Dynamic Dendronized Polymers for Effective siRNA Delivery to Pancreatic Islets

描述(申请人提供)：该项目集合了加州大学欧文分校和希望之城糖尿病研究中心的实验室的技术，设计了一种新的动态树枝状聚合物(Denpols)家族，用于安全有效地将siRNA输送到胰岛，以抑制胰岛细胞凋亡，提高胰岛移植的结果。尽管RNAi在治疗方面具有巨大的潜力，但缺乏安全有效的递送工具阻碍了siRNA的临床前景。虽然在非病毒载体的开发上已经做出了相当大的努力，但很少有系统进入临床试验，也没有一个系统获得FDA的批准。在总结前人研究成果的基础上，我们提出了一种安全有效的siRNA动态递送系统。DENPOL的设计提供了一个前所未有的机会，将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/响应功能结合在一起，以优化siRNA传递的有效性。PI和CO-I实验室合作的初步结果表明，DENPOL系统在将siRNA导入分离的细胞(NIH3T3)和整个大鼠胰岛方面表现出最低的细胞毒性和高效率。在初步研究成果的基础上，提出了实现本研究总体目标的三个具体目标。目标1将集中于设计、合成和表征齿极库 具有可控的成分和结构。分子参数的优化将是系统的和组合的变化。目的研究AIM 1中合成的致密物与siRNA的络合作用及其对体外培养细胞(NIH 3T3、大鼠INS-1和小鼠NIT-1)的影响，以确定毒性最小、转染率最高的最佳结构/组成。最后，在目标3中，将通过将分离的胰岛暴露在培养中以及通过大鼠胰腺的体外灌流来研究有希望的denpol候选sirna转基因，以确定最佳的 安全有效地将抗BBC3 siRNA送入胰岛以抑制胰岛细胞凋亡的密度和条件。选择胰岛作为疾病治疗模型是基于两个主要原因：(1)RNAi治疗，如抗BBC3 siRNA，具有增加胰岛产量和改善胰岛移植效率的巨大潜力。我们迫切需要将siRNA安全有效地运送到胰岛以改善胰岛移植的结果；以及(2)胰岛应该作为一个很好的模型系统来测试我们的密极细胞的体内转染效率和安全性，因为像大多数体内组织一样，胰岛是由紧密结合的细胞有序组成的。该项目的成功演示将引入新一代siRNA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.