg, 7mg / kg, 8 It may be administered in doses of mg / kg, 9 mg / kg, 10 mg / kg, or more. In this context, TNF-alpha inhibitors can be administered at a dose of approximately 3 mg / kg. The pha inhibitor may be administered in the same solution (e.g., buffer) as the pancreatic islet cells. In other embodiments... In this case, TNF-alpha inhibitors and pancreatic islet cells may be administered separately.

[0080]

[0102] In some cases, this disclosure describes the treatment of insulin resistance or deficiency. This provides a method for performing the following in non-porcine mammals: insulin resistance or deficiency symptoms. The conditions include type 1 or type 2 diabetes, monogenetic diabetes syndrome (neonatal diabetes and early-onset adult diabetes). Diabetes mellitus (such as type 1 diabetes [MODY]), exocrine pancreatic diseases (such as cystic fibrosis and pancreatitis), or medications. Alternatively, chemical-induced diabetes (use of glucocorticoids, treatment for HIV / AIDS or This may include post-organ transplantation, etc. In some embodiments, insulin resistance or deficiency When the deficiency includes type 1 or type 2 diabetes, mammals have fasting plasma glucose levels, Results from oral glucose tolerance tests or specific clinical criteria such as HbA1c may be shown. Several implementations In terms of morphology, insulin resistance or deficiency can manifest as unstable diabetes, asymptomatic hypoglycemia, and severe Enhanced risk factors present in mammals, such as a degree of hypoglycemic episode or glycemic instability. It may include.

[0081]

[0103] Non-porcine mammalian species may be primate species. In some embodiments, Non-porcine mammalian species are non-human primates. Non-human primates include marmosets (Callit). richidae (marmosets and tamarins), Cebidae (New World monkeys), Cercopithecidae (Old World monkeys), Cheirogaleidae e) (Dwarf lemurs and mouse lemurs), Daubentoniidae (Aye-aye) (Insect), Galagonidae (bush babies and galagos), Hominidae (Including great apes), Hylobatidae (gibbons and small apes), Indridae (Indri, Sifaka and related species), Lemuridae )(Brown lemur), Loridae (Loris), Megaladapidae (Megalad The families of lemurs (Apidae) and tarsiidae (Tarsiidae) This includes, but is not limited to, Lee, any of the various classifications of extant non-human primates or This includes all extant non-human primates. The term "non-human primate" refers to all extant non-human primates. Non-human primates and their groups classified according to any or all of the various classifications of primates. It includes. For example, Wilson and Reeder (1993) found that mega from the family Lemuridae. The family Megaladapidae was divided into the family Loridae and the family Galagonidae. (The former spelling is Loridae, not Lorisidae), Hi The family Hominidae includes great apes. Wilson, DE and DM Reeder. 1993. Mamm al Species of the World, A Taxonomic and Geographic Reference. 2nd edition. Smit Hsonian Institution Press, Washington. Anderson and Jones (1984) currently The order of primates is divided into two suborders: Strepsirhini and Haplorhini. They are separated. Thorington, RW, Jr. and S. Anderson. 1984. Primates. Pp. 187-217 i n Anderson, S. and JK Jones, Jr. (eds). Orders and Families of Recent Mammals of the World. John Wiley and Sons, NY. Strepsirhines are mainly... This includes arboreal species with primitive characteristics, but at the same time several species with specific lifestyles. Having undergone extreme differentiation, the Haplorhini suborder is a so-called "higher" primate, and further They can be divided into two main groups: Platyrrhini and Catarrhini. Platyrrhine is characterized by a flat nose, an outward-facing nasal opening, and three small teeth in the upper and lower jaws. Molars, having presupromalis with three or four major tooth apices, New World (Cebiidae (Cebi) It is found only in the dae and marmosets (Callitrichidae). The suborder Catarrhini is contiguous with each other. It has a pair of close, downward-facing nasal openings and typically has two premolars and four apical teeth on each jaw. They have prefrontal molars and belong to the Old World families (Cercopithecidae, Hylobatidae). It is found only in the families Hominidae (and ae). Most primates live in tropical or subtropical regions. However, some species also inhabit temperate regions. With the exception of a few terrestrial species, primates They are arboreal. Some species feed on leaves or fruits, while others are insectivorous or carnivorous. Myers, P. 1999. “Primates” (On-line), Animal Diversity Web. Accessed Aug. 26, 20 See .

[0082]

[0104] In some embodiments, the non - porcine mammalian species is Homo Sapiens is.

[0083]

[0105] A method of treating insulin - resistant or deficient states in a non - porcine mammal that requires it may include administration or transplantation of any of the compositions, cells, organs or tissues described herein. In some cases, when a cell composition is administered, the composition is administered centrally and, for example, is administered via the internal jugular vein or the portal vein of the non - porcine mammal.

[0084]

[0106] In some cases, prior to treatment with a composition, cell, organ or tissue, the non - porcine mammal is undergoing an induction immunosuppression regimen. The induction regimen is a therapeutically effective dose of antithymocyte globulin, anti - CD40 antibody, anti - CD20 antibody before administering the cell, tissue or organ, rapalog, calcineurin inhibitor, ganciclovir or its prodrug, anti - histamine agent or corticosteroid, which may be included.

[0085]

[0107] In some cases, after treatment with a composition, cell, organ or tissue, the non - porcine mammal is undergoing a maintenance immunosuppression regimen. The maintenance regimen is a therapeutically effective dose of anti - CD40 antibody rapalog, calcineurin inhibitor and ganciclovir or a prodrug of ganciclovir which may be included.

[0086]

[0108] In some cases, after treatment with a composition, cell, organ or tissue, the non - porcine mammal​ The substance receives a supportive insulin regimen. The supportive insulin regimen is administered to the aforementioned cells and groups. After administration of tissue, organ, or composition, an effective dose of intermediate-acting or long-acting insulin is administered. Analogues (e.g., insulin glargine, insulin detemir, or NPH insulin) It may include.

