PROJECT SUMMARY Prostate cancer (PC) is the most common cancer in men, and the second leading cause of cancer death among men in the United States. There is a critical unmet need to develop novel, efficacious therapeutics for PC. NUAgo Therapeutics, Inc. is developing an RNA-interference-based therapeutic for PC based on research conducted at Northwestern University into a naturally occurring anti-cancer mechanism. This research identified a class of short (19-22 nt long) double-stranded (ds) interfering RNA molecules (here termed “sRNAs”) that potently target genes that are essential for cancer cell survival. These sRNAs are derived from CAG trinucleotide repeat (TNR) sequences that are found in genes associated with several neurodegenerative diseases, including huntingtin (the cause of Huntington’s disease) and the androgen receptor (AR) gene (the cause of spinobulbar muscular atrophy [SBMA]/Kennedy disease). Both diseases are associated with reduced cancer incidence. Evidence indicates that CAG expansions lead to increased production of short interfering RNAs that bind to complementary transcripts and downregulate their expression. sRNAs containing the CAG repeat (“sCAG”) are extremely toxic to cancer cells by targeting highly expressed CUG repeat-containing genes, providing both a mechanistic explanation for the reduced incidence of cancer in patients with certain neurodegenerative diseases and a novel therapeutic approach for cancer treatment. PC is a particularly attractive target for this novel therapeutic approach. The number of CAG repeats in the AR gene is inversely correlated with PC incidence, the aggressiveness of the disease, and the risk of distant metastasis. Longer CAG repeats thus appear to have an anti-tumor effect, suggesting susceptibility of PC cells to sCAG. The goal of this STTR project is to develop an sCAG-based therapeutic, which will be delivered to cells using lipopolyplex (LPP) nanoparticles, to treat PC. In Aim 1, the uptake and activity of sCAG-LPP (“NU002”) will be evaluated in mouse models of prostate cancer, quantifying both sCAG delivery and silencing activity in PC cells in vivo. Uptake and activity of the NU002 and a non-toxic control sRNA will be evaluated in both tumor and normal cells in four mouse models, including two xenograft models of PC and a syngeneic PC model. Uptake will be monitored by fluorescently labeling the sRNAs, while silencing activity will be measured using a fluorescent reporter gene that includes a corresponding target sequence. RNA sequencing will be used to measure the expression of target genes in tumors. In Aim 2, the anti-tumor effects of NU002 will be characterized in vivo in the same four xenograft and syngeneic models as in Aim 1, measuring the effect on excised tumor size at 4-6 weeks post-treatment. Histological and liver enzyme analyses will be used to examine toxicity in normal tissues, and RNA sequencing will again be performed on tumor and normal tissue samples to quantif...