APOPTOSIS:A MEANS OF IMMUNE REGULATION TO TREAT DIABETES

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DESCRIPTION (provided by applicant): The main objective of this grant is to induce apoptosis specifically in auto and alloreactive lymphocytes by activating endogenous caspases. Physical elimination of these lymphocytes in NOD mouse is expected to abrogate the autoimmune process in prediabetic animals and to prevent islet allograft rejection in diseased animals, leading to the prevention and treatment of diabetes. Type I insulin-dependent diabetes mellitus (IDDM) is a chronic autoimmune disorder that affects a large number of people worldwide. Advances in understanding of the molecular and cellular basis of IDDM pathogenesis and improvements in medical practice have resulted in effective therapeutic approaches for the prevention of IDDM. Despite advances in medical therapies, IDDM remains a major cause of long-term morbidity and mortality. It is the single most common cause of renal failure requiring transplantation. Transplantation of whole pancreas or purified islets containing insulin-producing beta-cells promises an efficient approach to achieving euglycemia in IDDM patients. However, immune rejection limits long-term graft survival. Induction of islet-specific tolerance without the requirement for chronic immunosuppression would, therefore, offer a major advancement. T cells, directed at unique pancreatic beta-cell antigens, play the most critical role in islet cell destruction and development of IDDM. In this grant application, we propose to induce apoptosis in autoreactive and alloreactive lymphocytes by IL-2R-targeted delivery of caspase-activating molecules to treat diabetes. Apoptosis is the most potent physiological cell death that regulates immune homeostasis and tolerance to self-antigens. Dysregulation of the apoptotic process leads to autoimmunity, including diabetes. Caspases are the most important molecules that regulate the apoptotic machinery. Caspases are synthesized as inactive proenzymes in all the cells of the body. The activation of one caspase molecule is sufficient to commit the cell to death since it can activate the other caspases (>13 in mammals) by transcatalysis. Once activated, caspases override all the regulatory processes and commit the cell to death. Inasmuch as activated autoreactive and alloreactive T cells express high affinity IL-2R, we hypothesize that these cells can be targeted for apoptosis using IL-2R chimeric with caspase-activating proteins. Physical elimination of auto and alloreactive T cells is expected to result in the prevention of disease in prediabetic and treatment in diabetic animals transplanted with islet allografts. This approach may have direct clinical application as chimeric proteins can easily be generated in large quantities using recombinant DNA technology. The Specific Aims of this proposal include: 1) constructing recombinant genes encoding IL-2 chimeric with caspase-activating molecules and assessing chimeric proteins for specific apoptosis of autoreactive and alloreactive lymphocytes in vitro; 2) testing chimeric proteins in vivo for the prevention of diabetes in prediabetic NOD mice; 3) investigating the ability of chimeric proteins to induce tolerance to autoantigens and alloantigens for the prevention of islet allograft rejection and recurrence of autoimmunity.