UMMS Affiliation

Horae Gene Therapy Center; Department of Microbiology and Physiological Systems

Publication Date

2019-12-16

Document Type

Article

Disciplines

Congenital, Hereditary, and Neonatal Diseases and Abnormalities | Genetic Phenomena | Genetics and Genomics | Hematology | Hemic and Lymphatic Diseases | Therapeutics

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

Rights and Permissions

© The Author(s). 2019 Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

DOI of Published Version

10.1186/s13059-019-1907-9

Source

Genome Biol. 2019 Dec 16;20(1):276. doi: 10.1186/s13059-019-1907-9. Link to article on publisher's site

Journal/Book/Conference Title

Genome biology

Related Resources

Link to Article in PubMed

PubMed ID

31843008

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

Share

COinS