Improved in vivo delivery of siRNA

描述（由申请人提供）：本提案的总体目标是开发新型试剂，以有效地将功能性siRNA递送到动物体内进行基因沉默实验。制药和生物技术公司正在积极测试siRNA作为治疗剂并验证药物靶点。不幸的是，siRNA在动物中的递送通常不是有效的，并且已经证明难以将siRNA递送到一些治疗上重要的细胞类型和组织。siRNA体内应用的商业成功取决于新递送方法的开发。拟议的研究将导致开发改进的基于抗体的缀合物，以将siRNA靶向过表达适当细胞表面抗原的细胞。将开发两种不同类型的免疫缀合物。第一种免疫缀合物类型通过将大的重组RNA结合蛋白化学交联至抗体来产生。siRNA通过RNA结合蛋白与靶向抗体非共价结合。第二种类型是通过使用含有内部细胞不稳定结构的异双功能交联剂将功能性修饰的siRNA直接连接到抗体以促进siRNA在内化后的释放来产生。第一阶段的具体目标：1.开发新型免疫缀合物以显著增加抗体/siRNA复合物中每个抗体分子负载的siRNA的量。第二章：免疫缀合物使用可切割接头将siRNA缀合至抗体以产生在血浆中稳定但在细胞中有效释放功能性siRNA的免疫缀合物。3.使用目的1和2中产生的新型免疫缀合物，将功能性siRNA特异性递送至过表达适当细胞表面抗原的培养细胞和小鼠异种移植物中的肿瘤细胞中。 使用荧光测量直接比较每种免疫缀合物的siRNA结合能力和稳定性。还将使用qRT-PCR测试免疫缀合物将功能性siRNA递送到靶细胞中以沉默特定基因的能力。 在第二阶段，我们将扩展和调整该技术，以与设计用于体内应用的化学修饰的siRNA一起工作。 叙述：siRNA在下调靶基因表达方面的多功能性和效力使其成为研究哺乳动物细胞培养系统中基因功能的优选工具。由于这一成功，siRNA的治疗潜力正在动物疾病模型和人类中积极研究。不幸的是，目前在动物中递送siRNA的方法非常低效且难以控制。在开发出更有效的递送模式之前，治疗性siRNA的前景将无法实现。因此，我们概述了一个有效的研究计划，以开发改进的体内siRNA递送方法。我们的策略涉及使用基于抗体的递送剂来靶向和递送siRNA到特定的细胞类型中，如果成功的话，将极大地推进siRNA在体内的递送，以用于理解和治疗癌症。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop novel reagents to efficiently deliver functional siRNA into animals for gene silencing experiments. Pharmaceutical and biotechnology companies are actively testing siRNAs as therapeutic agents and to validate drug targets. Unfortunately siRNA delivery in animals is usually not efficient and it has proven difficult to deliver siRNA to some therapeutically important cell types and tissues. The commercial success of in vivo applications of siRNA hinges on the development of new delivery methods. The proposed research will result in the development of improved antibody-based conjugates to target siRNAs to cells overexpressing the appropriate cell surface antigen. Two different types of immunoconjugates will be developed. The first immunoconjugate type is produced by chemically crosslinking large recombinant RNA binding proteins to antibodies. The siRNA noncovalently associates with the targeting antibody via the RNA binding protein. The second type is produced by directly attaching functional modified siRNAs to antibodies using heterobifunctional crosslinking agents containing internal cell labile structures to promote release of the siRNA after internalization. Specific aims for Phase I: 1. Develop novel immunoconjugates to significantly increase the amount of siRNA loaded per antibody molecule in the antibody/siRNA complex. 2: Covalently conjugate siRNA to antibodies using cleavable linkers to produce immunoconjugates which are stable in plasma but efficiently release functional siRNA in cells. 3. Use the novel immunoconjugates produced in Aims 1 and 2 to specifically deliver functional siRNA into cultured cells overexpressing the appropriate cell surface antigen and into tumor cells in mouse xenografts. The siRNA binding capacity and stability of each of immunoconjugates will be directly compared using fluorescence measurements. The ability of the immunoconjugates to deliver functional siRNA into targeted cells to silence specific genes will also be tested using qRT-PCR. In Phase II, we will expand and adapt the technique to work with chemically modified siRNAs designed for in vivo applications. Narrative: The versatility and potency of siRNA in downregulating target gene expression has made it a preferred tool to study gene function in mammalian cell culture systems. As a result of this success, the therapeutic potential of siRNA is being actively investigated in animal disease models and in humans. Unfortunately, the current methods to deliver siRNA in animals are very inefficient and difficult to control. The promise of therapeutic siRNA will not be realized until more efficient modes of delivery are developed. Therefore, we outline an efficient research plan to develop improved methods of in vivo siRNA delivery. Our strategy involves the use of antibody-based delivery agents to target and deliver siRNA into specific cell types and if successful will greatly advance the delivery of siRNA in vivo for understanding and treating cancer.