Applicability of reference cells in lentiviral vector integration site detection with different methods
10.3760/cma.j.cn112309-20220407-00101
- VernacularTitle:慢病毒载体整合位点检测方法的系统适应性对照品初步研究
- Author:
Xiaoya ZHOU
1
;
Fangying JIA
;
Xueling WU
;
Kehua ZHANG
;
Shufang MENG
Author Information
1. 中国食品药品检定研究院生物制品检定所细胞资源保藏研究中心，国家卫生健康委员会生物技术产品检定方法及其标准化重点实验室，北京 100050
- Keywords:
Lentiviral vector;
Integration site;
ddPCR;
High-throughput sequencing;
Optical genome mapping
- From:
Chinese Journal of Microbiology and Immunology
2023;43(10):791-801
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To evaluate the feasibility of 8E5 cells and CD19-CAR-Jurkat cells used as reference cells in the detection of lentiviral vector integration sites with different methods.Methods:Single clones of 8E5 cells and CD19-CAR-Jurkat cells were selected using limiting dilution method. Digital PCR was established to detect the copy number of HIV-1 in 8E5 cells and the copy number of CAR in CD19-CAR-Jurkat cells. High-throughput sequencing techniques (whole-genome resequencing, modified genome sequencing and probe hybridization capture) were used to detect integration sites in 8E5 cells and CD19-CAR-Jurkat cells, and optical genome mapping (OGM) technology was used for further confirmation.Results:Three clones of 8E5-D8 cells and six clones of CD19-CAR-Jurkat 2-6 cells were selected using the limiting dilution method. 8E5-D8 and CD19-CAR-Jurkat 2-6 were chosen as candidate cells based on their gene copy numbers detected by digital PCR and flow cytometry. These cells were then expanded and cryopreserved. Digital PCR showed that 8E5-D8 cells contained approximately 1 copy per cell, while CD19-CAR-Jurkat 2-6 cells contained approximately 13 copies per cell. High-throughput sequencing revealed one integration site in 8E5 cells and 13 integration sites in CD19-CAR-Jurkat cells, which matched the copy number detection results. All these integration sites were further confirmed at the submicroscopic level of chromosomes using OGM.Conclusions:Based on the insertion copy numbers and integration sites, 8E5-D8 cells and CD19-CAR-Jurkat 2-6 cells could be used as reference cells in further development of methods for detecting integration sites in CAR-T cell lentiviral vectors.
