한빛사 논문
Anna Jua,1, Sung Won Leeb,1, Young Eun Leea,c, Ki-Cheol Hana, Jin-Chul Kimd, Sang Chul Shina, Hyun Jung Parkb, Eunice EunKyeong Kima, Seokmann Hongb,*, Mihue Janga,*
a Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul, 02792, Republic of Korea
b Department of Integrative Bioscience and Biotechnology, Institute of Anticancer Medicine Development, Sejong University, Seoul, 05006, Republic of Korea
c Department of Life Sciences, College of Life Sciences and Biotechnology, Korea University, Seoul, 136-701, Republic of Korea
d Natural Constituents of Research Center, Natural Products Research Institute, Korea Institute of Science and Technology (KIST), Gangneung, Republic of Korea
*Corresponding author : Seokmann Hong, Mihue Jang
1Anna Ju and Sung Won Lee contributed equally to this work.
Abstract
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.
Keywords : CRISPR/Cas9; Cas9 RNP; Multiplexed gene editing; Suspension cells; Immune checkpoints; Cancer immunotherapy
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