Gene Therapy for Islet Cell Transplantation

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DESCRIPTION (provided by applicant) The overall objective of this pilot and feasibility project is to develop an ex vivo genetic modification strategy that will constrain autoimmunity and alloimmunity and facilitate permanent islet graft survival without exogenous immunosuppressive therapy. The non-obese diabetic (NOD) mouse will be used as the islet graft recipient in view of the similarities in islet destructive mechanisms in the NOD mouse and type 1 diabetic patients. A multi-pronged ex vivo genetic modification strategy will be explored to constrain the immune and the non-immune destructive forces directed at the islets. We hypothesize that the immune destructive forces will be blocked by ex vivo genetic modification of islets and/or DCs to express transforming growth factor-beta1 (TGF-beta1). We postulate that the islets will be protected from non-immune injury by islet expression of the anti-apoptotic gene Bcl-2 or the angiogenic gene vascular endothelial growth factor (VEGF). Specific Aim 1. To investigate the efficacy of the immunoregulatory gene TGF-beta1 to facilitate permanent engraftment of mouse islets. Islets will be ex vivo genetically modified to express TGF-beta1, and the ability of this transgene to prevent islet destructive autoimmunity (NOD-SCm islets to NOD recipients), alloimmunity (C57BL6 islets to BALB/c recipients) or autoimmunity plus alloimmunity (C57BIJ6 islets to NOD recipients) will be examined. We will also determine whether ex vivo genetic modification engenders tolerance to allogeneic islet grafts. Specific Aim 2. To determine the efficacy of angiogenic and anti-apoptotic genes to facilitate islet graft survival. Islets will be ex vivo modified to express VEGF or Bcl-2 and transplanted along with DCs modified to express TGF-beta1 The allogeneic donor-recipient combinations will be the same as in Specific Aim 1. Specific Aim 3. To identify the cellular, cytokine and molecular mechanisms associated with islet graft rejection or acceptance. The islet graft recipient will be studied for: (1) emergence of donor antigen specific hyporesponsiveness (precursor frequencies of helper and cytotoxic T cells); (2) polarization of T cell cytokine production from a TH1 profile to a TH2 and/or TH3 profile; (3) alterations in glutamic acid decarboxylase (GAD) antigen specific T cell response; and (4) emergence of regulatory cells that constrain diabetogenic T cells from inducing diabetes following adoptive transfer. We will also determine whether successful ex vivo genetic modification strategies constrain intragraft expression of mRNA encoding cytotoxic attack molecules (e.g., perforin) and proinflammatory cytokines (e.g., interferon-gamma) while stimulating the expression of mRNA encoding anti-inflammatory cytokines (e.g., interleukin-4).