Gene Therapy for Islet Cell Transplantation

描述（由申请人提供） 这个试点和可行性项目的总体目标是开发一个前 体内遗传修饰策略，将限制自身免疫， 同种异体免疫和促进永久胰岛移植物存活， 免疫抑制治疗非肥胖糖尿病（NOD）小鼠将用作 胰岛移植受者在胰岛破坏的相似性， NOD小鼠和1型糖尿病患者的发病机制。一个多管齐下的前任 将探索体内遗传修饰策略以限制免疫 以及针对胰岛的非免疫破坏力。我们假设 免疫破坏力将被体外遗传物质阻断， 修饰胰岛和/或DC以表达转化生长因子-β 1 （TGF-β 1）。我们假设，胰岛将受到保护， 抗凋亡基因Bcl-2或血管生成因子的胰岛表达损伤 基因血管内皮生长因子（VEGF）。具体目标1。探讨 免疫调节基因TGF-β 1促进永久性 小鼠胰岛的移植。胰岛将被离体遗传修饰， 表达TGF-β 1，这种转基因阻止胰岛细胞增殖的能力， 破坏性自身免疫（NOD受体的NOD-SCm胰岛），同种异体免疫 （C57BL6胰岛移植给BALB/c受体）或自身免疫加同种异体免疫（C57BIJ6 胰岛）进行检查。我们还将确定是否有前 体内遗传修饰产生对同种异体胰岛移植物的耐受性。 具体目标2。确定血管生成和抗凋亡的功效， 促进胰岛移植物存活的基因。将对胰岛进行体外修饰， 表达VEGF或Bcl-2，并与修饰成表达VEGF或Bcl-2的DC一起沿着移植 TGF-β 1同种异体供体-受体组合将与 具体目标1。 具体目标3。确定细胞、细胞因子和分子机制 与胰岛移植物排斥或接受有关。胰岛移植受体 将研究：（1）供体抗原特异性低反应性的出现 （辅助和细胞毒性T细胞的前体频率）;（2） 从TH1谱到TH2和/或TH3谱的细胞细胞因子产生;（3） 谷氨酸脱羧酶（GAD）抗原特异性T细胞 反应;和（4）限制糖尿病性T细胞的调节细胞的出现 细胞在过继转移后诱导糖尿病。 我们还将确定成功的体外基因改造 限制移植物内编码细胞毒性攻击的mRNA表达的策略 分子（例如，穿孔素）和促炎细胞因子（例如， 干扰素-γ），同时刺激编码 抗炎细胞因子（例如，白细胞介素-4）。

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).