title: 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.affiliation: Department of Physiology and Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea.
authors.forename: Chul-Yong
authors.initials: CY
authors.lastname: Park
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, Seoul 151-742, Korea; Department of Chemistry, Seoul National University, Seoul 151-742, Korea.
authors.forename: Duk Hyoung
authors.initials: DH
authors.lastname: Kim
authors.affiliation: Laboratory of Stem Cell Biology, Division of Biotechnology, College of Life Science and Biotechnology, Korea University, Seoul 136-713, Korea.
authors.forename: Jeong Sang
authors.initials: JS
authors.lastname: Son
authors.affiliation: Department of Physiology and Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea.
authors.forename: Jin Jea
authors.initials: JJ
authors.lastname: Sung
authors.affiliation: Laboratory of Stem Cell Biology, Division of Biotechnology, College of Life Science and Biotechnology, Korea University, Seoul 136-713, Korea.
authors.forename: Jaehun
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Chemistry, Hanyang University, Seoul 133-791, Korea.
authors.forename: Sangsu
authors.initials: S
authors.lastname: Bae
authors.affiliation: Laboratory of Stem Cell Biology, Division of Biotechnology, College of Life Science and Biotechnology, Korea University, Seoul 136-713, Korea.
authors.forename: Jong-Hoon
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Physiology and Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea. Electronic address: dwkim2@yuhs.ac.
authors.forename: Dong-Wook
authors.initials: DW
authors.lastname: Kim
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, Seoul 151-742, Korea; Department of Chemistry, Seoul National University, Seoul 151-742, Korea. Electronic address: jskim01@snu.ac.kr.
authors.forename: Jin-Soo
authors.initials: JS
authors.lastname: Kim
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D007446:Chromosome Inversion / Q000235:genetics*; D002999:Clone Cells; D005167:Factor VII / Q000235:genetics*; D006367:HeLa Cells; D006467:Hemophilia A / Q000235:genetics*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data
chemical_list: D005167:Factor VII