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. 2015 Mar;23(3):523-32.
doi: 10.1038/mt.2014.234. Epub 2014 Dec 10.

Correction of Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients Through Genomic Excision of Exon 51 by Zinc Finger Nucleases

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Free PMC article

Correction of Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients Through Genomic Excision of Exon 51 by Zinc Finger Nucleases

David G Ousterout et al. Mol Ther. .
Free PMC article

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.

Figures

Figure 1
Figure 1
Design of ZFNs targeted to exon 51. ZFN pairs (shown as blocks) were designed as a panel of targets across exon 51 and the flanking introns. ZFN, zinc finger nuclease.
Figure 2
Figure 2
Screening for active eMA ZFNs using an episomal reporter assay. All ZFNs used the wild-type FokI nuclease domain. (a) Schematic of single-stranded annealing assay to detect ZFN activity. Each target site was cloned between a split luciferase reporter with flanking homology on either side of each target sequence. Luciferase expression occurs when a ZFN pair successfully recognizes and cleaves its cognate site in the reporter, causing single-strand annealing and recombination of an active luciferase gene. (b) Activity of different combinations of eMA ZFN pairs in HEK293T cells compared to cells transfected only with the reporter plasmid. eMA, extended Modular Assembly; ZFN, zinc finger nuclease.
Figure 3
Figure 3
Evaluation of selected ZFNs in human cells. (a) All CoDA ZFNs and selected eMA ZFNs were transfected into wild-type myoblasts (10 μg of each monomer expression plasmid), and endogenous gene editing activity was measured at 3 days posttransfection by the Surveyor assay. (b) Activity of ZFN pairs that showed measurable activity in a was also determined at 10 days posttransfection to assess stability and survival of modified cells. The ratio of gene editing activity at 3 and 10 days was calculated from the data in a and b. n.d., not detected. n.q., not quantified. (c) Results of a cytotoxicity assay based on retention of GFP expression in HEK293T cells after transfection with the indicated nucleases and a GFP reporter. Percentage survival was calculated as the ratio of percent GFP-positive cells at days 2 and 5 posttransfection and normalized to transfection in the absence of nucleases. CoDA, Context-Dependent Assembly; eMA, extended Modular Assembly; GFP, green fluorescent protein; ZFN, zinc finger nuclease.
Figure 4
Figure 4
Restoration of the dystrophin reading frame in DMD patient myoblasts. (a) Schematic of strategy to delete exon 51 from the dystrophin gene locus. DZF-1 and DZF-9 flank the 5′ splice acceptor site of exon 51, which is removed after genomic deletion. P1/P2: primers used for detection of the genomic deletion by PCR in (c). (b) Gene modification activities of DZF-1 L6/R6 and DZF-9 as measured by the Surveyor assay 3 days after electroporation of 10 µg of each monomer expression cassette into DMD patient cells. (c) End-point genomic PCR across the deleted locus in human HEK293T or DMD myoblasts 3 days after treating cells with the indicated pair of nucleases. (d) Sanger sequencing of the PCR product from genomic DNA of a genetically corrected clonal cell population. Underlined sequences show target half-sites for the indicated ZFN target site. (e) End-point RT-PCR analysis of mRNA from control wild-type and untreated or a genetically corrected clonal population of DMD myoblasts after differentiation into myotubes. (f) Sanger sequencing of this PCR band showed the expected junction of exons 47 and 52. (g) Dystrophin expression as detected by western blot with antibodies to detect the C-terminus (NCL-DYS2) or rod domain (MANDYS8) in each of the indicated cell populations. All samples shown were run together on the same blot and cropped postimaging to remove extraneous lanes. However, different exposure times for the NCL-Dys2 western images were used to image DMD myoblasts and the genetically corrected clones compared to control samples to compensate for overexposure of control protein. The images for MANDYS8 and GAPDH are the same exposure time for all samples. DMD, Duchenne muscular dystrophy; ZFN, zinc finger nuclease.
Figure 5
Figure 5
Cell implantation and dystrophin expression in vivo. Untreated or genetically corrected human Δ48–50 DMD myoblasts carrying a background deletion of exons 48–50 were injected into the hind limbs of immunodeficient mice and assessed for human-specific protein expression in muscle fibers after 4 weeks posttransplantation. Serial cryosections were stained with antihuman spectrin, which is expressed by both uncorrected and corrected myoblasts that have fused into mouse myofibers or anti-human dystrophin antibodies as indicated. DMD, Duchenne muscular dystrophy.
Figure 6
Figure 6
Evaluation of ZFN off-target effects in human cells. Human DMD myoblasts were electroporated with ten micrograms of DNA constructs encoding either DZF-1 L6/R6 or DZF-9. After 3 days, genomic DNA was analyzed by the Surveyor assay to measure activity at eight different potential off-target loci for (a) DZF-1 L6/R6 or (b) DZF-9 predicted by the PROGNOS algorithm. Asterisks indicate detectable Surveyor cleavage products. DMD, Duchenne muscular dystrophy; ZFN, zinc finger nuclease.

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