[0087]

[0109] Treatment of insulin resistance or deficiency in non-porcine mammals that require it. The methods used in this context may include the administration of a specific islet equivalent dose (IEQ per kg). For reference, islet equivalence (IEQ) is the same as a single spherical islet with a diameter of 150 μm. It is defined as IEQ (Huang et al. Cell Transplant 2018 Jul: 27 (7): 1017-26), The islet equivalent dose is the IEQ per kg of non-porcine mammalian body weight of the recipient. In this case, the dosage is at least about 1,000 IEQ per kg of body weight of a non-porcine mammal. (IEQ / kg), 2,000IEQ / kg, 3,000IEQ / kg, 4,000IE Q / kg, 5,000IEQ / kg, 6,000IEQ / kg, 7,000IEQ / kg , 8,000IEQ / kg, 9,000IEQ / kg, 10,000IEQ / kg, 11 ,000IEQ / kg, 12,000IEQ / kg, 13,000IEQ / kg, 14, 000IEQ / kg, 15,000IEQ / kg, 16,000IEQ / kg, 17,0 00IEQ / kg, 18,000IEQ / kg, 19,000IEQ / kg, 20,00 The amount may be 0 IEQ / kg or more. In some cases, the dose may be up to about 20 ,000IEQ / kg, 19,000IEQ / kg, 18,000IEQ / kg, 17, 000IEQ / kg, 16,000IEQ / kg, 15,000IEQ / kg, 14,0 00IEQ / kg, 13,000IEQ / kg, 12,000IEQ / kg, 11,00 0IEQ / kg, 10,000IEQ / kg, 9,000IEQ / kg, 8,000IE Q / kg, 7,000IEQ / kg, 6,000IEQ / kg, 5,000IEQ / kg , 4,000IEQ / kg, 3,000IEQ / kg, 2,000IEQ / kg, 1,0 It may be 00IEQ / kg or less. In one example, the dose is measured in the body of a non-porcine mammal. It could be at least 5,000 IEQ per kilogram of weight.

[0088]

[0110] In some embodiments, this disclosure relates to transplantation into non-porcine mammalian recipients. Previously, we provided a method to improve the yield of pancreatic islets from pig donors. The method involves pancreatic organoids. To provide a method for culturing the organoid in the presence of an effective concentration of a caspase inhibitor. This may include continuing the culture in the presence of an effective concentration of corticosteroids. Noids, in the presence of a caspase inhibitor, for at least 30 minutes, at least 60 minutes, at least 90 minutes, at least 2 hours, at least 4 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 90 hours, at least 120 hours, at least 180 hours, at least 360 hours, less Organoids can be cultured for at least 720 hours or longer. In the presence of tycosteroids, after treatment with caspase inhibitors, at least 1, at least at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, At least 8, at least 9, at least 10, at least 11, at least 12, at least 13 or at least 14 days. Pancreatic organoids can be isolated from pig animals before the 7th day. The caspase inhibitor can be Z-VAD-FMK, Z-LEHD-FMK, Z-IETD-FMK, Emricasan, Z-VEIDFMK, Z-DEV D-CMK, MX1122, M867, MMPSI, isatin sulfonamide, Boc- Asp-FMK, VX-166, Q-VD-OPh or IDN-6556. The corticosteroid can be methylprednisolone. The organoids can be cultured in the presence of nicotinamide or a metabolically acceptable analog thereof.

Example

[0089] Example Example 1. Composition and Characterization Evaluation of Transgenic Pig Animals and Endothelial Cells Derived Therefrom [[ID=2'4]]

[0111] Using NHEJ via CRISPR-Cas9, three major carbohydrate-producing glycosyltransferases / gly cosylhydrolase genes GGTA1, CMAH and B4GALNT2 were functionally knocked out in primary fibroblast cells of Bama mini pigs. Next, 12 human transgenes (CD46, CD55, CD59, CD39 , CD47, A20, PD-L1, HLA-E, B2M, THBD, TFPI, HO-1 ) were integrated into a single multi -transgene cassette of the pig genome via random integration via the PiggyBAC transposon to generate "4-7", a 3KO / 12TG cell (used to generate pigs via somatic cell nuclear transfer (SCNT)). Wild-type pig ear Fibroblasts first target the GGTA, CMAH, and B4GALNT2 genes. b)(i) PiggyBac transposer case (i i) A payroll that holds a transgenic construct containing one or more of the 12 human transgenes. Both the transplasmid and the transgene were electroporated. The transgene was the desired ubiquitous Placed within four different cistrons having a general promoter or a tissue-specific promoter The transgenes within each cistron were expressed in a similar molar ratio to ensure that they were ribosomal. It was separated by humskip 2A peptide. Furthermore, ubiquitous chromatin opener By introducing a combination of cis-elements such as the gu-element (UCOE), the introduced gene Insulators that prevent silencing and feature strong polyadenylation sites and terminators. By introducing a motor, interactions between introduced genes and between introduced genes and adjacent chromosomes are minimized. It was kept to a minimum. Single-cell clones of fibroblasts were generated, and fragment analysis / whole-genome sequencing was performed. Screening is performed to identify clones with the desired genome modification, and then the desired modification is applied to... Using a cloned pig as a donor, we created a live pig using SCNT (Sclerotic Cell Transplantation).

[0090]

[0112] The expression level of the transgene is determined by qPCR, and the integration site is inverse P Determined using CR-based junction capture, protein levels are determined by fireflies. The results were determined by photoactivated cell sorting (FACS). The results are shown in Table 1B below. It will be translated.

[0091] [Table 3]

[0092]

[0113] Table 1B shows the results of immunohistochemistry (IHC) staining of 4-7 pig tissues. It shows the expression of various introduced genes.

[0093] [Table 4]

[0094]

[0114] Functional characteristics of the results of gene knockout / knock-in

[0115] GGTA / CMAH / B4Gal

[0116] Pre-formed antibodies that bind to wild-type pig tissue are the primary antibodies against xenotransplantation. These three genes are considered to be early immunological barriers, and these antibodies target them. It has been confirmed that it is significantly involved in the production of heterologous antigens (Byrne 2014, Lai 2002, Lutz 2013, Martens 2017, Tseng 2006). Therefore, loss of function of these genes, This is predicted to significantly reduce the binding of pre-formed anti-porcine antibodies to the endothelium of the porcine graft. This was done. This involves targeting pig umbilical vein endothelial cells (called "4-7"), and using GGTA1, CMA Binding of host antibodies to H, including B4GalNT2 knockout (see Figure 1) This was confirmed by flow cytometry results showing a decrease, demonstrating a reduction in antibody binding. Therefore, genetically modified pig endothelial cells were incubated with pooled human serum, and Combined human IgM and IgG were detected with a conjugated secondary anti-human antibody, and then analyzed by flow cytometry. The analysis was conducted in contrast to wild-type pig umbilical vein endothelial cells (PUVECs) (red contour plot). When the three genes were eliminated, antibody binding was significantly reduced (comparing the blue and yellow contour plots). compared with approximately 1 log reduction in coupling).

