A Single CRISPR-Cas9 Deletion Strategy that Targets the Majority of DMD Patients Restores Dystrophin Function in hiPSC-Derived Muscle Cells

Courtney S Young; Michael R Hicks; Natalia V Ermolova; Haruko Nakano; Majib Jan; Shahab Younesi; Saravanan Karumbayaram; Chino Kumagai-Cresse; Derek Wang; Jerome A Zack; Donald B Kohn; Atsushi Nakano; Stanley F Nelson; M Carrie Miceli; Melissa J Spencer; April D Pyle

doi:10.1016/j.stem.2016.01.021

A Single CRISPR-Cas9 Deletion Strategy that Targets the Majority of DMD Patients Restores Dystrophin Function in hiPSC-Derived Muscle Cells

Cell Stem Cell. 2016 Apr 7;18(4):533-40. doi: 10.1016/j.stem.2016.01.021. Epub 2016 Feb 11.

Authors

Courtney S Young¹, Michael R Hicks², Natalia V Ermolova³, Haruko Nakano⁴, Majib Jan⁵, Shahab Younesi⁵, Saravanan Karumbayaram⁶, Chino Kumagai-Cresse³, Derek Wang⁷, Jerome A Zack⁸, Donald B Kohn⁹, Atsushi Nakano¹⁰, Stanley F Nelson¹¹, M Carrie Miceli⁹, Melissa J Spencer¹², April D Pyle¹³

Affiliations

¹ Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA; Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Neurology, University of California, Los Angeles, CA 90095, USA.
² Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
³ Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Department of Neurology, University of California, Los Angeles, CA 90095, USA.
⁴ Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA 90095, USA.
⁵ Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA; CIRM Bridges Program, California State University, Northridge, CA 91330, USA.
⁶ Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
⁷ Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
⁸ Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
⁹ Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA; Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
¹⁰ Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA; Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA 90095, USA.
¹¹ Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA; Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Human Genetics, University of California, Los Angeles, CA 90095, USA.
¹² Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA; Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Neurology, University of California, Los Angeles, CA 90095, USA. Electronic address: mspencer@mednet.ucla.edu.
¹³ Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA; Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA. Electronic address: apyle@mednet.ucla.edu.

Abstract

Mutations in DMD disrupt the reading frame, prevent dystrophin translation, and cause Duchenne muscular dystrophy (DMD). Here we describe a CRISPR/Cas9 platform applicable to 60% of DMD patient mutations. We applied the platform to DMD-derived hiPSCs where successful deletion and non-homologous end joining of up to 725 kb reframed the DMD gene. This is the largest CRISPR/Cas9-mediated deletion shown to date in DMD. Use of hiPSCs allowed evaluation of dystrophin in disease-relevant cell types. Cardiomyocytes and skeletal muscle myotubes derived from reframed hiPSC clonal lines had restored dystrophin protein. The internally deleted dystrophin was functional as demonstrated by improved membrane integrity and restoration of the dystrophin glycoprotein complex in vitro and in vivo. Furthermore, miR31 was reduced upon reframing, similar to observations in Becker muscular dystrophy. This work demonstrates the feasibility of using a single CRISPR pair to correct the reading frame for the majority of DMD patients.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Animals
CRISPR-Cas Systems / genetics*
Dystrophin / deficiency
Dystrophin / genetics
Dystrophin / metabolism*
Gene Deletion*
Gene Editing / methods*
Humans
Induced Pluripotent Stem Cells / cytology
Induced Pluripotent Stem Cells / metabolism*
Induced Pluripotent Stem Cells / pathology
Mice
Mice, SCID
Muscle, Skeletal / cytology
Muscle, Skeletal / metabolism*
Muscle, Skeletal / pathology
Muscular Dystrophy, Duchenne / genetics*
Muscular Dystrophy, Duchenne / metabolism
Muscular Dystrophy, Duchenne / pathology

Substances

Dystrophin

Abstract

Publication types

MeSH terms

Substances

Grants and funding