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

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

doi:10.1016/j.stem.2015.07.001

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

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

Department of Biotechnology

Research output: Contribution to journal › Article › peer-review

233 Citations (Scopus)

Abstract

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.

Original language	English
Article number	1805
Pages (from-to)	213-220
Number of pages	8
Journal	Cell Stem Cell
Volume	17
Issue number	2
DOIs	https://doi.org/10.1016/j.stem.2015.07.001
Publication status	Published - 2015 Aug 6

Bibliographical note

Funding 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.

Publisher Copyright:
© 2015 Elsevier Inc.

ASJC Scopus subject areas

Molecular Medicine
Genetics
Cell Biology

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10.1016/j.stem.2015.07.001

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title = "Functional Correction of Large Factor VIII Gene Chromosomal Inversions in Hemophilia A Patient-Derived iPSCs Using CRISPR-Cas9",

abstract = "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.",

author = "Park, {Chul Yong} and Kim, {Duk Hyoung} and Son, {Jeong Sang} and Sung, {Jin Jea} and Jaehun Lee and Sangsu Bae and Kim, {Jong Hoon} and Kim, {Dong Wook} and Kim, {Jin Soo}",

note = "Funding 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. Publisher Copyright: {\textcopyright} 2015 Elsevier Inc.",

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AU - Lee, Jaehun

AU - Bae, Sangsu

AU - Kim, Jong Hoon

AU - Kim, Dong Wook

AU - Kim, Jin Soo

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N2 - 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.

AB - 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.

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

Abstract

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