Normalizing aberrant metabolism in ovarian cancer by a unique drug delivery system

使用小干扰RNA(SiRNA)的基因沉默是一种可行的治疗方法，但在翻译方面受到限制。 由于缺乏有效的交付系统。开发高效无毒的递送系统将翻译siRNA- 以治疗学为基础的临床研究。在这里，使用体外细胞培养和体内动物模型，我们建议开发 一种新型的siRNA递送系统，用于有效的基因沉默和治疗应用。 我们最近报道了MICU1，一种线粒体内膜蛋白，其功能是一种代谢 促进卵巢癌糖酵解和治疗抵抗的开关。不幸的是，缺乏药理作用 体内沉默MICU1的抑制剂和有效策略对未来的临床提出了重大挑战 MICU1靶向治疗的翻译。因此，MICU1可以作为一个新的治疗靶点来验证 我们新的siRNA传递平台的沉默和治疗效果，并提供了正常化的机会 代谢异常是导致治疗抵抗的原因。因此，我们计划开发一种金纳米颗粒(AuNP)- 用于siRNA传递的基于脂质体的制剂(AuroLiposome)在体内有效地沉默MICU1。 为了在体内有效地沉默MICU1，我们开发了基于DOPC-DOTAP的常规 纳米脂质体siRNA递送平台(MICU1 siRNA-CLP)。有趣的是，AuNP(20 nm尺寸)掺杂 制剂(MICU1 siRNA-AuroLps)在沉默MICU1方面显示出增强的效果，所需时间降低3-4倍 SiRNA浓度高于MICU1 siRNA-CLP或商业上可用的转染剂，如 HiPerfect、RNAiMax和Lipofetamine3000。克隆生长分析中反映了增强的沉默；MICU1 SiRNA-AuroLPs对HGSOCs克隆生长的抑制作用(~90%)高于MICU1 siRNA-CLPs(~50%)或 HiPerfect(~30%)。重要的是，与MICU1 siRNA-AuroLps相比，MICU1 siRNA-AuroLps更有效地抑制肿瘤生长(~75% 对MICU1 siRNA-CLP(~35%)。重要的是，使用化学抑制剂，我们证明了AuNP的掺入 转换型MICU1 siRNA-CLP细胞内摄取途径的研究 介导的内吞作用主要通过空泡摄取途径进行。因此，我们假设AuNP在 纳米脂质体制剂触发AuroLipoome(AuroLps)的空泡摄取导致降解减少 在溶酶体中增加siRNA-AuroLps，从而增强沉默效果。我们将使用下面的特定目标来测试 提出假设，实现总体目标； 目的：探讨金掺杂增强基因沉默效应的机制。 目的2：测定优化后纳米制剂的药代动力学、生物分布和毒性。 目的：检测专利异种移植物(PDX)和同基因小鼠模型的治疗效果。 该项目的成功完成将为任何体外和体内的 体内基因沉默的应用和将异常代谢正常化的潜在可翻译策略 克服对高级别浆液性卵巢癌的治疗阻力。

Gene silencing using small interfering RNA (siRNA) is a viable therapeutic approach but, limited in translation due to lack of effective delivery systems. Developing effective and non-toxic delivery system will translate siRNA- based therapeutics to clinics. Here, using in vitro cell culture and in vivo animal models, we propose to develop a new type of siRNA delivery system for effective gene silencing and therapeutic applications. We recently reported that MICU1, a mitochondrial inner membrane protein, functions as a metabolic switch that promotes glycolysis and therapy resistance in ovarian cancer. Unfortunately, lack of pharmacological inhibitors and effective strategies to silence MICU1 in vivo posit a significant challenge against future clinical translation of MICU1-targeted therapy. Therefore, MICU1 could serve as a new therapeutic target to validate silencing and therapeutic efficacy of our new siRNA delivery platform and provides opportunity to normalize aberrant metabolism responsible for therapy resistance. Hence, we plan to develop a gold nanoparticle (AuNP)- based liposomal formulation (AuroLiposome) for siRNA delivery to effectively silence MICU1 in vivo. To effectively silence MICU1 in vivo we have developed DOPC-DOTAP based conventional nanoliposomal siRNA delivery platforms (MICU1 siRNA-cLPs). Interestingly, AuNP (20 nm size)-doped formulation (MICU1 siRNA-AuroLPs) exhibited enhanced efficacy in silencing MICU1, requiring 3-4-fold lower siRNA concentrations than MICU1 siRNA-cLPs or commercially available transfection reagents such as Hiperfect, RNAiMax and Lipofectamine 3000. Enhanced silencing was reflected in clonal growth assays; MICU1 siRNA-AuroLPs inhibited clonal growth of HGSOCs more efficiently (~90%) than MICU1 siRNA-cLPs (~50%) or Hiperfect (~30%). Importantly MICU1 siRNA-AuroLPs inhibited tumor growth more effectively (~75%) compared to MICU1 siRNA-cLPs (~35 %). Importantly, using chemical inhibitors we showed that incorporation of AuNP switched intracellular uptake pathway of MICU1 siRNA-cLPs from a combination of clathrin and caveolar mediated endocytosis to mostly caveolar uptake pathway. Hence, we hypothesize that incorporation of AuNP in nanoliposomal formulation triggers caveolar uptake of AuroLiposome (AuroLPs) resulting in reduced degradation of siRNA-AuroLPs in lysosome and thus enhancing silencing efficacy. We will use specific aims below to test the hypothesis and accomplish overall objectives; Aim1: Determining mechanisms of enhanced gene silencing efficacy due to gold doping. Aim 2: Determining pharmacokinetics, biodistribution and toxicity of the optimized nanoformulation. Aim 3: Determining therapeutic efficacy in patent derived xenografts (Pdx) and syngeneic mouse model. Successful completion of the project will provide a generalized siRNA delivery approach for any in vitro and in vivo gene silencing applications and a potential translatable strategy to normalize aberrant metabolism to overcome therapy resistance against high grade serous ovarian cancer.