Functional Correction of Large Factor VIII Gene Chromosomal Inversions in Hemophilia A Patient-Derived iPSCs Using CRISPR-Cas9.

Abstract

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.

• Authors: Chul-Yong Park, Duk Hyoung Kim, Jeong Sang Son, Jin Jea Sung, Jaehun Lee, Sangsu Bae, Jong-Hoon Kim, Dong-Wook Kim, Jin-Soo Kim
• Journal: Cell stem cell (Cell Stem Cell) Vol. 17 Issue 2 Pg. 213-20 (Aug 06 2015) ISSN: 1875-9777 [Electronic] United States
• PMID: 26212079 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2015 Elsevier Inc. All rights reserved.

Chemical References

• Factor VII

Topics

• Animals
• Base Sequence
• CRISPR-Cas Systems (genetics)
• Chromosome Inversion (genetics)
• Clone Cells
• Factor VII (genetics)
• HeLa Cells
• Hemophilia A (genetics)
• Humans
• Induced Pluripotent Stem Cells (metabolism)
• Mice
• Molecular Sequence Data