136 Extracellular vesicle-based delivery of Cas9/sgRNA for targeting ACSL4 to inhibit castration-resistant prostate cancer

Objectives/Goals: We aim to develop and optimize extracellular vesicle (EV)-mediated delivery of Cas9/single-guide RNA (sgRNA) ribonucleoprotein (RNP) complex to target ACSL4, a key driver of dysregulated fatty acid metabolism in androgen receptor (AR)-independent prostate cancer, and to evaluate its therapeutic potential in vitro and in vivo. Methods/Study Population: We examined ACSL4 gene editing using lentiviral delivery and EV-mediated delivery of Cas9/sgRNA constructs. In HEK-293T cells, we optimized EV treatment frequency and concentration. In PC3 cells, we analyzed gene editing efficiency in detached cell population in comparison to attached cell population. Editing was determined by next generation sequencing. To examine EV-mediated gene editing in vivo, we employed a xenograft mouse model produced by subcutaneous injection of PC3 cells. EVs were administered by intratumoral injection three times a week for three weeks, and tumor growth, histology, and gene editing were assessed. Future studies will include patient-derived xenograft models to evaluate translational relevance. Results/Anticipated Results: Lentiviral delivery achieved up to 36% ACSL4 gene editing. EV-mediated delivery showed dose-dependent gene editing in HEK-293T cells, which contained up to 13% editing. Gene editing was enriched in detached PC3 cells, reaching a maximum editing efficiency of 47%, potentially contributing to cell death. Attached PC3 cells showed 1-2% editing and reduced proliferation after EV treatment. In the ongoing xenograft mouse model experiments, EV treatment is expected to decrease tumor growth, reduce proliferation, and induce apoptosis, confirmed by histology and sequencing. Further optimization of EV dosing and delivery aims to further enhance editing efficiency and therapeutic impact. Discussion/Significance of Impact: EV-mediated delivery of Cas9/sgRNA RNP to target ACSL4, a key metabolic driver of aggressive disease, represents a novel therapeutic strategy for treatment-resistant prostate cancer. Continued work on this nonviral platform will enable targeted, tumor-specific editing while minimizing safety concerns associated with viral delivery.