Abstract 2584: Exosome molecular reference standards (Exo-Ref) generated from CRISPR/Cas9 engineered cell Lines for oncology diagnostic applications
Authors: Mani Vigneshwaran , Thakker Suhani , Sengupta Debapriya , Kondapalli Sravanthi , Roma Gianluca , Zheng Qi , Saddar Sonika 
Published: August 2019

Abstract
Introduction: Exosomes (commonly known as extracellular vesicles) have generated tremendous interest for the therapeutic and diagnostic applications for oncology and other diseases. Specifically, exosome molecular cargo (proteins, nucleic acid, and small molecules) profiling have been subject of intense research for potential biomarkers for cancer. Currently, there exist only few exosomal nucleic acid (ExoRNA) biomarkers that have been realized for cancer diagnostics and treatment monitoring. This is due to the lack of exosome reference standards for assay development, assay performance validation, and interpretation of results. Exo-Ref products are ideal reference standards, since they mimic physical properties and genomic composition of exosomes isolated from patient biofluids. Further, Exo-Ref are biologically relevant, reproducible, and uses engineered cell lines that have previously demonstrated a source of reference materials (ctDNA, FFPE, and cells). We have developed Exo-Ref featuring RNA SNPs including point mutations in EGFR (c.2369C&gt;T, c.2573T&gt;G), NRAS (c.181C&gt;A), and PIK3A(c.1633G&gt;A). Herein we present the isolation of exosomes from engineered cell lines and characterization of exo-RNA SNPs transcripts using RT-digital PCR and Next Generation Sequencing (NGS). The validation of variants using the two methods show a great promise for Exo-Ref products for oncology exosome-based diagnostic applications. Methods: CRISPR/Cas9 targeting reagents were transfected into either HCT116 or RKO cell line. Exosomes are produced by culturing the cells in exosome free culture media. Exosomes were then isolated from ExoRNeasy kit (Qiagen). For genetic analysis, exo-RNA was isolated from using trizol/membrane filter using the ExoRNeasy kit and allelic rare mutations in RNA were verified by RT dPCR and validated by NGS. Results: Engineered, homozygous clones were conclusively identified by Sanger Sequencing. We were able to reproducibly achieve fragmentation profile of exo-RNA centered at approximately 25bp. Digital RT-PCR validated cellular and exosome mutant transcripts. Targeted variants including EGFR-T790M, EGFR-L858R, PIK3CA-E45K, NRAS-Q61K showed measurable copies of EV-RNA and cell-RNA from engineered cells. Digital PCR verified variants in exo-RNA was also detected by NGS and were consistent with dPCR. NGS sequence validation of exo-RNA transcript were verified at 100% mutation frequency. Conclusion CRISPR/Cas9 engineered cell lines are biologically relevant materials and allow for a reproducible and renewable source of Exo-RNA reference standards for assay development and quality control for use in clinical exosome-based diagnostic workflows.
Citation Format: Vigneshwaran Mani, Suhani Thakker, Debapriya Sengupta, Sravanthi Kondapalli, Gianluca Roma, Qi Zheng, Sonika Saddar. Exosome molecular reference standards (Exo-Ref) generated from CRISPR/Cas9 engineered cell Lines for oncology diagnostic applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2584.