Abstract 2868: A novel fusogenic peptide for delivery of bioactive siRNAs targeting CSNK2A1 in ovarian cancer cells in vitro

Abstract

Abstract Introduction: Ovarian cancer recurrence and survival rates have remained stagnant over time with current therapeutics displaying low efficacy and often rendered ineffective by degradation. This work presents novel fusogenic peptides that will electrostatically complex with siRNAs and cause a pH-responsive insertion into the endosomal membrane, resulting in destabilization and breakdown allowing for release of siRNAs into the cytoplasm of ovarian cancer cells. Delivery of bioactive siRNAs targeting the CSNK2A1 gene in ovarian cancer cells is hypothesized to reduce cell viability, migration, and invasion. Experimental Procedures: Peptide formulations were complexed with non-targeting siRNAs (siNT) and CSNK2A1 siRNAs (siCSNK2A1) at various N:P ratios for all procedures. Analysis of complex formation was completed through gel-shift assays and dynamic light scattering. After characterization of the complexes, each formulation without siRNA was delivered to OVCAR-3 and CAOV-3 cells for cytotoxicity analysis measured via MTS assay. Observation of cellular uptake and endosomal escape was completed through fluorescent imaging after delivering each peptide formulation complexed with Cy3-labeled siNT (Cy3-siNT) and performing early endosome antigen-1 staining. To confirm bioactivity of targeted siRNAs, complexes with siCSNK2A1 were delivered and gene silencing was analyzed via western blotting. Results: Complexation of siRNAs with each formulation was achieved at N:P ratios of 40:1 and greater. Each of the three peptide formulations were able to form uniform nanocomplexes, at 195 nm, 111 nm, and 59 nm with 8.25 mV, -14.56 mV, and -18.42 mV surface charges respectively, and did not display vehicle cytotoxicity at an 80:1 ratio. Each peptide also mediated siRNA uptake into ovarian cancer cells, with fluorescent siRNA signal increasing with increasing N:P ratios. Of note, we observed that Cy3-siRNA appeared separate from the endosome, indicative of endosomal escape, which was seen in several peptide formulations with both CAOV-3 and OVCAR-3 cell lines. Confirmation of bioactive delivery of siCSNK2A1 is shown through western blot imaging, demonstrating knockdown of CSNK2A1 in comparison to cells treated with siNT alone or left untreated. Conclusion: Our novel fusogenic peptides have shown the ability to complex with and deliver siRNAs into ovarian cancer cells. Each peptide mediates endosomal escape activity, with varying degrees of effectiveness, and silencing of CSNK2A1 as measured by western blot. Further study into downstream effects of CSNK2A1 is ongoing. Future work includes optimization of a single fusogenic sequence for optimal delivery of siRNAs and development of a novel targeting peptide to increase cancer targeting and uptake efficiency. Acknowledgements: This research was supported in part by the National Science Foundation EPSCoR Program under NSF Award # OIA-1655740. Citation Format: Timothy Samec, Jessica Boulos, Serena Gilmore, Angela Alexander-Bryant. A novel fusogenic peptide for delivery of bioactive siRNAs targeting CSNK2A1 in ovarian cancer cells in vitro [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2868.