Thursday, April 25, 2024
4/25/2024
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.
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