Dynamic Dendronized Polymers for Effective siRNA Delivery to Pancreatic Islets

描述（由申请人提供）：该项目汇集了加州大学欧文分校和City of Hope糖尿病研究中心实验室的技能，设计了一种新的动态树枝化聚合物（denpols）家族，用于安全有效地将siRNA递送到胰岛中，以抑制胰岛细胞凋亡并增强胰岛移植的结果。尽管RNAi具有巨大的治疗潜力，但缺乏安全有效的递送载体阻碍了siRNA的临床前景。虽然已经针对非病毒载体的开发做出了相当大的努力，但是很少有系统已经进展到临床试验，并且没有一个获得FDA批准。基于以往研究的经验教训，在这里，我们提出了一种新的动态denpol系统的安全和有效的siRNA交付。denpol设计提供了一个前所未有的机会，结合联合收割机的多价性和构象灵活性，精确的结构控制和组合多样性，以及多种动态/响应功能，以优化siRNA递送的有效性。PI和co-I实验室之间合作的初步结果表明，denpol系统在将siRNA转染到分离的细胞（NIH 3 T3）和整个大鼠胰岛中时表现出最小的细胞毒性和高效率。在初步成功的基础上，提出了三个具体目标，以实现本研究的总体目标。目标1将集中在设计，合成和表征的一个图书馆denpols 具有受控的组成和结构。denpols的分子参数将系统地和组合地变化以进行优化。目的2将研究siRNA复合和体外转染到分离的细胞（NIH 3 T3和大鼠INS-1和小鼠NIT-1）中用于目的1中合成的denpols，由此将确定具有最小毒性和最佳转染效率的最佳结构/组成。最后，在目标3中，将通过将分离的胰岛暴露于培养物中以及通过大鼠胰腺的离体灌注来研究有希望的denpol候选物用于siRNA转染，以确定最佳的转染条件。 denpols和安全有效地将抗-BBC 3 siRNA递送到胰岛中以抑制胰岛凋亡的条件。选择胰岛作为疾病治疗模型来评估所提出的denpol载体有两个主要原因：（1）RNAi治疗，如抗BBC 3 siRNA，具有增加胰岛产量和提高胰岛移植效率的巨大潜力。迫切需要安全有效地将siRNA递送到胰岛以改善胰岛移植结果;和（2）胰岛应该作为测试我们的denpols的体内转染效率和安全性的优良模型系统，因为像大多数体内组织一样，胰岛由有序的紧密结合的细胞组成。该项目的成功演示将引入新一代siRNA递送合成载体，改善胰岛移植的结果，并有助于开发1型糖尿病的治疗方法。

DESCRIPTION (provided by applicant): This project brings together the skills of laboratories at UC-Irvine and at City of Hope Diabetes Research Center to design a new family of dynamic dendronized polymers (denpols) for safe and effective delivery of siRNA into pancreatic islets to suppress the islet cell apoptosis and enhance outcome of islet transplantation. Despite the tremendous potential of RNAi for therapeutics, the lack of safe and effective delivery vehicles hampers the clinical promise of siRNA. While considerable efforts have been directed towards the development of non-viral vectors, few systems have progressed into clinical trials and none has received FDA approval. Based on lessons learned from previous studies, here we propose a novel dynamic denpol system for safe and effective siRNA delivery. The denpol design provides an unprecedented opportunity to combine multivalency and conformational flexibility, precise structural control and combinatorial diversity, and multiple dynamic/responsive features to optimize the effectiveness for siRNA delivery. The preliminary results from the collaboration between the PI and co-I's labs have shown that the denpol system exhibits minimal cytotoxicity and high efficiency in transfecting siRNA into both isolated cells (NIH 3T3) and whole rat islets. Built upon the initial successes, three specific aims are proposed to achieve the overall goal of this study. Aim 1 will focus on the design, synthesis, and characterization of a library of denpols with controlled composition and structure. The molecular parameters for the denpols will be systematically and combinatorial varied for optimization. Aim 2 will investigate siRNA complexation and in vitro transfection to isolated cells (NIH 3T3 and rats INS-1 and mouse NIT-1) for the denpols synthesized in Aim 1, from which the optimal structure/composition with minimal toxicity and optimal transfection efficiency will be determined. Finally, in Aim 3, the promising denpol candidates will be investigated for siRNA transfection by exposing isolated islets in culture as well as through ex vivo perfusion of the rat pancreas to determine the optimal denpols and conditions for safe and effective delivery of anti-BBC3 siRNA into islets to suppress islets apoptosis. Islets are chosen as the disease treatment model to evaluate the proposed denpol vectors for two major reasons: (1) RNAi treatment, such as anti-BBC3 siRNA, has great potential to increase islet yield and improve islet transplantation efficiency. There is an urgent need for safe and effective delivery of siRNA to islets to improve islets transplantation outcome; and (2) Islets should serve as an excellent model system to test in vivo transfection efficiency and safety of our denpols, because like most in vivo tissues, islets are composed of tightly bound cells in an orderly fashion. Successful demonstration of this project will introduce a new generation of synthetic vectors for siRNA delivery and improve the outcome of islets transplantation and help developing treatment for type 1 diabetes.