Improved in vivo delivery of siRNA

描述(由申请人提供)：这项提案的总体目标是开发新的试剂，以有效地将功能性siRNA输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siRNAs作为治疗剂并验证药物靶点。不幸的是，在动物体内的siRNA传递通常是不有效的，并且已经被证明很难将siRNA传递到一些具有治疗意义的细胞类型和组织。SiRNA在体内应用的商业成功取决于新的递送方法的开发。这项拟议的研究将导致开发改进的基于抗体的结合物，以靶向过度表达适当细胞表面抗原的细胞的siRNAs。将开发两种不同类型的免疫结合物。第一种免疫结合物类型是通过将大的重组RNA结合蛋白与抗体化学交联而产生的。SiRNA通过RNA结合蛋白与靶向抗体非共价结合。第二类是通过使用含有细胞内部不稳定结构的异双功能交联剂将功能性修饰的siRNA直接连接到抗体上，以促进内化后siRNA的释放。第一阶段的具体目标：1.开发新的免疫结合物，显著增加抗体/siRNA复合体中每个抗体分子的siRNA负载量。2：使用可切割的连接物将siRNA与抗体共价偶联，产生在血浆中稳定但在细胞中有效释放功能性siRNA的免疫偶联物。3.使用AIMS 1和AIMS 2中生产的新型免疫结合物，将功能性siRNA特异性地输送到过度表达适当细胞表面抗原的培养细胞和小鼠移植瘤的肿瘤细胞中。每种免疫结合物的siRNA结合能力和稳定性将通过荧光测量直接进行比较。免疫结合物将功能siRNA输送到靶细胞以沉默特定基因的能力也将使用qRT-PCR进行测试。在第二阶段，我们将扩展和调整这项技术，以用于体内应用的化学修饰的siRNA。 叙述：siRNA在下调靶基因表达方面的多功能性和有效性使其成为研究哺乳动物细胞培养系统中基因功能的首选工具。由于这一成功，正在积极研究siRNA在动物疾病模型和人类中的治疗潜力。遗憾的是，目前在动物体内传递siRNA的方法效率很低，而且很难控制。在开发出更有效的递送方式之前，治疗性siRNA的前景将无法实现。因此，我们概述了一项有效的研究计划，以开发改进的体内siRNA传递方法。我们的策略包括使用基于抗体的递送剂来靶向并向特定类型的细胞递送siRNA，如果成功，将极大地促进体内siRNA的递送，以了解和治疗癌症。

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.