Ju, Anna Lee, Sung Won Lee, Young Eun Han, Ki-Cheol Kim, Jin-Chul Shin, Sang Chul Park, Hyun Jung Kim, Eunice EunKyeong Hong, Seokmann Jang, Mihue 2024-01-19T19:03:35Z 2024-01-19T19:03:35Z 2021-09-04 2019-10 0142-9612 https://pubs.kist.re.kr/handle/201004/119525 Genetically engineered cells via CRISPR/Cas9 system can serve as powerful sources for cancer immunotherapeutic applications. Furthermore, multiple genetic alterations are necessary to overcome tumor-induced immune-suppressive mechanisms. However, one of the major obstacles is the technical difficulty with efficient multiple gene manipulation of suspension cells due to the low transfection efficacy. Herein, we established a carrier-free multiplexed gene editing platform in a simplified method, which can enhance the function of cytotoxic CD8(+) T cells by modulating suspension cancer cells. Our multiple Cas9 ribonucleoproteins (RNPs) enable simultaneous disruption of two programmed cell death 1 (PD-1) ligands, functioning as negative regulators in the immune system, by accessing engineered Cas9 proteins with abilities of complexation and cellular penetration. In addition, combination with electroporation enhanced multiple gene editing efficacy, compared with that by treatment of multiple Cas9 RNPs alone. This procedure resulted in high gene editing at multiple loci of suspension cells. The treatment of multiple Cas9 RNPs targeting both ligands strongly improved Th1-type cytokine production of cytotoxic CD8(+) T cells, resulting in synergistic cytotoxic effects against cancer. Simultaneous suppression of PD-L1 and PD-L2 on cancer cells via our developed editing system allows effective anti-tumor immunity. Furthermore, the treatment of multiple Cas9 RNPs targeting PD-L1, PD-L2, and TIM-3 had approximately 70-90% deletion efficacy. Thus, our multiplexed gene editing strategy endows potential clinical utilities in cancer immunotherapy. English ELSEVIER SCI LTD TUMOR-CELLS PD-L1 CANCER BLOCKADE LIGANDS IMMUNOTHERAPY INTERNALIZATION PEMBROLIZUMAB EXPRESSION RESPONSES A carrier-free multiplexed gene editing system applicable for suspension cells Article 10.1016/j.biomaterials.2019.119298 1 BIOMATERIALS, v.217 BIOMATERIALS 217 scie scopus 000477786200032 2-s2.0-85068263394 Engineering, Biomedical Materials Science, Biomaterials Engineering Materials Science Article TUMOR-CELLS PD-L1 CANCER BLOCKADE LIGANDS IMMUNOTHERAPY INTERNALIZATION PEMBROLIZUMAB EXPRESSION RESPONSES CRISPR/Cas9 Cas9 RNP Multiplexed gene editing Suspension cells Immune checkpoints Cancer immunotherapy