Skip to main content

Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases

Publication ,  Journal Article
Ousterout, DG; Kabadi, AM; Thakore, PI; Perez-Pinera, P; Brown, MT; Majoros, WH; Reddy, TE; Gersbach, CA
Published in: Molecular Therapy
March 5, 2015

Duchenne muscular dystrophy (DMD) is caused by genetic mutations that result in the absence of dystrophin protein expression. Oligonucleotide-induced exon skipping can restore the dystrophin reading frame and protein production. However, this requires continuous drug administration and may not generate complete skipping of the targeted exon. In this study, we apply genome editing with zinc finger nucleases (ZFNs) to permanently remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript. This approach can restore the dystrophin reading frame in ∼13% of DMD patient mutations. Transfection of two ZFNs targeted to sites flanking the exon 51 splice acceptor into DMD patient myoblasts led to deletion of this genomic sequence. A clonal population was isolated with this deletion and following differentiation we confirmed loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin protein expression. Furthermore, transplantation of corrected cells into immunodeficient mice resulted in human dystrophin expression localized to the sarcolemmal membrane. Finally, we quantified ZFN toxicity in human cells and mutagenesis at predicted off-target sites. This study demonstrates a powerful method to restore the dystrophin reading frame and protein expression by permanently deleting exons.

Duke Scholars

Altmetric Attention Stats
Dimensions Citation Stats

Published In

Molecular Therapy

DOI

EISSN

1525-0024

ISSN

1525-0016

Publication Date

March 5, 2015

Volume

23

Issue

3

Start / End Page

523 / 532

Related Subject Headings

  • Biotechnology
  • 3206 Medical biotechnology
  • 3202 Clinical sciences
  • 3105 Genetics
  • 11 Medical and Health Sciences
  • 10 Technology
  • 06 Biological Sciences
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Ousterout, D. G., Kabadi, A. M., Thakore, P. I., Perez-Pinera, P., Brown, M. T., Majoros, W. H., … Gersbach, C. A. (2015). Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases. Molecular Therapy, 23(3), 523–532. https://doi.org/10.1038/mt.2014.234
Ousterout, D. G., A. M. Kabadi, P. I. Thakore, P. Perez-Pinera, M. T. Brown, W. H. Majoros, T. E. Reddy, and C. A. Gersbach. “Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases.” Molecular Therapy 23, no. 3 (March 5, 2015): 523–32. https://doi.org/10.1038/mt.2014.234.
Ousterout DG, Kabadi AM, Thakore PI, Perez-Pinera P, Brown MT, Majoros WH, et al. Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases. Molecular Therapy. 2015 Mar 5;23(3):523–32.
Ousterout, D. G., et al. “Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases.” Molecular Therapy, vol. 23, no. 3, Mar. 2015, pp. 523–32. Scopus, doi:10.1038/mt.2014.234.
Ousterout DG, Kabadi AM, Thakore PI, Perez-Pinera P, Brown MT, Majoros WH, Reddy TE, Gersbach CA. Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases. Molecular Therapy. 2015 Mar 5;23(3):523–532.

Published In

Molecular Therapy

DOI

EISSN

1525-0024

ISSN

1525-0016

Publication Date

March 5, 2015

Volume

23

Issue

3

Start / End Page

523 / 532

Related Subject Headings

  • Biotechnology
  • 3206 Medical biotechnology
  • 3202 Clinical sciences
  • 3105 Genetics
  • 11 Medical and Health Sciences
  • 10 Technology
  • 06 Biological Sciences