[0095]

[0117] Complement regulatory proteins (CD46, CD55, and CD59)

[0118] To maintain the function of the pig graft and protect the donor organ from complement-mediated toxicity. Therefore, human complement regulatory proteins were overexpressed. To put it simply, genetically modified Porcine fibroblasts and porcine splenocytes were incubated with 25% human complement for 1 hour. The cells were stained with propidium iodide and analyzed by flow cytometry to quantify cell death. (See Figure 2). The left panel of Figure 2 shows the assay workflow, and the right panel... Nell's human umbilical vein after incubation with various concentrations of human complement ("HC") Endothelial cells ("HUVEC"), transgenic 4-7 pig umbilical vein endothelial cells ("4- Either "7PUVEC" or normal pig umbilical vein endothelial cells ("WT PUVEC") This is a chart showing death. Cells 4-7 that possess all three transgenes are human HUVE Similar to C cells, they showed a dramatic reduction in death in response to human complement compared to normal porcine counterparts. vinegar.

[0096]

[0119] Coagulation response genes

[0120] When angiogenic WT pig organs are transplanted into humans, pre-formed antibodies are used. Complement and innate immune cells may induce endothelial cell activation, potentially leading to coagulation and inflammation. There is incompatibility between coagulation regulatory factors from porcine endothelial cells and human blood, resulting in abnormal platelets. This leads to activation and thrombin formation, exacerbating the injury. Furthermore, between pigs and humans Incompatibility of coagulation regulatory factors (e.g., tissue factor pathway inhibitors, TFPIs) leads to exogenous factors Coagulation regulation becomes ineffective.

[0097]

[0121] To address these heterocoagulation problems, a) Human CD39 (coagulation cascade) a) ADP hydrolase that counteracts the thrombotic effect of ADP in the cerebrospinal fluid, and b) human TFPI Both of these factors (factors that move to the cell surface after endothelial cell activation) are overexpressed in 4-7PUVEC. Next, various in vitro and ex vivo assays were performed to determine if these transgenes We verified its ability to function correctly and regulate platelets and the coagulation cascade. Figure 3 shows 4-7 CD39 expression / functionality in lancegenic endothelial umbilical vein pig cells (PUVECs) The results of the analysis conducted to verify this are shown. The chart shows HUVEC, 4-7PUV Colorimetric CD39 ADP hydrolysis-based activity enhancement performed in EC or WT PUVEC The results for Sei are shown. Cells 4-7 showed enhanced CD39 activity, which is due to the transgenetic effect. This suggests that the child is functionally and overexpressing. In vitro ADPase biochemistry Issey showed significantly higher CD in 4-7 PUVEC compared to WT PUVEC or HUVEC. 39 showed activity. Similarly, activated 4-7PUVEC effectively bound to human Xa. It exhibits the ability to neutralize, thereby mitigating coagulation, and thrombin-antithrombin (TA) T) Complex formation can be reduced (Figure 5). Humans co-cultured with 4-7PUVEC The ex vivo coagulation assay using whole blood (Figure 5) shows minimal TAT (thrombin-antibody). Rhombin was formed, and the level of TAT formation was similar to that of HUVEC. As shown (Figure 5), this suggests that 4-7PUVEC enhances coagulation compatibility with human factors. They are doing it.

[0098]

[0122] Figure 6 shows the effects of these gene modifications on platelet activation. The results of the assay performed are shown. Figure 6 shows the results of the assay performed with 4-7 transgenic cells. The results of the platelet lysis assay are shown. HUVEC, 4-7PUVEC, or WT PUVEC and After incubation for 45 or 60 minutes, the remaining platelets (with outlines drawn) are extracted from human blood. FACS traces quantifying the number of clusters are shown. Cells 4-7 are from porcine W. The number of remaining platelets relative to T ECs continues to increase, which indicates that HUVEC cells and i This is equivalent to the percentage of remaining platelets after incubation.

[0099]

[0123] HLA components (HLA-E / B2M)

[0124] Killer cell suppression receptors (KIRs) on natural killer (NK) cells Linking of MHC I on target cells inhibits NK cell-mediated target cell death. (Pig M) HCI cannot transmit signals via human NK KIR, therefore, porcine cells It is susceptible to target cell killing by NK cells. To overcome NK-mediated cell death To achieve this, human HLA-E, which links to the human NK KIR receptor, was overexpressed in pig cells. Furthermore, a human copy of the MHC heterodimerization partner B2M was also overexpressed.

[0100]

[0125] Next, a functional assay was performed to determine that endothelial cells 4-7 produced NK cells due to genetic modification. We verified its resistance to cell death mediated by WT PUVEC, 4-7PUVEC, and HUVEC was targeted for killing by human NK cells in an in vitro assay (Figure 7). Figure 7 shows that 4-7PUVEC exhibits significantly lower NK-mediated cytotoxicity than its wild-type counterpart. This is demonstrated by an independent two-tailed Student t-test.

[0101] Example 2. Isolation of pancreatic islets from transgenic animals

[0126] Transgenic male Bama miniature pig (produced as in Example 1) The infant was anesthetized and subjected to laparotomy and bloodletting 0-7 days after birth. The pancreas was removed and sterile tissue was used with a scalpel. Under certain conditions, the pancreatic fragments were cut into small pieces. The pancreatic fragments were subjected to collagenase V digestion (1 mg / ml), and the gas Transfer to a permeable culture bag (OriGen PermaLife® cell culture bag) and transport to the culture facility. The pancreatic islets were kept at 22-24°C for transport. R3-IGF, ascorbic acid, hEGF, heparin, D-glucose, nicotinamide D, 10% porcine serum, 50 μM IBMX, 120 μM amikacin and 60 μM ampicillin EGM-2 containing phosphorus, EGM-2 medium + corticosteroid (EGM-2 + 1 μM methyl phosphate) In either tilprednisolone or Ham's F-10 medium, for 7 days, The cells were cultured in a tub or petri dish. Islet equivalency (IEQ) was measured over 7 days during the culture period. This was then graphed (see Figures 14, 15, and 16). Corticosteroids The EGM-2 medium was associated with improved islet yield among the three conditions.

