Abstract 6241: PAX8-directed nanotherapeutics for high-grade serous ovarian carcinoma

Abstract

Abstract Objective: High-grade serous ovarian cancer (HGSOC) is the most common epithelial ovarian cancer subtype. Targeted therapy for HGSOC is challenging because the disease is characterized by copy number variation rather than recurrent somatic mutations. HGSOC displays well-conserved epigenetic features such as expression of transcription factor PAX8. We propose employing epigenetic therapies that capitalize on interactions between PAX8 and specific DNA-binding sites to develop potential therapeutic approaches to target HGSOC. Our hypothesis is to use a biomimetic approach wherein PAX8 is induced to transcribe an ectopically transfected CRISPR-base editor (BE)-guide RNA (gRNA) plasmid containing a PAX8 binding site in its promoter. The gRNA will impart a dominant-negative allele of Polo Like Kinase 1 (PLK1) which has been shown to be synergistically lethal with deficient tp53 in cancer cells. In the presence of PAX8, the plasmid will be transcribed, while in the absence of PAX8, the plasmid is degraded, with minimal toxicity observed in healthy cells. Methods: PAX8-expressing ovarian cancer cell lines including COV362, Kuramochi, OVCAR8 and OVSAHO or PAX8-negative control cell lines including JEG3, MCF-7, HeLa and HEK293T were transfected with polyplexes bearing the plasmid of interest. Polyplex diameter, polydispersity indices, and surface zeta potentials were measured on a Malvern Zetasizer. Polyplexes were imaged for uptake and functionality using fluorescence microscopy and Amnis ImageStream. Polyplex toxicity was assessed in vitro using cell viability assays. Preliminary in vivo experiments to demonstrate luciferase plasmid-bearing polyplexes have been performed in NOD-SCID mice. To monitor in vivo transfection efficiencies, we will deliver fluorescently labeled plasmids or luciferase-encoding plasmids to test bio-distribution and pharmacokinetics. Results: We have synthesized sub-200nm (diameter) polyplexes using the polyamine PPLG-g-azidopropylamine (PPLG) and BE or gRNA plasmids. Using -/-PLK1 gRNA, we observed that the BE and the gRNA can be efficiently packaged into two distinct polymeric nanoplexes and co-delivered to ovarian cancer cells or control cells to impart a dominant-negative mutant of PLK1, which is also synthetically lethal with mutated tp53, as observed from cancer cell death compared to controls. Ongoing work includes repetitive polyplex injections in vitro and in vivo, quantifying percentage of total sequencing reads with target base pair conversions after treatment, determination of plasmid loading efficiency, and testing gRNAs against other targets for efficacy in ovarian cancer targeting. Conclusion: Well-defined lineage transcription factors have the potential to serve as novel targets for gene-editing therapies in ovarian cancer and other malignancies. Combining nanotechnology and gene editing can improve specificity toward ovarian cancer cells as well as incorporate precision therapy aimed at the underlying genetic architecture of this disease. Citation Format: Akshaya Chandrasekaran, Mariam Ahmed, Wade Wang, Paula T. Hammond, Kevin M. Elias. PAX8-directed nanotherapeutics for high-grade serous ovarian carcinoma [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 6241.