Systemic Delivery of Synthesized Aptamer-siRNA Chimeras Results in Prostate-Targeted Radiosensitization

Abstract

Our previously published work using an in vitro transcribed PSMA aptamer with a shRNA targeting DNA-PK demonstrated enhanced radiation response in human prostate cancer (PCa) cell lines and xenografts following direct tumor injection. While important, a systemically-delivered sensitizing chimera would have even greater clinical impact. As such, we developed and tested next generation chemically synthesized aptamer-siRNA chimeras in both PCa cell lines and xenografts with systemic intravenous (IV) delivery and externally-applied radiation. PSMA aptamer-siRNAs were chemically synthesized. Cell culture utilized two human PCa cell lines: LNCaP-Mluc (PSMA+), and PC-3 (PSMA-). Quantitative RT-PCR, Cell-Titer Blue assay, luciferase assay, xenograft experiments, radiation, and immunohistochemistry (IHC) were performed in vitro or in vivo for evaluating target knockdown and radiosensitization. Systemic administration of chimeras was performed by tail vein injections on Days 1 and 2 with or without radiation on Day 4. DNA-PK mRNA and protein levels were reduced in PSMA-expressing LNCaP cells using the PSMA aptamer-siDNA-PK chimera (60% and 53% reduction, respectively) compared to a PSMA aptamer-siControl chimera (p < 0.05). Treatment of PSMA expressing PCa cells with aptamer-siDNA-PK chimera and radiation revealed increased cell death when compared to a PSMA aptamer-siControl not capable of DNA-PK knockdown (p < 0.005). DNA-PK mRNA knockdown following systemic administration of aptamer-siDNA-PK was 67% (p < 0.05) and IHC confirmed a decrease of 30% in DNA-PK staining with DNA-PK chimera treatment compared to control (p < 0.005), which resulted in significant tumor growth delay when combined with radiation. Tissue type or cancer cell specific radiosensitization has the potential to substantively improve tumor control while also decreasing radiation-induced side effects. Exploiting cell surface targeted RNA aptamers designed to deliver siRNAs is one powerful path to this goal. Our previous work demonstrated that intratumoral injections of an in vitro transcribed PSMA aptamer-shDNA-PK chimera reduces DNA-PK mRNA and protein resulting in enhanced therapeutic response to radiation. To our knowledge, the data presented here represent the first data to demonstrate that chemically synthesized PSMA aptamer-siDNA-PK chimeras can also be constructed, be systemically delivered, successfully target PSMA-expressing PCa, drive reductions in DNA-PK mRNA and protein levels, culminating in radiation sensitization. These data support continued development of targeted RNA chimeras and testing in clinical trials.