[0102]

[0127] In some cases, pancreatic fragments sediment after mechanical or enzymatic digestion (for example) It can be purified by (Ficol gradient sedimentation). In other cases, such purification by sedimentation A thrombotomy may or may not be necessary. In such cases, a pancreatic thrombotomy is performed. The tissue can be cultured before transplantation (e.g., in a culture dish for about 7 days), during which time non-islet cells (e.g., (Exocrine cells) may die.

[0103]

[0128] Example 3. Analysis of pancreatic islets from transgenic animals

[0129] From normal and transgenic Bama miniature pigs as in Example 2 After isolating pancreatic islet cells, the assay was performed on 4-7 endothelial cells as in Example 1. Similar to the experimental scheme, we evaluated the heterocompatibility aspects of 4-7 islet cells.

[0104]

[0130] Firstly, we evaluate the effects of gene modifications to regulate platelets and coagulation cascades. Experiments were conducted to evaluate the results. Figure 11 shows the results obtained using islet cells isolated as in Figure 9 / Example 2. The results of the platelet lysis or TAT complex formation assays performed are shown. Negative control (NC) As shown in Figure 6, the study was performed using human umbilical vein endothelial cells (HUVECs), either in the WT or 4-7 pancreatic islets. Platelet lysis assay (left panel) and HUVEC NC, WT, or 4-7 islets were performed. A chart showing the TAT complex formation assay, as shown in Figure 5, is presented. Pancreatic islets 4-7 are When incubated with human blood components, it reduces platelet lysis and TAT complex formation. show.

[0105]

[0131] Secondly, the short-term IBMIR response of the human immune system to transplanted pig tissue. Experiments were conducted to evaluate the effects of gene modification to regulate [the gene]. Figure 12 shows the same result as in Figure 9. Immediate blood-mediated inflammatory response (IBMI) was performed using human blood in 4-7 pancreatic islets obtained from the sea. R) The results of the assay are shown. Briefly, human whole blood is used as disclosed herein. Incubate with porcine islet cells, then contact between human whole blood and porcine islet cells. Check for coagulation or blood clots that are at least partially caused by (or interactions) In some cases, the size and / or weight of the blood clot was measured. 4-7 pancreatic islet sections were used on human pancreatic islets. Antibodies (IgG and IgM, left panel) and complement (C3a and IgM) after incubation with blood. Figure 9 shows an IHC micrograph at 200x magnification, illustrating the staining of the lesion (C4d, right panel). As shown in 4-7, pancreatic islet cells show staining related to IgG, IgM, C3a and C4d and This shows a reduction in lesions, which indicates a decrease in islet cells (IBMIRs) and a decrease in death at the time of the first transplant. This suggests that resistance to it has been strengthened.

[0106]

[0132] Thirdly, to evaluate the effects of gene modification to regulate human neutrophil activity The experiment was conducted to determine the number of neutrophils remaining in human whole blood cultured from 4-7 pancreatic islets. Figure 13 shows the number of neutrophils remaining in human whole blood cultured from 4-7 pancreatic islets. The results show that when 4-7 islets are incubated with human whole blood, they produce more neutrophils compared to WT islets. It became clear that a large number of them remain.

[0107] Example 4. Transplantation of pancreatic islets into recipient mice

[0133] To test the function of 4-7 porcine transgenic pancreatic islet cells during xenotransplantation. A STZ-based mouse diabetic islet adoptive transfer model was established. Using this model procedure... Figure 19 shows an example of blood glucose levels in the mice used. This model uses the toxin (streptozotosh). Using STZ, pancreatic islet cells in immunodeficient mice were killed, resulting in a dramatic increase in blood glucose levels. Pancreatic islet cell transplantation leads to normalization of blood glucose levels within approximately 60 days after transplantation.

[0108]

[0134] To evaluate the effectiveness of 4-7 islet cells in the treatment of diabetes, Treatment involves a single dose of streptozotocin (STZ, 125 mg / kg), followed by... With a washout period of 3 days, n=12 NCD(NOD-Prkdc em2 6Cd52 Il2rg em26Cd22 (NjuCrl) Initially induced in mice, 3 mice Untreated mice of the same age were kept as the target group. Next, the untreated mice and STZ-treated mice were selected. Three of our mice were subjected to a sham transplant surgery, and three of the STZ mice were subjected to the procedure shown in Figure 9 / Example 2. Three of the STZ mice were administered isolated wild-type porcine pancreatic islets (3000IEQ), Figure 9 / Example 2 shows isolated 4-7 transgenic porcine pancreatic islets (3000IEQ). ) was administered. If pancreatic islets were transplanted, they were transplanted under the left renal capsule. Brief explanation Then, mix or disperse the 4-7 pancreatic islet cells in a solution (e.g., buffer solution) using a syringe and softener. It was injected subcapsulate via a tube (for example, slowly).

[0109]

[0135] Figure 20 shows 4-7 porcine transgenic pancreatic islets provided herein. Pancreatic islet-like cell clusters (NICCs) containing cells were administered (as a T1D rodent model). This shows the blood glucose levels of NCG mice. NICCs containing wild-type porcine islet cells were used as a control. Various amounts of NICC were used. NICC was implanted into NCG mice: 4000IEQ, 2000IEQ. EQ and 1000IEQ. Data compared WT porcine islet cells with 4-7 porcine trans cells. We have shown similar effectiveness in controlling elevated blood glucose levels in mice using genic islet cells. The results show that 4-7 transgenic islet cells from pigs function in vivo approximately 2 weeks after transplantation. They started doing this (for example, in controlling blood sugar levels).

[0110]

[0136] In some cases, the abnormal proliferation of transplanted pig cells lasts longer (for example, It may be monitored for a period of 5, 6, 7, 8, 9, 10, 11, or 12 months or longer. In some cases, human adult pancreatic islet cells are positive for, for example, at clinical human adult pancreatic islet treatment doses. It can be used as a reference. In some cases, the non-obese diabetic (NOD) T1D mouse model It can be used as a secondary in vivo model. In some cases, its suitability and safety For testing, porcine transgenic pancreatic islet cells may be administered by intra-portal vein injection. ru.

