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系统递送到靶细胞。靶向内化细胞表面表位的抗体是siRNA全身递送的有希望的靶向药物。最近，利用几种抗体片段成功实现了体内基因沉默，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能力作为siRNA的靶向剂。因此，最适合功能性siRNA递送的抗体可能最好是通过功能测定来鉴定。目前，还没有对一组抗体的靶向和递送潜力进行系统的评估。此外，没有抗体介导的siRNA递送到前列腺癌的研究。结合噬菌体抗体展示和激光捕获显微解剖，我们先前已经确定了一组内化单链可变片段（scFvs），其靶向临床代表的前列腺癌细胞表面抗原。这些scFvs之所以被选中，是因为它们能够原位结合肿瘤细胞，并介导高效和特异性的细胞内有效载荷递送，因此是siRNA递送的有希望的候选者。我们建议利用这组scFvs开发靶向体内siRNA递送载体，目的是开发纳米靶向siRNA治疗前列腺癌。目的1：开发基于内化抗体的纳米siRNA递送复合物，并评估其体外沉默基因表达的能力。我们建议修改这些内部化的scFvs，赋予它们siRNA结合功能。为了促进核内体的逃逸，我们建议在靶向复合物中引入一个额外的内溶性片段。我们将利用这些纳米siRNA靶向复合物评估siRNA的体外传递和基因沉默。目的2：实现siRNA在前列腺癌细胞体内的系统性靶向递送。在体外研究之后，将选择最有效的scFv/siRNA靶向复合物用于前列腺癌异种移植的体内研究。在系统给药后，将在体内评估它们沉默报告基因以及参与前列腺癌生长和稳态的基因的能力。

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.