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Background: Pig islet xenotransplantation is a potential treatment for type 1 diabetes. We have shown that maintenance immunosuppression is required to protect genetically modified (GM) porcine islet xenografts from T cell‐mediated rejection in baboons. Local expression of a depleting anti‐CD2 monoclonal antibody (mAb) by the xenograft may provide an alternative solution. We have previously reported the generation of GGTA1 knock‐in transgenic pigs expressing the chimeric anti‐CD2 mAb diliximab under an MHC class I promoter (MHCIP). In this study, we generated GGTA1 knock‐in pigs in which MHCIP was replaced by the β‐cell‐specific porcine insulin promoter (PIP), and compared the pattern of diliximab expression in the two lines. Methods: A PIP‐diliximab knock‐in construct was prepared and validated by transfection of NIT‐1 mouse insulinoma cells. The construct was knocked into GGTA1 in wild type (WT) porcine fetal fibroblasts using CRISPR, and knock‐in cells were used to generate pigs by somatic cell nuclear transfer (SCNT). Expression of the transgene in MHCIP‐diliximab and PIP‐diliximab knock‐in pigs was characterised at the mRNA and protein levels using RT‐qPCR, flow cytometry, ELISA and immunohistochemistry. Islets from MHCIP‐diliximab and control GGTA1 KO neonatal pigs were transplanted under the kidney capsule of streptozotocin‐diabetic SCID mice. Results: NIT‐1 cells stably transfected with the PIP‐diliximab knock‐in construct secreted diliximab into the culture supernatant, confirming correct expression and processing of the mAb in β cells. PIP‐diliximab knock‐in pigs showed a precise integration of the transgene within GGTA1. Diliximab mRNA was detected in all tissues tested (spleen, kidney, heart, liver, lung, pancreas) in MHCIP‐diliximab pigs, but was not detectable in PIP‐diliximab pigs. Likewise, diliximab was present in the serum of MHCIP‐diliximab pigs, at a mean concentration of 1.8 μg/mL, but was not detected in PIP‐diliximab pig serum. An immunohistochemical survey revealed staining for diliximab in all organs of MHCIP‐diliximab pigs but not of PIP‐diliximab pigs. Whole genome sequencing (WGS) of a PIP‐diliximab pig identified a missense mutation in the coding region for the dixilimab light chain. This mutation was also found to be present in the fibroblast knock‐in clone used to generate the PIP‐diliximab pigs. Islet xenografts from neonatal MHCIP‐diliximab pigs restored normoglycemia in diabetic immunodeficient mice, indicating no overt effect of the transgene on islet function, and demonstrated expression of diliximab in situ. Conclusion: Diliximab was widely expressed in MHCIP‐diliximab pigs, including in islets, consistent with the endogenous expression pattern of MHC class I. Further investigation is required to determine whether the level of expression in islets from the MHCIP‐diliximab pigs is sufficient to prevent T cell‐mediated islet xenograft rejection. The unexpected absence of diliximab expression in the islets of PIP‐diliximab pigs was probably due to a mutation in the transgene arising during the generation of the knock‐in cells used for SCNT. [ABSTRACT FROM AUTHOR]

Intrahepatic islet transplantation for type 1 diabetes is limited by the need for multiple infusions and poor islet viability posttransplantation. The development of alternative transplantation sites is necessary to improve islet survival and facilitate monitoring and retrieval. We tested a clinically proven biodegradable temporizing matrix (BTM), a polyurethane-based scaffold, to generate a well-vascularized intracutaneous "neodermis" within the skin for islet transplantation. In murine models, BTM did not impair syngeneic islet renal-subcapsular transplant viability or function, and it facilitated diabetes cure for over 150 days. Furthermore, BTM supported functional neonatal porcine islet transplants into RAG-1−/− mice for 400 days. Hence, BTM is nontoxic for islets. Two-photon intravital imaging used to map vessel growth through time identified dense vascular networks, with significant collagen deposition and increases in vessel mass up to 30 days after BTM implantation. In a preclinical porcine skin model, BTM implants created a highly vascularized intracutaneous site by day 7 postimplantation. When syngeneic neonatal porcine islets were transplanted intracutaneously, the islets remained differentiated as insulin-producing cells, maintained normal islet architecture, secreted c-peptide, and survived for over 100 days. Here, we show that BTM facilitates formation of an islet-supportive intracutaneous neodermis in a porcine preclinical model, as an alternative islet-transplant site. Article Highlights: Human and porcine pancreatic islets were transplanted into a fully vascularized biodegradable temporizing matrix (Novosorb) that creates a unique intracutaneous site outside of the liver in a large-animal preclinical model. The intracutaneous prevascularized site supported pancreatic islet survival for 3 months in a syngeneic porcine-transplant model. Pancreatic (human and porcine) islet survival and function were demonstrated in an intracutaneous site outside of the liver for the first time in a large-animal preclinical model. [ABSTRACT FROM AUTHOR]

