PROJECT SUMMARY Like the challenges and skepticism that faced the antibody therapeutics field over a decade ago, RNA therapeutics is facing the same. And, like the antibody therapeutics field, we are beginning to realize the clinical impact of RNA therapeutics amiss these challenges. This is most clearly highlighted with the recent approval of two mRNA vaccines to prevent against SARS-CoV-2 and the first three FDA approved RNAi drugs targeted to the liver. Unfortunately, RNA-based drugs targeted to cancer cells is lagging behind, even with countless years of work that has revealed the power of using RNAi for treating oncological diseases. Lack of success in this space is attributed to inability to deliver RNAi safely and effectively. We previously developed a method that can safely deliver therapeutic microRNAs (miRNAs) to tumors that overexpress the folate receptor. However, the anti-tumor response was short-lived due to instability of the miRNA and poor pharmacokinetics, necessitating frequent dosing. To overcome these insufficiencies requires a stabilized miRNA that retains targeting activity. Recently we screened a panel of fully modified versions of miR-34a (FM-miR-34a) and identified one with >400- fold increased stability and outstanding in vivo efficacy when conjugated to folate. Treatment of mice implanted with breast cancer xenografts with folate-FM-miR-34a resulted in complete cures in two out of six mice and significant tumor regression in the remaining four. Based on this exciting data, here we propose to advance FM- miR-34 forward in two ways. In Aim 1 we will evaluate the activity, efficacy, and safety profile of FM-miR-34a in in vivo models of lung and prostate cancer. FM-miR-34a will be conjugated to: i) folate for delivery to lung cancer, and ii) PSMA-617, a ligand that targets prostate specific membrane antigen (PSMA) for delivery to prostate cancer. In Aim 2 we propose to capitalize on the stability afforded by FM-miR-34 to increase the circulation ½ time of folate-FM-miR-34 and PSMA-617-FM-miR-34a though incorporating an albumin binding moiety (ABM) into the ligands. Using these ligands we will evaluate serum albumin binding and stability of the new ligands. We will also verify that conjugation to ABM does not alter the activity of miR-34a nor cell binding and internalization kinetics. Finally, we will assess in vivo distribution of ligand-ABM-miR-34a conjugates. At the completion of this work we expect to have an all-encompassing miRNA delivery vehicle that can target a stabilized tumor suppressive RNAs specifically to NSCLC and prostate cancer. We will also have new ligands with increased circulation ½ life. The data obtained will ultimately have a significant impact in cancer treatment by providing new opportunities to advance the next phase of miRNA-based therapeutics. While proposed for NSCLC and prostate cancer, based on the utility of miR-34a for treating other cancers and overexpression of the folate receptor alone o...