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，但在翻译上受到限制 由于缺乏有效的运输系统。开发有效和无毒的递送系统将siRNA- 基于临床的疗法。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，MICU 1是一种线粒体内膜蛋白，具有代谢功能， 卵巢癌中促进糖酵解和治疗抵抗的开关。不幸的是，缺乏药理学 抑制剂和有效的策略，沉默MICU 1在体内是一个重大的挑战，对未来的临床 MICU 1靶向治疗的翻译。因此，MICU 1可以作为一个新的治疗靶点， 我们的新siRNA递送平台的沉默和治疗功效，并提供了使 导致治疗抵抗的异常代谢。因此，我们计划开发一种金纳米粒子（AuNP）- 用于siRNA递送的基于脂质体的制剂（AuroLiposome），以在体内有效地沉默MICU 1。 为了在体内有效地沉默MICU 1，我们开发了基于DOPC-DOTAP的常规方法， 纳米脂质体siRNA递送平台（MICU 1 siRNA-cLP）。有趣的是，AuNP（20 nm尺寸）掺杂的 制剂（MICU 1 siRNA-AuroLP）在沉默MICU 1方面表现出增强的功效，需要3-4倍的低表达量。 siRNA浓度高于MICU 1 siRNA-cLP或市售转染试剂， Hyperfect、RNAiMax和Lipofectamine 3000。增强的沉默反映在克隆生长测定中; MICU 1 siRNA-AuroLP比MICU 1 siRNA-cLP（~50%）或MICU 2 siRNA-cLP更有效地抑制HGSOC的克隆生长（~90%）。 Hyperfect（~30%）。重要的是，MICU 1 siRNA-AuroLPs与对照组相比更有效地抑制肿瘤生长（约75%）。 MICU 1 siRNA-cLP（约35%）。重要的是，使用化学抑制剂，我们表明， 从网格蛋白和小窝的组合转换MICU 1 siRNA-cLP的细胞内摄取途径 介导的内吞作用转变为主要的小窝摄取途径。因此，我们假设AuNP的掺入可能是一个潜在的原因。 纳米脂质体制剂触发AuroLiposome（AuroLP）的小窝摄取，导致降解减少 在溶酶体中的siRNA-AuroLPs，从而增强沉默功效。我们将使用下面的具体目标来测试 提出假设并完成总体目标; 目的1：确定金掺杂增强基因沉默功效的机制。 目的2：确定优化的纳米制剂的药代动力学、生物分布和毒性。 目的3：确定在专利衍生的异种移植物（Pdx）和同基因小鼠模型中的治疗功效。 该项目的成功完成将为任何体外和体内的siRNA递送提供一种通用的方法。 体内基因沉默的应用和潜在的翻译策略，以正常化异常代谢， 克服对高级别浆液性卵巢癌的治疗抗性。

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.