Ontology highlight
ABSTRACT: Background
Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin (Alb). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%).Results
We report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely reconstituting serum F8 activity in a mouse model of hemophilia A after hydrodynamic injection of Cas9-sgAlb and B domain-deleted (BDD) F8 donor plasmids. We find that the integration of a double-cut donor at the Alb locus in mouse liver is mainly through non-homologous end joining (NHEJ)-mediated knock-in. We then target BDDF8 to multiple sites on introns 11 and 13 and find that NHEJ-mediated insertion of BDDF8 restores hemostasis. Finally, using 3 AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and BDDF8 donor, we observe the same therapeutic effects. A follow-up of 100 mice over 1 year shows no adverse effects.Conclusions
These findings lay the foundation for curing hemophilia A by NHEJ knock-in of BDDF8 at Alb introns after AAV-mediated delivery of editing components.
SUBMITTER: Zhang JP
PROVIDER: S-EPMC6912951 | biostudies-literature | 2019 Dec
REPOSITORIES: biostudies-literature
Zhang Jian-Ping JP Cheng Xin-Xin XX Zhao Mei M Li Guo-Hua GH Xu Jing J Zhang Feng F Yin Meng-Di MD Meng Fei-Ying FY Dai Xin-Yue XY Fu Ya-Wen YW Yang Zhi-Xue ZX Arakaki Cameron C Su Ruijun Jeanna RJ Wen Wei W Wang Wen-Tian WT Chen Wanqiu W Choi Hannah H Wang Charles C Gao Guangping G Zhang Lei L Cheng Tao T Zhang Xiao-Bing XB
Genome biology 20191216 1
<h4>Background</h4>Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin (Alb). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%).<h4>Results</h4>We report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely recon ...[more]