Summary Hemophilia A is an X-linked genetic disorder caused by mutations in the F8 gene, which encodes the blood coagulation factor VIII. Almost half of all severe hemophilia A cases result from two gross (140-kbp or 600-kbp) chromosomal inversions that involve introns 1 and 22 of the F8 gene, respectively. We derived induced pluripotent stem cells (iPSCs) from patients with these inversion genotypes and used CRISPR-Cas9 nucleases to revert these chromosomal segments back to the WT situation. We isolated inversion-corrected iPSCs with frequencies of up to 6.7% without detectable off-target mutations based on whole-genome sequencing or targeted deep sequencing. Endothelial cells differentiated from corrected iPSCs expressed the F8 gene and functionally rescued factor VIII deficiency in an otherwise lethal mouse model of hemophilia. Our results therefore provide a proof of principle for functional correction of large chromosomal rearrangements in patient-derived iPSCs and suggest potential therapeutic applications.
Bibliographical noteFunding Information:
D.-W.K. was supported by grants from the National Research Foundation of Korea (the Bio and Medical Technology Development Program, 2012M3A9B4028631 and 2012M3A9C7050126) and from the Korean Ministry of Health and Welfare (A120254). J.-S.K. was supported by a grant from IBS (IBS-R021-D1). J.-H.K. was supported by a grant from the National Research Foundation of Korea (2012M3A9C7050139). D.-W.K, J.-S.K, C.-Y.P, and D.H.K filed a patent application based on this work.
© 2015 Elsevier Inc.
ASJC Scopus subject areas
- Molecular Medicine
- Cell Biology