在晚期前列腺癌中使用RNA生物工程技术和定制化脂质纳米颗粒靶向PLXND1

Background: PLXND1 (Plexin D1) is a transmembrane receptor that plays critical roles in promoting neural lineage plasticity and driving resistance to enzalutamide therapy in prostate cancer. Despite its significance, PLXND1 has been considered "undruggable." siRNA-based therapeutics offer a strategy to silence disease-driving genes like PLXND1 but face limitations, including instability, poor cellular uptake, and off-target effects. Methods: We employed an RNA bioengineering approach using a tRNA/pre-miRNA-mir-34a scaffold to stabilize and express siRNAs targeting PLXND1 (BioRNA-siPLXND1). BioRNA/PLXND1-siRNA expression plasmids were constructed via molecular cloning, and the resulting BioRNA was purified and analyzed for quality, yield, and endotoxin levels. We tested BioRNA-siPLXND1 function in vitro and formulated it into lipid nanoparticles (LNPs) using two lipid components: DOPE and DOTAP. The stability, transfection efficiency, and safety of each formulation were evaluated in vitro and in vivo using prostate cancer cell lines and neuroendocrine prostate cancer (NEPC) patient-derived xenograft (PDX) organoids. Results: BioRNA-siPLXND1 achieved high yield and purity with low endotoxin levels. It efficiently silenced PLXND1 at the mRNA and protein levels, significantly inhibiting cell proliferation, colony formation, and organoid growth in vitro. Among the LNP formulations, LNP-DOTAP demonstrated higher transfection efficiency but greater cytotoxicity in normal cells, whereas LNP-DOPE showed a favorable safety profile and effective delivery. LNP-DOPE-loaded BioRNA-siPLXND1 remained stable for at least 7 days in vitro and suppressed tumor growth in NEPC PDX organoids. In vivo, LNP-DOPE accumulated in tumors within 2 hours, persisted for up to 4 days, and was predominantly retained in tumor tissue by day 7. Conclusions: Our study demonstrates the feasibility and therapeutic potential of a tRNA-based BioRNA platform for delivering PLXND1-targeting siRNAs in prostate cancer. LNP-DOPE serves as a safe and effective delivery system, offering a promising strategy for targeting "undruggable" oncogenes like PLXND1 in therapy-resistant prostate cancer. Financial support: This work was supported in part by grants from NIH/NCI R37CA249108 (Liu), R01CA251253 (Liu), R21CA277171 (Liu), Department of Defense HT9425-23-1-0144 (Liu), HT9425-23-1-0325 (Liu), HT9425-23-1-0324 (Dall'Era), and UC Davis Comprehensive Cancer Center Support Grant (CCSG) awarded by the National Cancer Institute (NCI P30CA093373).