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，即使引入了被称为埃德蒙顿方案的免疫抑制方案，胰岛素独立在大多数受者中也只是暂时的，因为在手术后早期就开始了显著的胰岛丢失。同种异体免疫排斥是胰岛死亡的主要原因之一，需要长期使用免疫抑制药物，这种药物对胰岛有毒性，并会导致严重的副作用，包括癌症和感染。此外，除了其显著的短期和长期副作用外，有直接证据表明，免疫抑制会导致自身反应性的重新出现，而TX本应治疗这种情况。迫切需要新的策略，通过为移植的胰岛提供替代防御方法来减少免疫抑制的剂量。RNA干扰是一种为治疗性基因沉默提供巨大潜力的技术，并可能 用于提高胰岛移植物移植后对损伤因素的抵抗力。我们之前已经证明了在免疫缺陷的啮齿动物移植前，siRNA结合的氧化铁磁性纳米颗粒(MN)探针用于将siRNA输送到胰岛的用途。针对导致胰岛损伤的基因的siRNA使这些基因沉默，并在移植前提供胰岛保护。通过使用磁性报告器，标记的胰岛可以通过活体磁共振成像(MRI)进行移植后的监测。这些在啮齿动物身上的初步研究表明，在沉默了与细胞凋亡和免疫排斥有关的基因后，移植结果有了显著的改善。在这项应用中，我们建议将这些研究扩展到下一个水平，并研究MN-siRNA在临床前具有免疫活性的非人类灵长类胰岛移植模型中的有效性。实验设计将包括合成针对caspase-3、caspase-8、Fas和Beta2-微球蛋白基因的MN-siRNA探针，然后将其与胰岛孵育。这将导致胰岛细胞的mRNA沉默和同时的磁性标记。用这些MN-siRNA探针(S)预先孵育的同种异体胰岛将被移植到糖尿病狒狒体内，在逐渐减少免疫抑制的同时，将使用磁共振成像对其进行长期监测。体内核磁共振成像将使我们能够跟踪移植物的结果，并将其与非操作的对照移植物进行比较。我们预计，对胰岛进行siRNA治疗将显著改善长期移植结果，并减少免疫抑制药的有效剂量。由于纳米粒子的模块设计，如有必要，可以将siRNA分子引入额外的靶点。

 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.