Transplantation of human embryonic stem cell (ESC) derived beta‐like cells and xenotransplantation of porcine islets are two potential cures for type 1 diabetes (T1D). We have previously reported the isolation of porcine ESCs and have also separately shown that islets from genetically modified pigs can effectively cure diabetes in a preclinical baboon model. Here we discuss the possibility of combining these technologies to produce gene‐edited porcine ESC‐derived islets as an alternative for treating T1D, which may offer several advantages compared with these approaches. [ABSTRACT FROM AUTHOR]

Xenotransplantation using porcine donors is rapidly approaching clinical applicability as an alternative therapy for treatment of many end-stage diseases including type 1 diabetes. Porcine neonatal islet cell clusters (NICC) have normalised blood sugar levels for relatively short periods in the preclinical diabetic rhesus model but have met with limited success in the stringent baboon model. Here we report that NICC from genetically modified (GM) pigs deleted for αGal and expressing the human complement regulators CD55 and CD59 can cure diabetes long-term in immunosuppressed baboons, with maximum graft survival exceeding 22 months. Five diabetic baboons were transplanted intraportally with 9,673 – 56,913 islet equivalents (IEQ) per kg recipient weight. Immunosuppression consisted of T cell depletion with an anti-CD2 mAb, tacrolimus for the first 4 months, and maintenance with belatacept and anti-CD154; no anti-inflammatory treatment or cytomegalovirus (CMV) prophylaxis/treatment was given. This protocol was well tolerated, with all recipients maintaining or gaining weight. Recipients became insulin-independent at a mean of 87 ± 43 days post-transplant and remained insulin-independent for 397 ± 174 days. Maximum graft survival was 675 days. Liver biopsies showed functional islets staining for all islet endocrine components, with no evidence of the inflammatory blood-mediated inflammatory reaction (IBMIR) and minimal leukocytic infiltration. The costimulation blockade-based immunosuppressive protocol prevented an anti-pig antibody response in all recipients. In conclusion, we demonstrate that genetic modification of the donor pig enables attenuation of early islet xenograft injury, and in conjunction with judicious immunosuppression provides excellent long-term function and graft survival in the diabetic baboon model. [ABSTRACT FROM AUTHOR]

Machine learning (ML)-workflows enable unprejudiced/robust evaluation of complex datasets. Here, we analyzed over 490,000,000 data points to compare 10 different ML-workflows in a large (N=11,652) training dataset of human pancreatic single-cell (sc-)transcriptomes to identify genes associated with the presence or absence of insulin transcript(s). Prediction accuracy/sensitivity of each ML-workflow was tested in a separate validation dataset (N=2,913). Ensemble ML-workflows, in particular Random Forest ML-algorithm delivered high predictive power (AUC=0.83) and sensitivity (0.98), compared to other algorithms. The transcripts identified through these analyses also demonstrated significant correlation with insulin in bulk RNA-seq data from human islets. The top-10 features, (including IAPP, ADCYAP1, LDHA and SST) common to the three Ensemble ML-workflows were significantly dysregulated in scRNA-seq datasets from Ire-1αβ-/- mice that demonstrate dedifferentiation of pancreatic β-cells in a model of type 1 diabetes (T1D) and in pancreatic single cells from individuals with type 2 Diabetes (T2D). Our findings provide direct comparison of ML-workflows in big data analyses, identify key elements associated with insulin transcription and provide workflows for future analyses. [ABSTRACT FROM AUTHOR]

