Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression
Year: 2022
Type: article
Abstract: Background Chimeric antigen receptor (CAR) T cells traditionally harbor viral vector-based sequences that encode the CAR transgene in the genome. These T cell products have yet to show consistent anti-tumor activity in patients with solid tumors. Further, viral vector manufacturing is resource intensive, suffers from batch-to-batch variability, and includes several animal components, adding regulatory and supply chain pressures. Methods Anti-GD2 CAR T cells were generated using CRISPR/Cas9 within nine days using recombinant Cas9 protein and nucleic acids, without any viral vectors or animal components. The CAR was specifically targeted to the T Cell Receptor Alpha Constant gene ( TRAC ). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, scRNA-seq, spectral cytometry, and ELISA assays. Functionality was evaluated in vivo in an NSG xenograft neuroblastoma model. Results In comparison to traditional retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC -targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory phenotype, and low tonic signaling prior to infusion arising in part from the the knockout of the TCR. Upon exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibited specific cytotoxicity against GD2+ cells in vitro and induced solid tumor regression in vivo , with robust homing, persistence, and low exhaustion against a human neuroblastoma xenograft model. Conclusions This proof-of-principle study leveraging virus-free genome editing technology could enable flexible manufacturing of clinically relevant, high-quality CAR T cells to treat cancers, including solid tumors.
Authors: Katherine P Mueller, Nicole J Piscopo, Matthew H Forsberg, Louise A Saraspe, Amritava Das, Brittany Russell, Madeline Smerchansky, Dan Cappabianca, Lei Shi, Keerthana Shankar, Lauren Sarko, Namita Khajanchi, Nina La Vonne Denne, Apoorva Ramamurthy, Adeela Ali, Cicera R Lazzarotto, Shengdar Q Tsai, Christian M Capitini, Krishanu Saha
Language: English
Topic: CAR-T cell therapy research
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,490
Funders: National Science Foundation, U.S. Department of Health and Human Services, American Association for Cancer Research, American Cancer Society, Wisconsin Alumni Research Foundation, Entertainment Industry Foundation, University of Wisconsin-Madison, University of Wisconsin Carbone Cancer Center, Midwest Athletes Against Childhood Cancer, National Institutes of Health, Stand Up To Cancer, National Human Genome Research Institute, National Cancer Institute, National Institute of General Medical Sciences, National Institute of Allergy and Infectious Diseases, Division of Graduate Education, Division of Chemical, Bioengineering, Environmental, and Transport Systems
Awards: Graduate Research Fellowship Program (GRFP), G1295252683, G1332332826, G1617846861, G1625130784, G1712026730, G1867304905, G2969218895, G3213234327, G3357929402, Combining hu14.18-IL2 and NK cell infusions to treat neuroblastoma, G388522380, G4004214011, G4305155978, Biotechnology Training Program, G4857410013, G5335812471, NSF Engineering Research Center for Cell Manufacturing Technologies (CMaT), BD FACSymphony High Parameter Flow Cytometer, HIV Protease Inhibitors and Hepatic Lipid Dysregulation, G5813506420, G6131128955, G6291298690, G7407334923, G7450968682, A novel human T-cell platform to define biological effects of genome editing, G7722500572, EAGER BIOMANUFACTURING: A Microscale Testbed to Assay and Manufacture CAR T-Cell Immunotherapies, G8838887969, G885864977, G9000352744, Biotechnology Training Program, UW Comprehensive Cancer Center Support, Assembly of Novel Gene Editing Particles to Understand Genome Surgery in Patient-Derived Cells, Institutional Training in the Genomic Sciences
OpenAlex ID: W4295022868