[0111]

[0137] Example 5. Transplantation of pancreatic islets into recipient monkeys

[0138] Testing the function of 4-7 pig transgenic pancreatic islet cells during xenotransplantation into primates. To test this, STZ-based NHP diabetic islet adoptive transplantation (using intra-portal islet cell transplantation) A model was established. The islets 4-7 and WT isolated as shown in Figure 9 / Example 2 were examined by ultrasound. These were implanted in cynomolgus monkeys via percutaneous transhepatic portal vein catheterization induced by [unspecified method]. The experimental scheme is shown in Figure 21.

[0112]

[0139] The immunosuppression protocol used for transplanting porcine cells was as follows:

[0113]

[0140] ATG is -7 days (±2 days), -6 days (±2 days), -4 days (±2 days) It is administered intravenously at a dose of 5 mg / kg, and phosphorus levels in the blood are reduced to less than 5% of the baseline level. If pocyte depletion was achieved, an additional ATG was administered on day -1.

[0114]

[0141] Anti-CD40 is administered on -4 days (±1 day), day 0, day 4, day 7, and day 10. It was administered intravenously on day 14, and thereafter, weekly doses of 50 mg / kg (initial dose) followed by 30 mg. It was administered at a dose of / kg.

[0115]

[0142] The anti-CD20 monoclonal antibody rituximab was administered at a dose of 375mg on day 0 (±2 days). If administered intravenously at a dose of g / m2 and the B cell count rises above 5% of baseline, The drug was administered repeatedly, up to every three months.

[0116]

[0143] Rapamycin and tacrolimus are 0.3 mg / kg QD and 0.0 mg / kg QD, respectively. The starting dose of 2 mg / kg BID is administered orally, starting on day 3 (±1 day), and plasma concentration is measured. It was adjusted accordingly.

[0117]

[0144] Ganciclovir should be started at a dose of 5 mg / kg from day -7 (±2 days). It was administered intramuscularly.

[0118]

[0145] To prevent infusion reactions, chlortrimethone 0.4 mg / kg IM and methicone are used. The prophylactic use of luprednisolone 10 mg / kg IV is for ATG, anti-CD40, and anti-CD2 0 It was administered before the previous dose.

[0119]

[0146] Supportive insulin administration to STZ-introduced animals to support their health is, It was provided as follows:

[0120]

[0147] Glargine insulin was administered in QDs, initially given as 2U QDs. The dosage is increased by 2 units if the FBG level is greater than 150 mg / dl, and by 100 mg When the value was less than / dl, the U decreased by 2U.

[0121]

[0148] Insulin was administered in BID in the morning and evening according to the recorded blood glucose levels of the animals. In the morning administration, insulin was not administered if the level was less than 200 mg / dl, and was administered between 200 and 350 mg / dl. For a level of g / dl, administer 4 units of insulin; for levels between 350 and 400 mg / dl, administer 6 units of insulin. Administer insulin, and if the level is 400-600 mg / dl, administer 8 U of insulin, and if it is 600 mg / dl For levels above 1, 10U of insulin was administered. For evening administration, levels below 300mg / dl were given If insulin is not administered, and the level is 300-350 mg / dl, administer 4 units of insulin, and 350 For levels ~400 mg / dl, administer 6 units of insulin; for levels 400-600 mg / dl, administer 8 units. Insulin was administered, and for levels exceeding 600 mg / dl, 10 units of insulin were administered.

[0122]

[0149] Pilot experiment using STZ diabetes induction protocol in monkeys (MB-1) This is shown in Figure 22, and the animals are managed according to the scheme in Figure 21. To measure functional output, animals were administered 1 ml / kg IV of 50% dextrose. Blood glucose, C-peptide, and insulin levels were evaluated. The data in Figure 22 are blood glucose levels after STZ treatment. The diabetes induction protocol was successful due to increased glucose levels and decreased levels of C-peptide and insulin. This demonstrates that further animal MA-1, MA-2, MB- 2. Induce diabetes in MC-1, MD-1, and ME-1 cells, and transfer the grafts according to Table 2 below. Transplanted. Post-transplant white blood cell count, lymphocyte count, CD4+ cell type, CD8+ cell type, B cells, N K cell and rapamycin levels were monitored in the animals (Figures 23, 24, and 25).

[0123]

[0150] Animals MA-1 and MA-2 subsequently confirmed the presence of transplanted 4-7 pancreatic islets. The immunohistochemistry of liver biopsies performed 12 hours and 1 month post-transplant was analyzed. Administer hematoxylin / eosin (for MA-1 and MA-2) and (for MA-2) Staining was performed for chromogranin A, a neuroepithelial marker that stains pancreatic islet cells, and the presence of islet-like structures was observed. The engraftment of 4-7 cells into the host and animal liver is shown (see Figure 26).

[0124]

[0151] To evaluate the function of non-human primate grafts over the long term, immunohistochemistry is used. Chemistry, blood glucose, C-peptide (both monkey and pig), and insulin levels are all listed in Table 2. The animals will continue to be monitored.

[0125] [Table 5]

[0126]