We have analysed insulin antibodies in 149 adults with type 1 diabetes and 2859 people without diabetes. We have determined that insulin antibody levels are higher in adults with, versus without, diabetes and that the levels are falling, and more patients are becoming antibody‐negative post islet cell transplantation. [ABSTRACT FROM AUTHOR]

Islet transplantation is an emerging therapy for type 1 diabetes and hypoglycemic unawareness. However, a key challenge for islet transplantation is cellular rejection and the requirement for long-term immunosuppression. In this study, we established a diabetic humanized NOD-scidIL2Rγnull (NSG) mouse model of T-cell-mediated human islet allograft rejection and developed a therapeutic regimen of low-dose recombinant human interleukin-2 (IL-2) combined with low-dose rapamycin to prolong graft survival. NSG mice that had received renal subcapsular human islet allografts and were transfused with 1 × 107 of human spleen mononuclear cells reconstituted human CD45+ cells that were predominantly CD3+ T cells and rejected their grafts with a median survival time of 27 days. IL-2 alone (0.3 × 106 IU/m2 or 1 × 106 IU/m2) or rapamycin alone (0.5-1 mg/kg) for 3 weeks did not prolong survival. However, the combination of rapamycin with IL-2 for 3 weeks significantly prolonged human islet allograft survival. Graft survival was associated with expansion of CD4+CD25+FOXP3+ regulatory T cells (Tregs) and enhanced transforming growth factor-β production by CD4+ T cells. CD8+ T cells showed reduced interferon-γ production and reduced expression of perforin-1. The combination of IL-2 and rapamycin has the potential to inhibit human islet allograft rejection by expanding CD4+FOXP3+ Tregs in vivo and suppressing effector cell function and could be the basis of effective tolerance-based regimens. [ABSTRACT FROM AUTHOR]

Simultaneous pancreas-kidney (SPK) transplantation remains the most effective treatment for providing consistent and long-term euglycemia in patients having type 1 diabetes with renal failure. Thrombosis of the pancreatic vasculature continues to contribute significantly to early graft failure and loss. We compared the rate of thrombosis to graft loss and systematically reviewed risk factors impacting early thrombosis of the pancreas allograft following SPK transplantation. We searched the MEDLINE, EMBASE, The Cochrane Library, and PREMEDLINE databases for studies reporting thrombosis following pancreas transplantation. Identified publications were screened for inclusion and synthesized into a data extraction sheet. Sixty-three studies satisfied eligibility criteria: 39 cohort studies, 22 conference abstracts, and 2 meta-analyses. Newcastle-Ottawa Scale appraisal of included studies demonstrated cohort studies of low bias risk; 1127 thrombi were identified in 15 936 deceased donor, whole pancreas transplants, conferring a 7.07% overall thrombosis rate. Thrombosis resulted in pancreatic allograft loss in 83.3% of reported cases. This review has established significant associations between donor and recipient characteristics, procurement and preservation methodology, transplantation technique, postoperative management, and increased risk of early thrombosis in the pancreas allograft. Further studies examining the type of organ preservation fluid, prophylactic heparin protocol, and exocrine drainage method and early thrombosis should also be performed. [ABSTRACT FROM AUTHOR]

