Imaging CRISPR-edited CAR-T cell therapies with optical and positron emission tomography reporters.

Rationale: Chimeric antigen receptor (CAR) T cell therapies have shown remarkable success in treating hematological cancers and are increasingly demonstrating potential for solid tumors. CRISPR-based genome editing offers a promising approach to enhance CAR-T cell potency and safety, yet challenges such as inefficient tumor homing and toxicities in normal tissues, limit broader adoption. Advanced imaging technologies, including bioluminescence imaging (BLI) and positron emission tomography (PET), provide real-time visualization of CAR-T cell behavior in vivo. Here, we developed Trackable Reporter Adaptable CRISPR-Edited CAR (tRACE-CAR) T cells, a modular system for site-specific integration of CARs and imaging reporters. Methods: The luciferase reporter AkaLuciferase (AkaLuc) or the human sodium iodide symporter (NIS) was cloned downstream of the CAR in adeno-associated virus (AAV) donors for imaging. CAR-reporter cassettes were inserted into the T-cell receptor α constant locus of primary human T cells via CRISPR editing and AAV transduction. Editing efficiency was assessed by flow cytometry. In vitro cytotoxicity was evaluated across multiple effector-to-target ratios. In vivo, BLI and PET imaging were used for tracking CAR-T cells in tumor-bearing immunodeficient mice. Results: T cell receptor (TCR) knockout efficiency exceeded 85% and CAR expression reached 70-80%. Reporter-engineered CAR-T cells exhibited significant cytotoxicity and outperformed naïve T cells. In vivo, AkaLuc BLI and (18)F-tetrafluoroborate PET enabled non-invasive tracking of viable CAR-T cells. Administration route (intravenous, peritumoral, or intraperitoneal) significantly influenced CAR-T cell distribution and therapeutic effectiveness. Conclusion: tRACE-CAR enables precise optical and PET tracking of CRISPR-edited CAR-T cells in models of leukemia and ovarian cancer, allowing dynamic, non-invasive monitoring of cell distribution in both tumors and off-target tissues. This imaging-enabled platform could lead to more personalized, effective CRISPR-edited CAR cell therapies.