Internalizing antibody-targeted nanosized siRNA therapeutics

描述（由申请人提供）：小干扰RNA（siRNA）的治疗应用需要有效和特异性的体内全身递送至靶细胞。靶向内化细胞表面表位的抗体是用于全身递送siRNA的有前景的靶向剂。最近，成功地在体内基因沉默已经实现了使用几个抗体片段，证明了这种方法的潜力。然而，由于结合表位、亲和力、内化活性和内化后亚细胞运输的差异，不同的抗体可能具有不同的充当siRNA靶向剂的能力。因此，对于功能性siRNA递送最佳的抗体可以最好地使用功能测定凭经验鉴定。目前，尚未对一组抗体的靶向和递送潜力进行系统评估。此外，尚未研究抗体介导的siRNA递送至前列腺癌。结合噬菌体抗体展示和激光捕获显微切割，我们以前已经确定了一个面板的内化单链可变片段（scFv）的目标临床代表前列腺癌细胞表面抗原。选择这些scFv是因为它们能够原位结合肿瘤细胞，并介导有效和特异性的细胞内有效载荷递送，因此是用于递送siRNA的有希望的候选者。我们建议利用这组scFv开发靶向体内siRNA递送载体，目标是开发针对前列腺癌的纳米靶向siRNA治疗剂。目的1：构建内化抗体介导的siRNA纳米复合物，并评价其体外沉默基因表达的能力。我们建议修饰这些内化scFv以赋予它们siRNA结合功能。为了增强内体逃逸，我们建议将额外的内体溶解部分引入靶向复合物。我们将使用这些纳米尺寸的siRNA靶向复合物评估siRNA递送和体外基因沉默。目的2：实现靶向系统性siRNA体内递送至前列腺癌细胞。在体外研究之后，将选择最有效的scFv/siRNA靶向复合物用于使用前列腺癌异种移植物的体内研究。全身给药后，将在体内评价它们沉默报告基因以及参与前列腺癌生长和体内平衡的基因的能力。 公共卫生相关性：我们先前已经鉴定了一组噬菌体抗体衍生的内化人单克隆抗体，其靶向临床代表的前列腺癌细胞表面抗原。我们建议利用这组抗体开发纳米级靶向小干扰RNA（siRNA）递送载体。这项研究将产生一组新的试剂，用于体内有效和特异性的siRNA递送，以沉默致病基因，并将促进基于纳米siRNA的前列腺癌治疗剂的开发。

DESCRIPTION (provided by applicant): Therapeutic application of small interfering RNA (siRNA) requires efficient and specific systemic delivery in vivo to target cells. Antibodies targeting internalizing cell surface epitopes are promising targeting agents for systemic delivery of siRNA. Recently, successful in vivo gene silencing has been achieved using several antibody fragments, demonstrating the potential of this approach. It is likely, however, that different antibodies will have varying abilities to act as targeting agents for siRNA, due to differences in binding epitopes, affinity, internalizing activity, and subcellular trafficking post-internalization. Thus, antibodies optimal for functional siRNA delivery may best be identified empirically using functional assays. Currently, no systematic evaluation of the targeting and delivery potential of a panel of antibodies has been done. In addition, no antibody-mediated siRNA delivery to prostate cancer has been studied. Combining phage antibody display and laser capture microdissection, we have previously identified a panel of internalizing single chain variable fragments (scFvs) that target clinically represented prostate cancer cell surface antigens. These scFvs were selected for their ability to bind to tumor cells in situ, and to mediate efficient and specific intracellular payload delivery, and thus are promising candidate for the delivery of siRNA. We propose to develop targeted in vivo siRNA delivery vehicles utilizing this panel of scFvs with the goal of developing nanosized targeted siRNA therapeutics against prostate cancer. Aim 1: To develop internalizing antibody-based nanosized siRNA delivery complexes and evaluate their ability to silence gene expression in vitro. We propose to modify these internalizing scFvs to impart them with siRNA binding functions. To enhance endosome escape, we propose to introduce an additional endosomolytic moiety to the targeting complex. We will evaluate siRNA delivery and gene silencing in vitro using these nanosized siRNA-targeting complexes. Aim 2: To achieve targeted systemic siRNA delivery in vivo to prostate cancer cells. Following the in vitro study, the most potent scFv/siRNA targeting complexes will be selected for in vivo study using prostate cancer xenografts. Following systemic administration, their ability to silence reporter genes as well as genes involved in prostate cancer growth and homeostasis will be evaluated in vivo. PUBLIC HEALTH RELEVANCE: We have previously identified a panel of phage antibody-derived; internalizing human monoclonal antibodies that target clinically represented prostate cancer cell surface antigens. We propose to develop nanosized targeted small interfering RNA (siRNA) delivery vehicles utilizing this panel of antibodies. This study will generate a panel of novel reagents for efficient and specific siRNA delivery in vivo to silence disease-causing genes, and will facilitate the development of nanosized siRNA-based prostate cancer therapeutics.