Understanding the immunological phenotype of transplant recipients is important to improve outcomes and develop new therapies. Immunophenotyping of whole peripheral blood (WPB) by flow cytometry is a rapid method to obtain large amounts of data relating to the outcomes of different transplant treatments with limited patient impact. Healthy individuals and patients with type 1 diabetes (T1D) enrolled in islet transplantation were recruited and WPB was collected. 46 fluorochrome-conjugated mouse-anti-human antibodies were used (43 of 46 antibodies were titrated). BD cytometer setup and tracking beads were used to characterize and adjust for cytometer performance. Antibody cocktails were pre-mixed <60 minutes before staining. Multicolour panels were designed based on fluorochrome brightness, antigen density, co-expression, and fluorochrome spillover into non-primary detectors in each panel on a 5 laser flow cytometer. WPB sample staining used 50–300 μl WPB for each panel and was performed within 2 hours of blood sample collection. Samples were acquired on a BD-LSRFortessa. The operating procedures, including specimen collection, antibody cocktails, staining protocol, flow-cytometer setup and data analysis, were standardized. The staining index of 43 antibodies and the spillover spreading matrix for each panel was calculated. The final concentrations for the 46 antibodies used was determined for staining of WPB samples. Absolute cell-count and 7 leukocyte profiling panels consisting of subsets and/or status of granulocytes, monocytes, dendritic, B, NK, and T cells including regulatory T cells (Tregs) and NKT were designed and established on a 5 laser BD-LSR Fortessa. 13 T1D patients, including 4 islet transplant recipients and 8 healthy controls, were evaluated. The ability to reproducibly measure immune subsets and immune-profiles of islet transplant patients up to 18 months post transplantation has been established as a tool to measure immune cell reconstitution after transplantation. [ABSTRACT FROM AUTHOR]

In the 2009 IXA consensus, the requirements for the quality and control of manufacturing of porcine islet products were based on the U.S. regulatory framework where the porcine islet products fall within the definition of somatic cell therapy under the statutory authority of the U.S. Food and Drug Administration ( FDA). In addition, porcine islet products require pre-market approval as a biologic product under the Public Health Services Act and they meet the definition of a drug under the Federal Food, Drug, and Cosmetic Act ( FD&C Act). Thus, they are subject to applicable provisions of the law and as such, control of manufacturing as well as reproducibility and consistency of porcine islet products, safety of porcine islet products, and characterization of porcine islet products must be met before proceeding to clinical trials. In terms of control of manufacturing as well as reproducibility and consistency of porcine islet products, the manufacturing facility must be in compliance with current Good Manufacturing Practices ( cGMP) guidelines appropriate for the initiation of Phase 1/2 clinical trials. Sponsors intending to conduct a Phase 1/2 trial of islet xenotransplantation products must be able to demonstrate the safety of the product through the establishment of particular quality assurance and quality control procedures. All materials (including animal source and pancreas) used in the manufacturing process of the porcine islet products must be free of adventitious agents. The final porcine islet product must undergo tests for the presence of these adventitious agents including sterility, mycoplasma (if they are cultured), and endotoxin. Assessments of the final product must include the safety specifications mentioned above even if the results are not available until after release as these data would be useful for patient diagnosis and treatment if necessary. In addition, a plan of action must be in place for patient notification and treatment in case the sterility culture results are positive. In terms of the characterization of porcine islet products and product release criteria, the information on the porcine islet products should be acquired from a sample of the final product to be used for transplantation and must include the morphology of the islets, specific identity, purity, viability, and potency of the product. In addition, information on the quantity of the islet products should also be provided in a standardized fashion and this should be in terms of islet equivalents and/or cell numbers. The current consensus was created to provide guidelines that manufacturing facilities may find helpful in the manufacture of and the release criteria for porcine islet products including encapsulated islets and combined islet products. Our intent with the above recommendations is to provide a framework for individual porcine islet manufacturing facilities to ensure a high level of safety for the initiation of Phase 1/2 clinical trials on porcine islet xenotransplantation. [ABSTRACT FROM AUTHOR]

Cell therapy for Type 1 diabetes (T1D) utilizing islet cell transplantation can successfully restore endogenous insulin production in affected patients. Islet cell engraftment and survival are conditional on the use of efficacious anti-rejection therapies and on the availability of healthy donor cells. The scarcity of healthy human donor pancreata is a limiting factor in providing sufficient tissue to meet the demand for islet transplantation worldwide. A potential alternative to the use of cadaveric human donor pancreases is the use of animal sourced islets. Pancreatic islets obtained from pigs have emerged as an alternative to human tissues due to their great availability, physiological similarities to human islets, including the time-tested use of porcine insulin in diabetic patients and the ability to genetically modify the donor source. The evolution of refined, efficacious immunosuppressive therapies with reduced toxicity, improvements in donor management and genetic manipulation of the donor have all contributed to facilitate long-term function in pre-clinical models of pig islet grafts in non-human primates. As clinical consideration for this option is growing, and trials involving the use of porcine islets have begun, more compelling experimental data suggest that the use of pig islets may soon become a viable, safe, effective and readily available treatment for insulin deficiency in T1D patients. [ABSTRACT FROM AUTHOR]

