Long-term correction of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration

Xi Chen; Xuran Niu; Yang Liu; Rui Zheng; Lei Yang; Jian Lu; Shuming Yin; Yu Wei; Jiahao Pan; Ahmed Sayed; Xueyun Ma; Meizhen Liu; Fengxiang Jing; Mingyao Liu; Jiazhi Hu; Liren Wang; Dali Li

doi:10.1016/j.jgg.2022.06.001

Long-term correction of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration

J Genet Genomics. 2022 Dec;49(12):1114-1126. doi: 10.1016/j.jgg.2022.06.001. Epub 2022 Jun 9.

Authors

Xi Chen¹, Xuran Niu¹, Yang Liu², Rui Zheng³, Lei Yang¹, Jian Lu¹, Shuming Yin¹, Yu Wei¹, Jiahao Pan¹, Ahmed Sayed⁴, Xueyun Ma¹, Meizhen Liu¹, Fengxiang Jing⁵, Mingyao Liu¹, Jiazhi Hu⁶, Liren Wang⁷, Dali Li⁸

Affiliations

¹ Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
² The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China.
³ Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China.
⁴ Biochemistry Laboratory, Chemistry Department, Faculty of Science, Assiut University, Assiut 71516, Egypt.
⁵ Shanghai Turtle Technology Co., Shanghai 201900, China.
⁶ The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China. Electronic address: hujz@pku.edu.cn.
⁷ Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China. Electronic address: lrwang@bio.ecnu.edu.cn.
⁸ Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China. Electronic address: dlli@bio.ecnu.edu.cn.

PMID: 35691554
DOI: 10.1016/j.jgg.2022.06.001

Abstract

CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications. However, it is still infeasible because homologous recombination (HR) is inefficient, especially for non-dividing cells. To overcome the challenge, we report that a homology-independent targeted integration (HITI) strategy is used for permanent integration of high-specificity-activity Factor IX variant (F9 Padua, R338L) at the albumin (Alb) locus in a novel hemophilia B (HB) rat model. The knock-in efficiency reaches 3.66%, as determined by droplet digital PCR (ddPCR). The clotting time is reduced to a normal level four weeks after treatment, and the circulating factor IX (FIX) level is gradually increased up to 52% of the normal level over nine months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through primer-extension-mediated sequencing (PEM-seq), no significant off-target effect is detected. This study not only provides a novel model for HB but also identifies a promising therapeutic approach for rare inherited diseases.

Keywords: Alb; CRISPR/Cas9-mediated HITI; FIX; Gene therapy; Hemophilia B.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Cas Systems / genetics
Factor IX / genetics
Factor IX / metabolism
Factor IX / therapeutic use
Genetic Therapy
Hemophilia B* / drug therapy
Hemophilia B* / therapy
Rats

Substances

Factor IX