siRNA Protection of Islet Grafts in Baboons

 描述（由申请人提供）：胰岛移植（Tx）对1型糖尿病患者具有挽救生命的潜力。然而，即使引入了称为埃德蒙顿方案的免疫抑制方案，由于在手术后早期开始的显著胰岛损失，大多数接受者的胰岛素依赖性仅是短暂的。同种异体免疫排斥是胰岛死亡的主要原因之一，需要永久使用免疫抑制药物，这些药物对胰岛有毒，并引起严重的副作用，包括癌症和感染。此外，除了其显着的短期和长期的副作用，有直接的证据表明，免疫抑制导致再次出现的自身反应性，条件Tx是应该treat. New战略，以减少剂量的免疫抑制提供替代防御方法移植胰岛是迫切需要的。RNA干扰是一种为治疗性基因沉默提供巨大潜力的技术， 用于提高移植后胰岛移植物对损伤因素的抵抗力。我们先前已经证明了siRNA缀合的基于氧化铁的磁性纳米颗粒（MN）探针用于在免疫缺陷啮齿动物中移植之前将siRNA递送至胰岛的效用。针对负责胰岛损伤的基因的siRNA使这些基因沉默，并在移植前提供胰岛保护。通过使用磁性报告，标记的胰岛可以在移植后通过体内磁共振成像（MRI）进行监测。这些在啮齿类动物中的初步研究显示，在沉默负责细胞凋亡和免疫排斥的基因后，移植结果显著改善。在本申请中，我们提出将这些研究扩展到下一个水平，并研究MN-siRNA在胰岛移植的临床前免疫活性非人灵长类动物模型中的有效性。实验设计将包括合成靶向caspase-3、caspase-8、Fas和β 2-微球蛋白基因的MN-siRNA探针，然后将其与胰岛孵育。这将导致mRNA沉默和胰岛细胞的同时磁性标记。将用这些MN-siRNA探针预孵育的同种异体胰岛移植到糖尿病狒狒中，使用磁共振成像长期监测，同时逐渐减少免疫抑制。体内MRI将使我们能够跟踪移植物的结果，并将其与未操作的对照移植物进行比较。我们预计，胰岛的siRNA治疗将允许显着改善长期移植结果和减少免疫抑制剂的有效剂量。由于纳米颗粒的模块设计，如果需要，可以引入针对额外靶标的siRNA分子。

 DESCRIPTION (provided by applicant): Pancreatic islet transplantation (Tx) has a lifesaving potential for Type 1 diabetes patients. However, even with introduction of the immunosuppressive regimen known as Edmonton Protocol, insulin independence was only transient in most recipients due to significant islet loss that starts early after the procedure. Allogeneic immune rejection, one of the major reasons for islet death, requires permanent use of immunosuppressive drugs, which are toxic to islets and cause serious side effects including cancer and infection. Moreover, in addition to its significant short and long-term side effects, there is direct evidence that immunosuppression leads to reemergence of autoreactivity, the condition that Tx is supposed to treat. New strategies to reduce the dose of immunosuppression by providing alternative defense approaches to transplanted islets are urgently needed. RNA interference is a technique that offers great potential for therapeutic gene silencing and could be employed for improving islet graft resistance to damaging factors after transplantation. We have previously demonstrated the utility of siRNA-conjugated iron oxide-based magnetic nanoparticle (MN) probes for siRNA delivery to pancreatic islets prior to transplantation in immunodeficient rodents. siRNA directed towards genes responsible for islet damage silenced these genes and provided islet protection prior to transplantation. By the use of a magnetic reporter, labeled pancreatic islets could be monitored after transplantation by in vivo magnetic resonance imaging (MRI). These preliminary studies in rodents showed significant improvement in graft outcome after the silencing of genes responsible for apoptosis and immune rejection. In this application we propose to extend these studies to the next level and investigate the effectiveness of MN-siRNA in a pre- clinical immunocompetent non-human primate model of islet transplantation. The experimental design will include synthesis of the MN-siRNA probes targeting caspase-3, caspase-8, Fas and beta2-microglobulin genes, which will then be incubated with pancreatic islets. This will lead to mRNA silencing and simultaneous magnetic labeling of islet cells. Allogeneic islets pre-incubated with these MN-siRNA probe(s) will be transplanted into diabetic baboons that will be monitored long-term using magnetic resonance imaging while tapering immunosupression. In vivo MRI will allow us to follow graft outcome and compare it with non- manipulated control grafts. We anticipate that siRNA treatment of the islets will allow for significant improvement in long-term graft outcome and reduction of the effective dose of immunosupressants. Due to the module design of the nanoparticles siRNA molecules to additional targets can be introduced if necessary.