Abstract 2582: Advancing liquid biopsies via integrated CRISPR-based extracellular vesicle RNA and protein analyses

Abstract BACKGROUND: We developed REPAIR, an integrated urine-based platform combining extracellular vesicle (EV) mRNA and protein profiling with CRISPR to enable non-invasive, multiparametric disease monitoring. Bladder cancer, our clinical use case, remains the costliest cancer to surveil and treat given the high mortality when advanced. Current surveillance of localized disease relies on invasive cystoscopy, lacking the molecular specificity for risk stratification and frequent treatment monitoring. METHODS: We significantly advanced our recent mRNA-focused CRISPR-Cas13 assay achieving sub-attomolar sensitivity and single-nucleotide resolution in plasma (Nature Biotech, 2025 [cover article]; PMID: 39375445), by: (1) integrating dual EV mRNA plus protein detection; (2) automating sample processing with innovative disc-based microfluidics we developed; and (3) translating to urine as the clinical matrix. As proof-of-concept, we designed REPAIR probes targeting EV proteins (CD63, EGFR, EpCAM, PD-L1) and mRNAs (GAPDH, LASS2, GALNT1) also relevant to bladder cancer. For protein detection, aptamers were used as affinity ligands. These aptamers were initially hybridized with complementary RNA sequences (initiator RNAs). When the aptamers bound to targets, the initiator RNAs were released and detected through REPAIR. A non-aptamer based strategy was employed for mRNA detection. Urine samples from 85 bladder cancer patients (mostly non-muscle invasive) undergoing treatment and 98 healthy controls were analyzed using the unified REPAIR protocol. RESULTS: Multiparametric profiling revealed significantly elevated marker expression in cancer versus control urine (p<0.05 for all markers). Integrated logistic regression combining protein and mRNA markers achieved 98% detection accuracy (AUC=0.95; 95% CI: 0.92-0.98). Single-marker analysis showed inferior performance, demonstrating the critical value of a multiparametric CRISPR-based EV approach. Integrated readouts were achieved within 60 minutes. CONCLUSION: REPAIR greatly facilitates simultaneous EV protein-RNA profiling in urine—a non-invasive, scalable liquid biopsy for bladder cancer surveillance and treatment monitoring. These findings potentially establish a nanotechnology-based platform for same-visit precision monitoring in a disease with an extraordinary economic burden and surveillance challenges. Current experiments in other malignancies where urine serves as a proximal fluid are ongoing. Citation Format: Cesar M. Castro, Jayeon Song, Hakho Lee. Advancing liquid biopsies via integrated CRISPR-based extracellular vesicle RNA and protein analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2582.