Surface Camouflaged and Genetically Engineered Pancreatic Islet Transplantation for the Treatment of Diabetes Mellitus

Abstract

Exogenous insulin therapy is currently used for the treatment of type 1 diabetes mellitus. However, insulin therapy induces unwanted complications such as hypoglycemia, allergy reaction, and so on. Recently, pancreatic islet transplantation is a promising strategy to treat type 1 diabetes, as it can achieve strict regulation of blood glucose level. However, the limited availability of cadaveric pancreas, donor shortage, immune rejection and autoimmunity are the major hurdles for successful islet transplantation. Especially, transplanted islets cell antigens are recognized by antibodies from recipients and it is the most difficult barrier to firm faces for successful islet transplantation. To overcome this problem, The 'Edmonton protocol' that is cocktailed immunosuppressive drug therapy has been used from 2000. Although known as one of the best accepted protocols outlined for islet transplantation in clinic to date, has shown lower than 10% only of its recipients achieving insulin independence ratio after 5 years of transplantation. Thus, a new immunoprotective remedy has been in needs to inhibit host immune reaction. In this study, islet donor shortage is one hurdles revealed from the past islet transplantation studies. At least 2-3 cadaveric pancreases were needed for treatment of one diabetic patients using islet transplantation. Recently, stem cell therapy and porcine islets transplantation have been accessed to solve this problem. However, it takes much time to apply these technologies to clinical study. Therefore, this study assessed that the newly designed exendin-4 (Ex-4) gene with highly releasable characteristics could enhance the beta cell function, thereby attenuating the essential islet mass required to cure diabetes. We constructed a lentiviral vector system encoding for a highly releasable secretion signal peptide, the peptide linked Ex-4 (SP-Ex-4) gene. After the transduction of lentivirus encoding for SP-Ex-4 (LV-SP-Ex-4) gene into the islets, the rate of insulin secretion was three-fold increased. In addition, 50 islets expressing Ex-4 were transplanted to cure the diabetic nude mice, whereas at least 150 untransduced islets had to be transplanted to cure the diabetic nude mice. When the transplanted islets were transplanted into diabetic immunocompetent mice, the survival time of islets was 18.0 ± 4.9 days

however, when the untransduced islets were transplanted, they were rejected within 10.0 ± 0.6 days. Therefore, the highly releasable Ex-4 could enhance the beta cell function with slightly enhanced viability of transplanted islets, presenting as a potential technology for overcoming islet shortage. Host immune reaction is another blockade for successful islet transplantation. Pancreatic islets were camouflaged with multi-branched PEG (6-arm-PEG-catechol) for minimizing of host immune reaction. It effectively inhibited xenogeneic immune reaction, because 6-arm-PEG-catechol could be more highly packed on the islet surface compared to linear PEG. Six-arm-PEG-catechol was covered the whole area of islet surface and surface camouflage technology did not affect the viability and functionality of islets. In addition, the synergistic effects of surface camouflage on immunoprotection for transplanted islets with low doses of immunosuppressive drugs, such as tacrolimus and anti-CD154 mAb, were established in the xenotransplantation model. When the recipients of 6-arm-PEG-catechol grafted islets were injected with 0.2 mg/kg of tacrolimus and 0.1 mg/mouse of anti-CD154 mAb, normal glucose level was maintained up to 50 days of transplantation without any fluctuation of glucose level. Therefore, a newly developed protocol using 6-arm-PEG-catechol with tacrolimus and anti-CD154 mAb would certainly be an effective combination therapy for the treatment of type 1 diabetes. To investigate the inhibition effects of pancreatic islet transplantation on the progression of obese type 2 diabetes, we analyzed the effects of surface camouflaged islet transplantation on delaying the disease progression in a db/db diabetic mouse model. Surface camouflaged islets using 6-arm-PEG-catechol were transplanted in db/db type 2 diabetic mice. The fat accumulation and toxicity in the liver, the expansion of islets in the pancreas, and the size change of abdominal adipocyte were analyzed. In addition, the blood glucose control, insulin levels and immunohistochemical staining of recovered tissues were analyzed after transplantation. Then co-administration of anti-CD154 mAb and tacrolimus (IT group) deterred the pathophysiological progression of obese type 2 diabetes. At day 3 of transplantation, the serum insulin concentration of IT group was increased compared to the control group, with the euglycemic control. The immunohistochemical studies demonstrated that the mass of 6-arm-PEG-catechol grafted islet was preserved in the transplantation site for 14 days

however, the intensity of insulin staining and serum insulin level was decreased with time. Therefore, transplantation of 6-arm-PEG-catechol grafted islets in the kidney capsule of db/db diabetic mice prevented the progression of obese type 2 diabetes and reduced the blood glucose level when immunosuppressive drugs were co-administered. Transplanted islets were eventually rejected by host immune reaction with time. Thus, repetitive islet transplantation is needed to treat type 1 diabetic patients experiencing graft rejection. The secondly transplanted islets might be rapidly rejected due to sensitized immune reaction of the humoral and cellular immunities induced by the first transplanted islets. Thus, we explored whether the incorporation of PEG on the surface of the islets can be an affordable immunoprotective remedy for repeated islet transplantation. Unmodified islets transplanted in combination with cyclosporin A (CsA) and anti-CD4 mAb (OX-38) into the sensitized recipients did not maintain a normal glucose level over 20 days. However, three of the five recipients became normoglycemic up to 30 days when PEGylated islets were transplanted in combination with CsA and anti-CD4 mAb. These results demonstrated that PEGylation alone was not an affordable immunoprotective method, but the combination of CsA and anti-CD4 mAb along with PEGylation showed a highly improved a synergic effects on the inhibition of sensitized host immune reactions. In conclusion, surface camouflage and genetically engineered pancreatic islets were effective for treating diabetes mellitus. In the future study, we are expecting to achieve prolonged normoglycemia in diabetes patients using porcine islets in clinical studies.