부산대학교 도서관

CAR T cell immunotherapies demonstrate great success for treating various types of hematological malignancies. Currently, CAR T cell products are individually designed for each antigen target. Using an adenine base editor (BE), we recently developed an epitope-masked chimeric antigen receptor approach targeting the pan-leukocyte antigen CD45 to generate a broadly applicable product effective against diverse hematologic malignancies. Intracellular delivery of gene editing reagents such as CRISPR BE is usually achieved with electroporation, a physical method that impairs T cell viability. We hypothesized that CRISPR BE delivered using lipid nanoparticle (LNP) could preserve T cell number and function, resulting in a superior product for ultimate clinical manufacturing, and sought to optimize a protocol for lentivirally transduced, BE CART cells. We evaluated combinations of LNP formulations, culture media compositions, and the order of genetic modifications to achieve high editing efficiency without impairing T cell viability, proliferative capacity, and CAR expression. The base editing components including the ABE8e base editor mRNA and the CD45 targeting sgRNA were encapsulated in LNPs using the NanoAssemblr instrument with high efficiency (98 ± 3%). Two days after stimulation, primary T cells were treated with LNPs or electroporated (EP) to deliver equivalent amounts (8ug) of gene editing cargo followed by lentiviral transduction of a 2nd generation 41BB costimulated CAR45. The optimized protocol led to efficient LNP-mediated bi-allelic base editing of the targeted CD45 epitope (82 ± 2%) and clinically relevant CAR45 expression (20 ± 12%). Transcriptomic analysis and cytokine profiling revealed higher cytotoxic ability of LNP-CART cells compared to EP-CART. Furthermore, the LNP-edited CAR45 T cells demonstrated enhanced proliferation capacity (about 2-fold compared to EP CART) and potent cytotoxicity against AML cells in co-culture. Here, we demonstrate that LNP-mediated delivery of base editors enables the safe and potent generation of universal CD45-redirected CAR T cells, while streamlines the manufacturing procedure and improves cell yield. [ABSTRACT FROM AUTHOR]