[0153] Preferred embodiments of the present invention have been shown and described herein, but It will be apparent to those skilled in the art that such embodiments are provided only as examples. Hereinafter, numerous variations, alterations, and substitutions will be conceivable to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in carrying out the invention. The following claims define the scope of the present invention, and the methods and structures within these claims, as well as their equivalents, are intended to be encompassed thereby. This application encompasses the following inventions. [Item 1] Isolated transgenic porcine pancreatic islet cells, (a) substantially lacking the enzymatic activity of at least one glycosyltransferase enzyme, wherein the glycosyltransferase enzyme is GGTA, B4GALNT2, or CMAH. (b) Expressing at least two polypeptide sequences derived from a non-porcine mammalian species, wherein the at least two polypeptide sequences include at least two of CD46, CD55, CD59, THBD, TFPI, CD39, B2M, HLAE, CD47, A20, PD-L1, FASL, or HO-1, and (c) Isolated transgenic porcine pancreatic islet cells exhibiting one or more of the following: reduced toxicity by complement derived from a non-porcine mammalian species, reduced induction of activated protein C coagulation derived from a non-porcine mammalian species, reduced induction of thrombin-antithrombin complex derived from a non-porcine mammalian species, or reduced toxicity by NK cells derived from a non-porcine species. [Item 2] Transgenic porcine pancreatic islet cells as described in Item 1, wherein the cells substantially lack the enzymatic activity of at least two or all three glycosyltransferase enzymes selected from GGTA, B4GALNT2, and CMAH. [Item 3] Transgenic porcine pancreatic islet cells as described in Item 1 or 2, wherein the cells express at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or all of the following: CD46, CD55, CD59, THBD, TFPI, CD39, B2M, HLAE, CD47, A20, PD-L1, FASL, or HO-1. [Item 4] Isolated transgenic porcine pancreatic islet cells, (a) substantially lacking the enzymatic activity of at least two glycosyltransferase enzymes, wherein the glycosyltransferase enzymes include at least two of GGTA, B4GALNT2, or CMAH. (b) Expressing a polypeptide sequence derived from a non-porcine mammalian species, wherein the polypeptide sequence is CD46, CD55, CD59, THBD, TFPI, CD39, B2M, HLAE, CD47, A20, PD-L1, FASL, or HO-1, and (c) Isolated transgenic porcine pancreatic islet cells exhibiting reduced toxicity by complement derived from a non-porcine mammalian species, reduced induction of activated protein C coagulation derived from a non-porcine species, reduced induction of thrombin-antithrombin complexes derived from a non-porcine species, or reduced toxicity by NK T cells derived from a non-porcine species. [Item 5] Transgenic porcine pancreatic islet cells as described in Item 4, wherein the cells substantially lack the enzymatic activity of GGTA, B4GALNT2, and CMAH. [Item 6] The cells mentioned above contain CD46, CD55, CD59, THBD, TFPI, and CD3 9. Transgenic porcine pancreatic islet cells as described in item 4 or 5, expressing at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or all of B2M, HLAE, CD47, A20, PD-L1, FASL, or HO-1. [Item 7] Transgenic porcine pancreatic islet cells expressing CD46, CD55, CD59, CD39, B2M, HLAE, and CD47, as described in any one of items 1 to 6. [Item 8] Transgenic porcine pancreatic islet cells according to any one of items 1 to 6, wherein the cells substantially do not express the one or more glycosyltransferase enzymes. [Item 9] Transgenic porcine pancreatic islet cells according to Item 8, wherein the cells contain frameshift mutations in one or more glycosyltransferase enzymes, causing premature termination of translation, thereby resulting in loss of activity of the glycosyltransferase enzymes. [Item 10] Transgenic porcine pancreatic islet cells according to any one of items 1 to 9, wherein one or more nucleic acid sequences encoding the one or more polypeptide sequences derived from a non-porcine mammalian species are inserted into the non-orthorhogus locus of the butorsolog. [Item 11] Transgenic porcine islet cells according to any one of items 1 to 10, wherein one or more nucleic acid sequences encoding the one or more polypeptide sequences derived from a non-porcine mammalian species are operably linked to a butorsolog non-orthologus promoter. [Item 12] Transgenic porcine islet cells according to item 11, wherein the non-orthologus promoter is a non-porcine promoter. [Item 13] Transgenic porcine pancreatic islet cells according to any one of items 1 to 12, wherein the pancreatic islet cells are obtained by deaggregation of porcine pancreas. [Item 14] Transgenic porcine pancreatic islet cells according to any one of items 1 to 13, wherein the islet cells are α cells, β cells, δ cells, ε cells, pancreatic polypeptide (PP) cells, or any combination thereof. [Item 15] Transgenic porcine pancreatic islet cells as described in any one of items 1 to 14, wherein the non-porcine mammalian species is a primate species. [Item 16] Transgenic porcine pancreatic islet cells according to any one of items 1 to 15, which, when transplanted into the non-porcine mammalian species, exhibit survival for more than 8 days. [Item 17] Transgenic porcine cells according to any one of items 1 to 16, wherein the cells exhibit reduced IBMIR compared to PBMCs isolated from the non-porcine mammalian species. [Item 18] A composition comprising a therapeutically effective amount of isolated porcine pancreatic islet cells as described in any one of items 1 to 17. [Item 19] The composition according to Item 18, further comprising heparin or a TNF-α inhibitor. [Item 20] The composition according to item 18 or 19, comprising at least about 12% to about 25% β cells or at least about 15% to about 30% α cells. [Item 21] The composition according to any one of items 18 to 20, wherein the composition is prepared according to any one of items 35 to 42. [Item 22] A method for treating insulin resistance or deficiency in a non-porcine mammal in need thereof, comprising administering to the mammal a therapeutically effective dose of isolated transgenic porcine pancreatic islet cells as described in any one of items 1 to 17 or a composition as described in any one of items 18 to 21. [Item 23] The method according to Item 22, comprising administering the cells to the central nervous system via the internal jugular vein or hepatic portal vein of the mammal. [Item 24] The method according to Item 22 or 23, wherein the insulin resistance state includes type 1 diabetes. [Item 25] The method according to Item 22 or 23, wherein the insulin resistance state includes type 2 diabetes. [Item 26] The method according to any one of Items 22 to 25, wherein the non-porcine mammal has received an induction regime comprising a therapeutically effective dose of anti-thymocyte globulin, anti-CD40 antibody, anti-CD20 antibody, rapalogue, calcineurin inhibitor, ganciclovir or its prodrug, antihistamine and corticosteroid before administration of the transgenic porcine pancreatic islet cells or the composition. [Item 27] ​​The method according to any one of items 22 to 26, comprising administering a therapeutically effective dose of an anti-CD40 antibody, a rapalog, a calcineurin inhibitor, and ganciclovir or a prodrug thereof after administration of the transgenic porcine pancreatic islet cells or the composition. [Item 28] The method according to any one of items 22 to 27, comprising administering a therapeutically effective dose of an intermediate-acting or long-acting insulin analog, insulin glargine, insulin detemir, or NPH insulin after administration of the transgenic porcine pancreatic islet cells or the composition. [Item 29] The method according to any one of Items 22 to 28, wherein the therapeutically effective dose is at least 5,000 IEQ per kg of body weight of a non-porcine mammal. [Item 30] Isolated porcine pancreatic islets containing isolated porcine pancreatic islet cells as described in any one of items 1-17. [Item 31] Isolated porcine pancreatic organoids containing isolated porcine pancreatic islet cells as described in any one of items 1-17. [Item 32] Isolated porcine pancreatic islets or isolated porcine pancreatic organoids according to Item 30 or 31, wherein the pancreatic islets or organoids substantially do not contain pancreatic exocrine cells. [Item 33] The pancreatic organoids are as follows: (a) Isolating the pancreas from neonatal piglets before the 7th day of neonatal life, and (b) Subjecting the pancreas to mechanical or enzymatic digestion to produce organoid fragments, and optionally, (c) Isolated porcine pancreatic organoids as described in item 31 or 32, prepared by purifying the organoid fragments of step (b) by Ficol gradient sedimentation. [Item 34] Isolated porcine pancreas containing porcine islet cells as described in any one of items 1-17. [Item 35] A method for improving the yield of pancreatic islets from a pig donor before transplantation to a non-porcine mammalian recipient, the following: (a) To provide pancreatic organoids from newly born piglets that have undergone a purification procedure, (b) Culturing the organoids in the presence of an effective concentration of a caspase inhibitor for at least 90 minutes after purification, and (c) Continue culturing for at least 7 days in the presence of an effective concentration of corticosteroids. A method that includes this. [Item 36] The purification procedure described above is (a) Isolating the pancreas from transgenic newborn piglets before the 7th day of neonatal life, and (b) Subjecting the pancreas to mechanical or enzymatic digestion to produce organoid fragments, and optionally, (c) Purification of organoid fragments from the digested pancreas by Ficol gradient sedimentation. The method described in item 35, including the method described in item 35. [Item 37] The method according to Item 35 or 36, wherein the newborn piglet is a transgenic pig comprising at least one porcine cell as described in any one of Items 1 to 17. [Item 38] The method according to any one of Items 35 to 37, wherein the caspase inhibitor is Z-VAD-FMK. [Item 39] The method according to any one of items 35 to 38, wherein the corticosteroid is methylprednisolone. [Item 40] The method according to any one of items 35-39, wherein the pancreatic organoid is cultured in the presence of IBMX, a phosphodiesterase inhibitor, or an adenosine receptor antagonist. [Item 41] The method according to any one of items 35 to 40, wherein the pancreatic organoid is cultured in the presence of nicotinamide or a metabolically acceptable analog thereof. [Item 42] A method for treating insulin resistance or deficiency in a non-porcine mammal in need thereof, wherein the organoid described in any one of items 35 to 41 is used according to the following criteria (a) Endotoxins less than approximately 5 EU / kg (b) Negative Gram staining, (c) A survival rate of over approximately 70%, or (d) Pancreatic islet concentration of approximately 20,000 IEQ / mL or more in total sedimentation A method comprising transplanting into a non-porcine mammal if any of the following conditions are met.

