Specific targeting of point mutations in EGFR L858R-positive lung cancer by CRISPR/Cas9.

Autor: Cheung AH; Department of Anatomical and Cellular Pathology, State Key Laboratory of Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China., Chow C; Department of Anatomical and Cellular Pathology, State Key Laboratory of Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China., Zhang J; Department of Anatomical and Cellular Pathology, State Key Laboratory of Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China.; Institute of Digestive Disease, Partner State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China.; Li Ka Shing Institute of Health Science, Sir Y.K. Pao Cancer Center, The Chinese University of Hong Kong, Hong Kong, China., Zhou Y; Department of Anatomical and Cellular Pathology, State Key Laboratory of Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China.; Institute of Digestive Disease, Partner State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China.; Li Ka Shing Institute of Health Science, Sir Y.K. Pao Cancer Center, The Chinese University of Hong Kong, Hong Kong, China., Huang T; Department of Anatomical and Cellular Pathology, State Key Laboratory of Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China.; Institute of Digestive Disease, Partner State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China.; Li Ka Shing Institute of Health Science, Sir Y.K. Pao Cancer Center, The Chinese University of Hong Kong, Hong Kong, China.; Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China., Ng KC; Department of Anatomical and Cellular Pathology, State Key Laboratory of Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China., Or TC; Department of Anatomical and Cellular Pathology, State Key Laboratory of Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China., Yao YY; Department of Anatomical and Cellular Pathology, State Key Laboratory of Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China., Dong Y; Institute of Digestive Disease, Partner State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China.; Department of Surgery, The Chinese University of Hong Kong, Hong Kong, China., Fung JM; Department of Anatomical and Cellular Pathology, State Key Laboratory of Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China., Xiong L; School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China., Chan AK; Department of Anatomical and Cellular Pathology, State Key Laboratory of Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China., Lung WR; Department of Anatomical and Cellular Pathology, State Key Laboratory of Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China., Kang W; Department of Anatomical and Cellular Pathology, State Key Laboratory of Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China. weikang@cuhk.edu.hk.; Institute of Digestive Disease, Partner State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China. weikang@cuhk.edu.hk.; Li Ka Shing Institute of Health Science, Sir Y.K. Pao Cancer Center, The Chinese University of Hong Kong, Hong Kong, China. weikang@cuhk.edu.hk.; Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China. weikang@cuhk.edu.hk., To KF; Department of Anatomical and Cellular Pathology, State Key Laboratory of Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China. kfto@cuhk.edu.hk.; Institute of Digestive Disease, Partner State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China. kfto@cuhk.edu.hk.; Li Ka Shing Institute of Health Science, Sir Y.K. Pao Cancer Center, The Chinese University of Hong Kong, Hong Kong, China. kfto@cuhk.edu.hk.; Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China. kfto@cuhk.edu.hk.
Jazyk: angličtina
Zdroj: Laboratory investigation; a journal of technical methods and pathology [Lab Invest] 2018 Jul; Vol. 98 (7), pp. 968-976. Date of Electronic Publication: 2018 May 10.
DOI: 10.1038/s41374-018-0056-1
Abstrakt: Cancer cells are defined genetically by the mutations they harbor, commonly single nucleotide substitutions. Therapeutic approaches which specifically target cancer cells by recognizing these defining genetic aberrations are expected to exhibit minimal side-effects. However, current protein-based targeted therapy is greatly limited by the range of genes that can be targeted, as well as by acquired resistance. We hypothesized that a therapeutic oligonucleotide-based strategy may address this need of specific cancer targeting. We used CRISPR/Cas9 system to target a commonly occurring EGFR point mutation, L858R, with an oligonucleotide guide that recognizes L858R as the suitable protospacer-adjacent motif (PAM) sequence for DNA cleavage. We found that this strategy, which utilized PAM to differentiate cancer mutation from normal, afforded high specificity to the extent of a single nucleotide substitution. The anti-L858R vehicle resulted in selective genome cleavage only in L858R mutant cells, as detected by Sanger sequencing and T7 Endonuclease I assay. Wild-type cells were unaffected by the same treatment. Digital PCR revealed 37.9 ± 8.57% of L858R gene copies were targeted in mutant. Only treated mutant cells, but not wild-type cells, showed reduction in EGFR expression and decreased cell proliferation. Treated mutant cells also formed smaller tumor load in vivo. This targeting approach is expected to be able to target a significant subset of the 15-35% cancer mutations with C > G, A > G, and T > G point mutations. Thus, this strategy may serve as a useful approach to target cancer-defining mutations with specificity, to the extent of differentiating the change of a single nucleotide.
Databáze: MEDLINE
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