{"doi":"10.1101/2025.06.06.658308","title":"Imaging CRISPR-Edited CAR-T Cell Therapies with Optical and Positron Emission Tomography Reporters","abstract":"Abstract 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 the potency and safety of CAR-T cells. However, several challenges persist, including inefficient tumor homing and treatment-related toxicities in normal tissues, which continue to hinder widespread adoption. Advanced imaging technologies, including bioluminescence imaging (BLI) and positron emission tomography (PET), provide real-time insights into CAR-T cell distribution and activity in vivo, both in preclinical models and in patients. 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) were cloned downstream of the CAR in adeno-associated virus (AAV) donors for BLI or PET tracking, respectively. CARs with imaging reporters were knocked into the TRAC locus of primary human T cells via CRISPR editing and AAV transduction. Editing efficiency was evaluated by flow cytometry and junction PCR. In vitro cytotoxicity was assessed by BLI using firefly luciferase (Fluc)-expressing cancer cells co-cultured with CAR-T cells at varying effector-to-target ratios. In vivo, BLI and PET imaging assessed CAR-AkaLuc and CAR-NIS T cell expansion and trafficking in Nod-SCID-gamma mice bearing xenograft tumors. Results T cell receptor (TCR) knockout efficiency exceeded 85%, with CAR expression observed in 70–80% of cells, depending on the reporter used. Reporter-engineered CAR-T cells retained functionality in vitro and exhibited significant cytotoxicity against target cancer cells, outperforming naïve T cells. In vivo, AkaLuc BLI and 18 F-tetrafluoroborate PET enabled non-invasive tracking of viable CAR-T cells. Notably, the route of administration (intravenous, peritumoral, or intraperitoneal) significantly influenced the distribution of CAR-T cells and their therapeutic effectiveness. Conclusion tRACE-CAR enabled precise optical and PET tracking of CAR-T cells in models of B cell leukemia and ovarian cancer, allowing dynamic, non-invasive monitoring of cell distribution in both tumors and off-target tissues. This imaging platform could lead to more personalized, effective CRISPR-edited CAR cell therapies.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":567779,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9468,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1248133,"name":"John J. Kelly","orcid":"0000-0002-3626-8629","position":1,"is_corresponding":false},{"id":1248132,"name":"Nourhan Shalaby","orcid":"0000-0001-9264-7191","position":2,"is_corresponding":false},{"id":1146164,"name":"Ying Xia","orcid":"0000-0002-0126-6594","position":3,"is_corresponding":false},{"id":1472344,"name":"Francisco Manuel Martinez-Santiesteban","orcid":null,"position":4,"is_corresponding":false},{"id":790474,"name":"Jasmine Lau","orcid":null,"position":5,"is_corresponding":false},{"id":1472345,"name":"Ivy Verriet","orcid":null,"position":6,"is_corresponding":false},{"id":1248135,"name":"Matthew S. Fox","orcid":"0000-0002-5121-4661","position":7,"is_corresponding":false},{"id":512065,"name":"Justin W. Hicks","orcid":"0000-0003-0269-9364","position":8,"is_corresponding":false},{"id":1248136,"name":"Jonathan D. Thiessen","orcid":"0000-0001-6132-3020","position":9,"is_corresponding":false},{"id":630570,"name":"John A. Ronald","orcid":"0000-0003-3665-173X","position":10,"is_corresponding":false},{"id":1248134,"name":"Rafael E. Sanchez‐Pupo","orcid":"0000-0002-6970-2137","position":0,"is_corresponding":true}],"reference_count":85,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:56:48.183032Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}