Claims

1. Isolated transgenic porcine pancreatic islet cells, (a) substantially lacking the enzymatic activity of at least one glycosyltransferase enzyme Here, the glycosyltransferase enzyme is GGTA, B4GALNT2 or It is CMAH, (b) Expressing at least two polypeptide sequences derived from a non-porcine mammalian species, where, Note: At least two polypeptide sequences are CD46, CD55, CD59, THBD, T FPI, CD39, B2M, HLAE, CD47, A20, PD-L1, FASL or H It includes at least two of O-1, and (c) Reduction of toxicity by complement derived from non-porcine mammalian species, Reduction of induction of activated protein C coagulation, thrombin-anti derived from non-porcine mammalian species One of the following: reduction of thrombin complex induction or reduction of toxicity by NK cells derived from non-porcine species. The above shows isolated transgenic porcine pancreatic islet cells.

2. The cells are selected from at least two of GGTA, B4GALNT2, and CMAH. Or, claim, the enzyme has substantially no enzymatic activity of all three glycosyltransferase enzymes. Transgenic porcine pancreatic islet cells as described in item 1.

3. The aforementioned cells are CD46, CD55, CD59, THBD, TFPI, CD39, B2M , HLAE, CD47, A20, PD-L1, FASL, or HO-1, at least three of these, At least four, at least five, at least six, at least seven, at least eight, At least nine, at least ten, at least eleven, at least twelve, or all of them Transgenic porcine pancreatic islet cells as described in claim 1 or 2.

4. Isolated transgenic porcine pancreatic islet cells, (a) Substantially lacking the enzymatic activity of at least two glycosyltransferase enzymes Here, the glycosyltransferase enzyme is GGTA, B4GALNT2 or Includes at least two CMAHs, (b) Expressing a polypeptide sequence derived from a non-porcine mammalian species, where the polypeptide sequence The columns are CD46, CD55, CD59, THBD, TFPI, CD39, B2M, HLA E, CD47, A20, PD-L1, FASL, or HO-1, and (c) Reduction of toxicity by complement derived from non-porcine mammalian species, activation of non-porcine species Reduction of rotin C coagulation induction, the non-porcine-derived thrombin-antithrombin complex The isolated cells demonstrate reduced induction or reduced toxicity by the non-porcine-derived NK T cells. Transgenic porcine pancreatic islet cells.

5. The aforementioned cells substantially lack the enzymatic activity of GGTA, B4GALNT2, and CMAH. The transgenic porcine pancreatic islet cells according to claim 4.

6. The aforementioned cells are CD46, CD55, CD59, THBD, TFPI, CD39, B2M , HLAE, CD47, A20, PD-L1, FASL or HO-1, at least two of these, At least three, at least four, at least five, at least six, at least seven, at least 8, at least 9, at least 10, at least 11, at least 1 Transgenic porcine pancreatic islet cells according to claim 4 or 5, expressing two or all of the cells.

7. It expresses CD46, CD55, CD59, CD39, B2M, HLAE, and CD47. or transgenic porcine pancreatic islet cells according to any one of claims 1 to 6.

8. The cells substantially express the one or more glycosyltransferase enzymes. Transgenic porcine pancreatic islet cells according to any one of claims 1 to 6, which are not possessed.

9. The cell is frame in one or more glycosyltransferase enzymes This includes a shift mutation that causes premature termination of translation, thereby altering the glycosyltrans Transgenic porcine pancreatic islet cells according to claim 8, wherein the activity of the ferase enzyme is lost.

10. One or more polypeptide sequences encoding one or more polypeptide sequences derived from non-porcine mammalian species Claims 1 to 1, in which a number of nucleic acid sequences are inserted into the non-orthologous locus of a butorsolog. Transgenic porcine pancreatic islet cells as described in any one of item 9.

11. One or more polypeptide sequences encoding one or more polypeptide sequences derived from non-porcine mammalian species Nucleic acid sequences are operably linked to a non-orthologous promoter of a butorsolog. transgenic porcine pancreatic islet cells according to any one of claims 1 to 10.

12. The non-orthologous promoter is a non-porcine promoter, according to claim 11. Transgenic porcine pancreatic islet cells.

