Ultrasound-activated microbubbles for targeted siRNA delivery to tumor

描述(申请人提供)：使用RNA干扰(RNAi)来沉默与疾病相关的特定基因的表达是医学上最有前途的新治疗范例之一。尽管RNAi，特别是小干扰RNA(SiRNA)在治疗癌症和其他广泛疾病方面显示出很大的前景，但由于RNA对血清核酸酶的脆弱性，容易引起非靶点效应，以及需要运送到靶细胞的胞浆中，RNAi的运送是一个挑战。因此，需要新的方法和载体来有效地将RNAi传递到靶组织。这项建议的目的是开发一种靶向传递siRNA的新方法，该方法利用超声(US)诱导携带siRNA的微泡(MB)振动所产生的独特生物效应。这些生物效应包括增强细胞膜对大分子的通透性，这被认为有助于siRNA内化。我们已经开发出一种具有声学活性的EGFR siRNA阳离子脂质MB载体，可以抑制小鼠鳞状细胞癌的生长。我们最近还设计了一种新型的亚微米聚合物MB，携带EGFR siRNA脂质体，在体外可以沉默EGFR的表达，并可能具有促进血管外siRNA转移的独特潜力。因此，我们将使用体内和体外鳞状细胞癌模型作为测试系统，测试这一总体假设，即通过这2 MB配方和US参数的最佳组合可以实现siRNA传递、基因沉默和治疗效果。提出了四个目标：(1)为了验证通过操纵MB化学和US参数可以提高siRNA对MB的负载效率和靶细胞摄取的假设，我们将在细胞培养中使用MB/US组合(“平台”)的矩阵来实验确定siRNA的负载和细胞内化。(2)为了验证我们的US-MB siRNA传递平台可诱导特异性基因沉默的假说，我们将使用AIM 1的平台测定体外EGFR沉默水平(并评估毒性)。(3)为了验证我们的US-MB siRNA治疗方法可在体内诱导治疗性基因沉默以及RNAi将以良好的毒性和生物分布抑制肿瘤生长的假设，我们将检测不同剂量的EGFR沉默在荷瘤小鼠中的作用，分析EGFR siRNA治疗对肿瘤生长的抑制作用，以及确定生物分布和毒性分布。(4)为了探讨US和MB介导的siRNA传递机制，我们将在显微镜下观察标记的siRNA的转运，重点是内吞和非内吞机制。这些研究将最终形成一种非侵入性的、有针对性的siRNA传递策略，这将促进RNAi的临床实施。重要的是，虽然我们建议的siRNA传递平台针对特定癌基因的mRNA(和后续蛋白质水平)，但我们的工作将建立一般原则，这些原则可以扩展到US-MB siRNA平台，用于图像引导的靶向基因沉默，用于其他疾病，特定基因沉默是一种治疗方法。

DESCRIPTION (provided by applicant): The use of RNA interference (RNAi) to silence the expression of specific genes associated with disease is one of the most promising new therapeutic paradigms in medicine. Although RNAi, and specifically small interfering RNA (siRNA), has shown much promise for the treatment of cancer and a broad spectrum of other diseases, delivery of RNAi is a challenge due to the vulnerability of RNA to serum nucleases, the propensity to cause off- target effects, and the requirement for delivery into the cytosol of target cells. Thus, new methods and vectors are needed to effectively deliver RNAi to target tissue. The objective of this proposal is to develop a new approach for targeted delivery of siRNA that capitalizes on the unique bioeffects that result from ultrasound (US)-induced vibrations of microbubles (MB) carrying siRNA. These bioeffects include enhanced cell membrane permeability to macromolecules, which is thought to facilitate siRNA internalization. We have developed an acoustically active cationic lipid MB carrier of EGFR siRNA that retards growth of murine squamous cell carcinomas. We have also recently designed a novel submicron polymer MB carrying EGFR siRNA-loaded liposomes that silences EGFR expression in vitro, and may have unique potential to facilitate extravascular siRNA transfer. Accordingly, we will test the overall hypothesis that siRNA delivery, gene silencing, and therapeutic effects can be achieved by optimal combinations of these 2 MB formulations and US parameters, using in vivo and in vitro models of squamous cell carcinoma as the test system. Four Aims are proposed: (1) To test the hypothesis that the loading efficiency of siRNA on MB and target cellular uptake can be increased by manipulation of MB chemistry and US parameters, we will experimentally determine the loading and cellular internalization of siRNA using a matrix of MB/US combinations ("platforms") in cell culture. (2) To test the hypothesis that our US-MB siRNA delivery platform induces specific gene silencing, we will determine levels of EGFR silencing in vitro (and assess toxicity) using platforms from Aim 1. (3) To test the hypothesis tha our US-MB siRNA theranostics approach induces therapeutic gene silencing in vivo and that the RNAi will suppress tumor growth with favorable toxicity and biodistribution, we will determine EGFR silencing at a variety of doses in tumor bearing mice, assay tumor growth inhibition upon EGFR siRNA treatment, and determine biodistribution and toxicity profiles. (4) To investigate the mechanisms of US and MB mediated siRNA delivery, we will microscopically observe the trafficking of labeled siRNA, focusing on endocytotic and endocytosis-independent mechanisms. These studies will culminate in a non-invasive, targeted siRNA delivery strategy that will facilitate the clinical implementation of RNAi. Importantly, while our proposed siRNA delivery platform targets mRNA (and subsequent protein levels) of a specific oncogene, our work will establish general principles that can be extended to US-MB siRNA platforms for image-guided targeted gene silencing in other diseases for which specific gene silencing represents a therapeutic approach.