Background Porcine neonatal islet-like cell clusters ( NICC) are being considered as a source of β-cell replacement. However, the lag time to full function due to hormonal immaturity remains a problem. This study aimed to determine whether time in culture was important for NICC function in vivo. Methods Neonatal islet-like cell clusters were isolated from piglets aged between 1 and 3 days, and cultured for up to 27 days post-isolation. Each week, NICC number, viability, and function were determined. Results Neonatal islet-like cell clusters cultured for 12, 19, and 27 days achieved normal blood glucose levels at 46 days (85% of animals), 32 days (100% of animals), and 35 days (81% of animals), respectively. By comparison, standard 6-day culture took a mean of 63 days to achieve normoglycemia in 35% of animals. Longer time in culture resulted in a significant loss of islet equivalent over time. However, insulin gene expression levels were significantly higher at days 12, 19, 27 compared to day 6. Glucagon gene expression was highest at day 12, and significantly higher than day 6 at all time points. Bcl-2 gene expression increased over time, and tissue factor ( TF) gene expression was highest on day 6 and then decreased over the remaining time points. Conclusion Culture of NICC for 12 days provides the best balance in vivo functional outcome for transplantation, shown by better reversal of diabetes, and higher levels of gene expression for insulin, glucagon and Bcl-2 and lower levels of TF expression with acceptable NICC number loss in terms of time and expense. [ABSTRACT FROM AUTHOR]

Beta cell replacement therapy has been proposed as a novel therapy for the treatment of type 1 diabetes. The proof of concept has been demonstrated with successful islet allotransplantation. Islet xenotransplantation has been proposed as an alternative, more reliable, and infinite source of beta cells. The advantages of islet xenotransplantation are the ability to transplant a well differentiated cell that is responsive to glucose and the potential for genetic modification which focuses the treatment on the donor rather than the recipient. The major hurdle remains overcoming the severe cellular rejection that affects xenografts. This review will focus on the major advances that have occurred with genetic modification and the successful therapeutic strategies that have been demonstrated in nonhuman primates. Novel approaches to overcome cell-mediated rejection including biological agents that target selectively costimulation molecules, the development of local immunosuppression through genetic manipulation, and encapsulation will be discussed. Overall, there has been considerable progress in all these areas, which eventually should lead to clinical trials. [ABSTRACT FROM AUTHOR]

The development of autoimmune disease type 1 diabetes (T1D) is determined by both genetic background and environmental factors. Environmental triggers include RNA viruses, particularly coxsackievirus (CV), but how they induce T1D is not understood. Here, we demonstrate that deletion of the transcription factor hypoxia-inducible factor-1α (HIF-1α) from β cells increases the susceptibility of non-obese diabetic (NOD) mice to environmentally triggered T1D from coxsackieviruses and the β cell toxin streptozotocin. Similarly, knockdown of HIF-1α in human islets leads to a poorer response to coxsackievirus infection. Studies in coxsackievirus-infected islets demonstrate that lack of HIF-1α leads to impaired viral clearance, increased viral load, inflammation, pancreatitis, and loss of β cell mass. These findings show an important role for β cells and, specifically, lack of β cell HIF-1α in the development of T1D. These data suggest new strategies for the prevention of T1D. • Type 1 diabetes is increasing worldwide, which must be due to environmental changes • Lack of β cell HIF1a increases risk of T1D after viral infection • β Cell HIF1a also decreases T1D after low doses of the β cell toxin streptozotocin • β Cell HIF1a is a major factor in determining whether insult leads to T1D or resolution Lalwani et al. describe a role for β cell hypoxia-inducible factor-1α (HIF1a) in determining whether β cell injury is followed by resolution and normal function or ongoing injury, autoimmunity, and type 1 diabetes. [ABSTRACT FROM AUTHOR]