13. The pancreatic islet cells are obtained by deaggregation of porcine pancreas, any one of claims 1 to 12. Transgenic porcine pancreatic islet cells as described.

14. The aforementioned pancreatic islet cells include alpha cells, beta cells, delta cells, epsilon cells, and pancreatic polyps. Peptide (PP) cells or any combination thereof, any one of claims 1 to 13 Transgenic porcine pancreatic islet cells as described in item 1.

15. The non-porcine mammal species is a primate species, according to any one of claims 1 to 14. Lancegenic porcine pancreatic islet cells.

16. If the aforementioned cells are transplanted into the aforementioned non-porcine mammalian species, they will survive for more than 8 days. Transgenic porcine pancreatic islet cells as described in any one of the requirements 1 to 15.

17. The aforementioned cells showed reduced IBMI compared to PBMCs isolated from the aforementioned non-porcine mammalian species. Transgenic pig cells according to any one of claims 1 to 16, wherein R is represented.

18. A therapeutically effective amount comprising isolated porcine pancreatic islet cells according to any one of claims 1 to 17 composition.

19. Claim 18 further comprises heparin or a TNF-alpha inhibitor. The composition described.

20. At least approximately 12% to 25% beta cells or at least approximately 15% to 30% A The composition according to claim 18 or 19, comprising Fa cells.

21. Claims 18-2, wherein the composition is prepared according to any one of claims 35-42. The composition according to any one of the items 0.

22. Treatment for insulin resistance or deficiency in non-porcine mammals requiring it. A method comprising, a therapeutically effective dose, of the isolated tortoise described in any one of claims 1 to 17. Lancegenic porcine pancreatic islet cells or the composition according to any one of claims 18 to 21 A method including administration to a mammal.

23. This includes administering the cells to the central nervous system via the internal jugular vein or hepatic portal vein of the mammal. The method described in item 22.

24. The method according to claim 22 or 23, wherein the insulin resistance state includes type 1 diabetes.

25. The method according to claim 22 or 23, wherein the insulin resistance state includes type 2 diabetes.

26. The non-porcine mammal is administered the transgenic porcine pancreatic islet cells or the composition. Before administering the therapeutically effective dose of anti-thymocyte globulin, anti-CD40 antibody, anti-CD20 antibody, and laparoscopic thymocyte globulin, anti-CD40 antibody, anti-CD20 antibody, laparoscopic thymocyte globulin, anti-CD40 antibody, anti-CD20 antibody, anti-thymocyte globulin, anti-CD40 antibody Log, calcineurin inhibitors, ganciclovir or its prodrugs, antihistamines and has received an induction regimen including a corticosteroid, any one of claims 22 to 25 The method described in item 1.

27. After administration of the transgenic porcine pancreatic islet cells or the composition, a therapeutically effective dose of anti-CD cells is administered. 40 antibodies, rapalogues, calcineurin inhibitors, and ganciclovir or its prodrugs The method according to any one of claims 22 to 26, comprising administering the following:

28. After administration of the transgenic porcine pancreatic islet cells or the composition, an intermediate dose of the therapeutically effective dose is administered. Insulin analogs for short-acting or long-acting insulin, insulin glargine, insulin detemir or The method according to any one of claims 22 to 27, which includes administering NPH insulin. Law.

29. The effective therapeutic dose is at least 5,000 IEQ per kg of body weight of a non-porcine mammal. The method according to any one of claims 22 to 28.

30. Isolated porcine pancreatic islet cells comprising isolated porcine pancreatic islet cells according to any one of claims 1 to 17 Pancreatic islets.

31. Isolated porcine pancreatic islet cells comprising isolated porcine pancreatic islet cells according to any one of claims 1 to 17 Pancreatic organoid.

32. Claim 30 or 3, wherein the pancreatic islets or organoids substantially do not contain pancreatic exocrine cells. Isolated porcine pancreatic islets or isolated porcine pancreatic organoids as described in 1.

33. The aforementioned pancreatic organoids are as follows: (a) Isolating the pancreas from neonatal piglets before the 7th day of neonatal life, and (b) Subjecting the pancreas to mechanical or enzymatic digestion to produce organoid fragments, Furthermore, by optional choice, (c) Purify the organoid fragments from step (b) by Ficol gradient sedimentation. An isolated porcine pancreatic organoid according to claim 31 or 32, prepared by...

34. Isolated porcine pancreas containing porcine islet cells according to any one of claims 1 to 17.

35. Methods to improve the yield of pancreatic islets from pig donors before transplantation into non-porcine mammalian recipients. And, as follows: (a) To provide pancreatic organoids from newly born piglets that have undergone a purification procedure, (b) In the presence of an effective concentration of a caspase inhibitor, for at least 90 minutes after the purification To culture the organoids over time, and (c) Continue culturing for at least 7 days in the presence of an effective concentration of corticosteroids. to do A method that includes this.

36. The aforementioned purification procedure, (a) Isolating the pancreas from transgenic newborn piglets before the 7th day of neonatal life, and (b) to subject the pancreas to mechanical or enzymatic digestion to produce organoid fragments, By choice, (c) Organoid fragments are purified from the digested pancreas by Ficol gradient sedimentation. thing The method according to claim 35, including the method described in claim 35.

37. The newborn piglet is at least one pig according to any one of claims 1 to 17 The method according to claim 35 or 36, wherein the transgenic pig contains cells.

38. The caspase inhibitor is Z-VAD-FMK, as per any one of claims 35 to 37. The method described in section [section number].

39. Any of claims 35 to 38, wherein the corticosteroid is methylprednisolone. The method described in item 1.

40. The pancreatic organoid is an IBMX, phosphodiesterase inhibitor, or adenosine receptor The method according to any one of claims 35 to 39, cultured in the presence of a somatic antagonist. 。

41. The pancreatic organoid, in the presence of nicotinamide or a metabolically acceptable analog thereof The method according to any one of claims 35 to 40, wherein the culture is performed in [a specific location].

42. Treatment for insulin resistance or deficiency in non-porcine mammals requiring it. A method wherein the organoid described in any one of claims 35 to 41 is used in the organ Noid meets the following criteria (a) Endotoxins less than approximately 5 EU / kg (b) Negative Gram staining, (c) A survival rate of over 70%, or (d) Pancreatic islet concentration of approximately 20,000 IEQ / mL or more in total sedimentation A method comprising transplanting into a non-porcine mammal if any of the following conditions are met.

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