Abstract
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.
Highlights
Engineered site-specific nucleases have broadly enabled the precise manipulation of DNA sequences in complex genomes.[1]
Design of zinc finger nucleases (ZFNs) targeted to exon 51 To identify ZFN pairs that are highly active, we created a large panel of ZFN pairs targeted across exon 51 of the dystrophin gene and its flanking introns with the goal of finding a combination of ZFN pairs to delete the entire exon or sequences important to its proper splicing in the resulting mRNA transcript (Figure 1, Supplementary Tables S1 and S2)
The extended Modular Assembly (eMA) approach is based on observations that ZFNs with four, five, or six zinc finger motifs in tandem are most likely to be
Summary
Engineered site-specific nucleases have broadly enabled the precise manipulation of DNA sequences in complex genomes.[1]. Oligonucleotide-based exon skipping is a powerful method to exclude specific exons and has been exploited to restore dystrophin expression by removing exons adjacent to genomic deletions and restoring the normal reading frame.[18] This strategy has predominantly been used to skip exon 51, which can address up to 13% of all DMD patient deletions.[19,20] this transient restoration requires regular administration of the exon skipping drug for the duration of treatment In contrast to this transient mRNA-targeted correction method, genome editing creates a stable change to the genome sequence of the cell that persists even after cell division. The introduction of random indels in the dystrophin gene results in heterogeneous changes to the final protein product that may Molecular Therapy vol 23 no. 3, 523–532 mar. 2015
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