Amelioration of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration
Amelioration of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration
Site-specific integration of exogenous gene through genome editing is a promising strategy for gene therapy. However, homology-directed repair (HDR) only occurring in proliferating cells is inefficient especially in vivo. To investigate the efficacy of Cas9-induced homology-independent targeted integration (HITI) strategy for gene therapy, a rat hemophilia B model was generated and employed. Through HITI, a DNA sequence encoding the last exon of rat Albumin (rAlb) gene fused with a high-specific-activity Factor IX variant (R338L) using T2A, was inserted into the last intron of rAlb via recombinant adeno-associated viral (rAAV). The knock-in efficiency reached up to 3.66% determined by ddPCR. The clotting time was reduced to normal level 4 weeks after treatment, and the circulating FIX level was gradually increased up to 52% of normal during 9 months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through PEM-seq, no significant off-targeting effect was detected. Moreover, this study provides a promising therapeutic approach for hereditary diseases.
Shao Yanjiao、Wei Yu、Sayed Ahmed、Li Dali、Chen Xi、Zheng Rui、Wang Liren、Yin Shuming、Yang Lei、Niu Xuran、Hu Jiazhi、Jing Fengxiang、Liu Meizhen、Liu Mingyao、Zhang Xiaohui、Ma Xueyun、Liu Yang、Pan Jiahao、Lu Jian
Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal UniversityShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal UniversityShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University||Biochemistry Laboratory, Chemistry Department, Faculty of Science, Assiut UniversityShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal UniversityShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal UniversityXinhua Hospital, Shanghai Jiao Tong University School of MedicineShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal UniversityShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal UniversityShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal UniversityShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal UniversityThe MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking UniversityShanghai Turtle Technology CoShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal UniversityShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal UniversityShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal UniversityShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal UniversityThe MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking UniversityShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal UniversityShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University
医学研究方法基础医学生物科学研究方法、生物科学研究技术
Hemophilia BGene therapyCRIPSR/Cas9HITIAlbuminPEM-seq
Shao Yanjiao,Wei Yu,Sayed Ahmed,Li Dali,Chen Xi,Zheng Rui,Wang Liren,Yin Shuming,Yang Lei,Niu Xuran,Hu Jiazhi,Jing Fengxiang,Liu Meizhen,Liu Mingyao,Zhang Xiaohui,Ma Xueyun,Liu Yang,Pan Jiahao,Lu Jian.Amelioration of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration[EB/OL].(2025-03-28)[2025-06-07].https://www.biorxiv.org/content/10.1101/2021.03.18.435908.点此复制
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