Internalizing antibody-targeted nanosized siRNA therapeutics

描述(申请人提供)：小干扰RNA(SiRNA)的治疗应用需要在体内有效和特定的系统递送到靶细胞。针对内化细胞表面表位的抗体是系统递送siRNA的很有前途的靶向剂。最近，利用几个抗体片段成功地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、内化活性和内化后的亚细胞运输的不同，不同的抗体可能具有不同的靶向siRNA的能力。因此，最适合功能性siRNA传递的抗体可能最好是使用功能分析进行经验性鉴定。目前，还没有对一组抗体的靶向性和递送潜力进行系统评估。此外，还没有抗体介导的siRNA传递给前列腺癌的研究。结合噬菌体抗体展示和激光捕获显微切割，我们先前已经确定了一组内化单链可变区(ScFv)，它们针对的是临床上代表的前列腺癌细胞表面抗原。这些单链抗体之所以被选中，是因为它们能够与肿瘤细胞原位结合，并介导有效和特异的细胞内有效载荷传递，因此有望成为siRNA传递的候选分子。我们建议利用这组单链抗体开发体内靶向siRNA递送载体，目标是开发抗前列腺癌的纳米靶向siRNA疗法。目的1：研制基于抗体内化的纳米siRNA递送复合体，并评价其体外沉默基因表达的能力。我们建议对这些内化单链抗体进行修饰，使其具有siRNA结合功能。为了增强内小体逃逸，我们建议在靶向复合体中引入一个额外的内溶部分。我们将使用这些纳米尺寸的siRNA靶向复合体来评估siRNA的传递和体外基因沉默。目的2：实现体内靶向前列腺癌细胞的系统性siRNA传递。在体外研究之后，最有效的scFv/siRNA靶向复合体将被选择用于前列腺癌异种移植瘤的体内研究。在全身给药后，他们沉默报告基因以及参与前列腺癌生长和动态平衡的基因的能力将在体内进行评估。 公共卫生相关性：我们之前已经确定了一组噬菌体抗体衍生的；内化针对临床上代表的前列腺癌细胞表面抗原的人类单抗。我们建议利用这一组抗体开发纳米级靶向小干扰RNA(SiRNA)递送载体。这项研究将产生一系列新型试剂，用于在体内高效和特异地传递siRNA，以沉默致病基因，并将促进基于纳米siRNA的前列腺癌治疗药物的发展。

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.