Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse

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

doi:10.1186/s13059-019-1907-9

Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse

Genome Biol. 2019 Dec 16;20(1):276. doi: 10.1186/s13059-019-1907-9.

Authors

Jian-Ping Zhang^{1

2}, Xin-Xin Cheng^{1

2}, Mei Zhao^{1

2}, Guo-Hua Li^{1

2}, Jing Xu^{1

2}, Feng Zhang^{1

2}, Meng-Di Yin^{1

2}, Fei-Ying Meng^{1

2}, Xin-Yue Dai^{1

2}, Ya-Wen Fu^{1

2}, Zhi-Xue Yang^{1

2}, Cameron Arakaki³, Ruijun Jeanna Su³, Wei Wen^{1

2}, Wen-Tian Wang^{1

2

4}, Wanqiu Chen⁵, Hannah Choi⁵, Charles Wang⁵, Guangping Gao⁶, Lei Zhang^{1

2

4

7

8}, Tao Cheng^{9

10

11

12

13}, Xiao-Bing Zhang^{14

15

16}

Affiliations

¹ State Key Laboratory of Experimental Hematology, Tianjin, 300020, China.
² Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, 300020, China.
³ School of Medicine, Loma Linda University, Loma Linda, CA, 92354, USA.
⁴ CAMS Key Laboratory of Gene Therapy for Blood Diseases, Tianjin, 300020, China.
⁵ Center for Genomics, School of Medicine, Loma Linda University, Loma Linda, CA, 92350, USA.
⁶ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, 01655, USA.
⁷ National Clinical Research Center for Blood Diseases, Tianjin, 300020, China.
⁸ Tianjin Key Laboratory of Gene Therapy for Blood Diseases, Tianjin, 300020, China.
⁹ State Key Laboratory of Experimental Hematology, Tianjin, 300020, China. [email protected].
¹⁰ Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, 300020, China. [email protected].
¹¹ National Clinical Research Center for Blood Diseases, Tianjin, 300020, China. [email protected].
¹² Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Tianjin, 300020, China. [email protected].
¹³ Department of Stem Cell & Regenerative Medicine, Peking Union Medical College, Tianjin, 300020, China. [email protected].
¹⁴ State Key Laboratory of Experimental Hematology, Tianjin, 300020, China. [email protected].
¹⁵ Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, 300020, China. [email protected].
¹⁶ School of Medicine, Loma Linda University, Loma Linda, CA, 92354, USA. [email protected].

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.

Keywords: CRISPR-Cas9; Genome editing; Hemophilia A; Knock-in; NHEJ.

Publication types

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

MeSH terms

Albumins / genetics
Animals
Codon, Terminator
DNA End-Joining Repair*
Factor VIII / genetics*
Gene Knock-In Techniques*
Genetic Therapy / methods*
Hemophilia A / therapy*
Mice

Substances

Albumins
Codon, Terminator
Factor VIII