Co‐Delivery of Multiple RNAs via Lipid Nanoparticles Enables Precise Gene Editing of CAR‐T Cells

Abstract Over the past decade, CAR‐T cell therapy has achieved remarkable success in treating hematological malignancies. However, traditional CAR‐T cell engineering employs viral vectors, which has several limitations. Additionally, the immunosuppressive tumor microenvironment, particularly mediated by the PD‐1/PD‐L1 pathway, significantly restricts CAR‐T cell efficacy. CRISPR/Cas9‐mediated PD‐1 knockout can enhance CAR‐T cell anti‐tumor activity, but traditional electroporation (EP) method often damages T cells. Herein, a novel lipid nanoparticles (LNPs)‐mediated delivery technology are introduced to engineer CAR‐T cells. The LNPs platform enables the simultaneous expression of CAR cassette and CRISPR/Cas9 gene editor in T cells via co‐delivery of multiplex RNAs (CD19 CAR mRNA+Cas9 mRNA+sgRNA targeting PD‐1). Importantly, LNPs exhibit higher transfection efficiency and superior cell viability compared to traditional electroporation method. The engineered CAR‐T cells with PD‐1 knockout, which express anti‐CD19 CAR, can specifically kill CD19+ Nalm‐6 tumor cells in vitro and display enhanced anti‐tumor activity in vivo. Furthermore, LNPs‐mediated co‐delivery of Cas9 mRNA and sgRNAs targeting PD‐1, TRAC, and B2M enables triple‐knockout of T cells with high editing efficiencies (76% for PD‐1, 86% for TRAC, and 80% for B2M), highlighting the ability for multiplex gene editing. This LNP‐mediated delivery strategy has great potentials for the development of safer and more efficacious CAR‐T cells.