Empowering cancer detection and monitoring with CampEx - CRISPR-accelerated molecular profiling of Extracellular vesicles

Extracellular vesicles (EVs) have emerged as potent analytes in liquid biopsy, presenting diverse molecular cargo that can potentially inform somatic mutations, resistance profiles, and tumor recurrence. However, EVs remain underutilized in clinical assays due to technical challenges. Tumor-derived EVs comprise a small fraction (<5%) of total circulating EVs, and the marker contents in EVs are often very low (e.g., mRNA). Consequently, current EV analyses necessitate large sample volumes (>2 mL plasma) and sophisticated resources. Innovation. We aim to address these limitations by advancing CampEx (CRISPR-accelerated molecular profiling of extracellular vesicles), an integrated platform for EV molecular analyses with exquisite sensitivity and precision. CampEx embodies our new breakthrough in CRISPR-based biosensing: CRISPR- associated (Cas) proteins first recognize their specific mRNA target, an event that triggers RNA replication and signal amplification. This unique mechanism enables CampEx to differentiate between single-nucleotide polymorphisms while achieving a detection limit in the sub-attomolar ranges. Goals. Our recent study validated the CampEx concept in pre-clinical and clinical samples. Building on these promising results, we aim to advance CampEx to a robust, clinically translatable diagnostic platform. We seek two complementary objectives: i) enhancing CampEx's analytical capabilities to enable high-throughput and comprehensive EV analysis and ii) rigorously evaluating EVs' value for precision oncology. Aim 1. We will expand CampEx's analytical targets (>40 markers). The mRNA detection panel will include i) key driver genes and mutations that account for >90% of cancer cases and ii) markers of chemotherapy resistance. Furthermore, we will incorporate protein detection into the CampEx assay flow, capitalizing on EVs' inherent advantage as multi- cargo carriers. We will validate the designed probes, adhering to regulatory (FDA) guidelines to obtain analytical statistics. Aim 2. We will implement a high-throughput, automated CampEx instrument capable of performing 96 parallel measurements. This new system will incorporate our novel "digital modulation" strategy to enhance the robustness of signal detection. Completing this aim will produce a practical system suitable for routine laboratory applications. Aim 3. We will apply CampEx to analyze clinical plasma samples for early tumor detection and treatment monitoring, focusing on two cancer types: ovarian cancer and colorectal cancer. This study will determine CampEx's clinical assay performance, including sensitivity, specificity, and correlation with clinical outcomes. To accomplish our goal, we have assembled an interdisciplinary team (biosensing, EV biology, clinical research, bioinformatics) and clinical resources. Completing this project will establish CampEx as an impactful platform, enabling multi-marker detection in a cohesive single platform. Ultimately, CampEx will expedite preclinical and clinical readouts, thus augmenting EVs' clinical utility in precision cancer care. Project Number: 1R01CA304204-01 | Fiscal Year: 2025 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Hakho Lee (+1 co-PI) | Institution: MASSACHUSETTS GENERAL HOSPITAL, BOSTON, MA | Award Amount: $2,855,619 | Activity Code: R01 | Study Section: Enabling Bioanalytical and Imaging Technologies Study Section[EBIT] View on NIH RePORTER: https://reporter.nih.gov/project-details/11202820