Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse
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 integrati...
Main Authors: | , , , , , , , , , , , , , , , , , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
BMC
2019-12-01
|
Series: | Genome Biology |
Subjects: | |
Online Access: | https://doi.org/10.1186/s13059-019-1907-9 |
id |
doaj-0175546634cc4b5da1856d314090ad7d |
---|---|
record_format |
Article |
spelling |
doaj-0175546634cc4b5da1856d314090ad7d2020-12-20T12:39:35ZengBMCGenome Biology1474-760X2019-12-0120111710.1186/s13059-019-1907-9Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouseJian-Ping Zhang0Xin-Xin Cheng1Mei Zhao2Guo-Hua Li3Jing Xu4Feng Zhang5Meng-Di Yin6Fei-Ying Meng7Xin-Yue Dai8Ya-Wen Fu9Zhi-Xue Yang10Cameron Arakaki11Ruijun Jeanna Su12Wei Wen13Wen-Tian Wang14Wanqiu Chen15Hannah Choi16Charles Wang17Guangping Gao18Lei Zhang19Tao Cheng20Xiao-Bing Zhang21State Key Laboratory of Experimental HematologyState Key Laboratory of Experimental HematologyState Key Laboratory of Experimental HematologyState Key Laboratory of Experimental HematologyState Key Laboratory of Experimental HematologyState Key Laboratory of Experimental HematologyState Key Laboratory of Experimental HematologyState Key Laboratory of Experimental HematologyState Key Laboratory of Experimental HematologyState Key Laboratory of Experimental HematologyState Key Laboratory of Experimental HematologySchool of Medicine, Loma Linda UniversitySchool of Medicine, Loma Linda UniversityState Key Laboratory of Experimental HematologyState Key Laboratory of Experimental HematologyCenter for Genomics, School of Medicine, Loma Linda UniversityCenter for Genomics, School of Medicine, Loma Linda UniversityCenter for Genomics, School of Medicine, Loma Linda UniversityHorae Gene Therapy Center, University of Massachusetts Medical SchoolState Key Laboratory of Experimental HematologyState Key Laboratory of Experimental HematologyState Key Laboratory of Experimental HematologyAbstract 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.https://doi.org/10.1186/s13059-019-1907-9Hemophilia ACRISPR-Cas9Genome editingKnock-inNHEJ |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Jian-Ping Zhang Xin-Xin Cheng Mei Zhao Guo-Hua Li Jing Xu Feng Zhang Meng-Di Yin Fei-Ying Meng Xin-Yue Dai Ya-Wen Fu Zhi-Xue Yang Cameron Arakaki Ruijun Jeanna Su Wei Wen Wen-Tian Wang Wanqiu Chen Hannah Choi Charles Wang Guangping Gao Lei Zhang Tao Cheng Xiao-Bing Zhang |
spellingShingle |
Jian-Ping Zhang Xin-Xin Cheng Mei Zhao Guo-Hua Li Jing Xu Feng Zhang Meng-Di Yin Fei-Ying Meng Xin-Yue Dai Ya-Wen Fu Zhi-Xue Yang Cameron Arakaki Ruijun Jeanna Su Wei Wen Wen-Tian Wang Wanqiu Chen Hannah Choi Charles Wang Guangping Gao Lei Zhang Tao Cheng Xiao-Bing Zhang Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse Genome Biology Hemophilia A CRISPR-Cas9 Genome editing Knock-in NHEJ |
author_facet |
Jian-Ping Zhang Xin-Xin Cheng Mei Zhao Guo-Hua Li Jing Xu Feng Zhang Meng-Di Yin Fei-Ying Meng Xin-Yue Dai Ya-Wen Fu Zhi-Xue Yang Cameron Arakaki Ruijun Jeanna Su Wei Wen Wen-Tian Wang Wanqiu Chen Hannah Choi Charles Wang Guangping Gao Lei Zhang Tao Cheng Xiao-Bing Zhang |
author_sort |
Jian-Ping Zhang |
title |
Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse |
title_short |
Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse |
title_full |
Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse |
title_fullStr |
Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse |
title_full_unstemmed |
Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse |
title_sort |
curing hemophilia a by nhej-mediated ectopic f8 insertion in the mouse |
publisher |
BMC |
series |
Genome Biology |
issn |
1474-760X |
publishDate |
2019-12-01 |
description |
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. |
topic |
Hemophilia A CRISPR-Cas9 Genome editing Knock-in NHEJ |
url |
https://doi.org/10.1186/s13059-019-1907-9 |
work_keys_str_mv |
AT jianpingzhang curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT xinxincheng curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT meizhao curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT guohuali curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT jingxu curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT fengzhang curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT mengdiyin curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT feiyingmeng curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT xinyuedai curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT yawenfu curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT zhixueyang curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT cameronarakaki curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT ruijunjeannasu curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT weiwen curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT wentianwang curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT wanqiuchen curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT hannahchoi curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT charleswang curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT guangpinggao curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT leizhang curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT taocheng curinghemophiliaabynhejmediatedectopicf8insertioninthemouse AT xiaobingzhang curinghemophiliaabynhejmediatedectopicf8insertioninthemouse |
_version_ |
1724376212592132096 |