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Efficient exon skipping of SGCG mutations mediated by phosphorodiamidate morpholino oligomers
Eugene J. Wyatt, … , Mayana Zatz, Elizabeth M. McNally
Eugene J. Wyatt, … , Mayana Zatz, Elizabeth M. McNally
Published May 3, 2018
Citation Information: JCI Insight. 2018;3(9):e99357. https://doi.org/10.1172/jci.insight.99357.
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Research Article Genetics Muscle biology

Efficient exon skipping of SGCG mutations mediated by phosphorodiamidate morpholino oligomers

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Abstract

Exon skipping uses chemically modified antisense oligonucleotides to modulate RNA splicing. Therapeutically, exon skipping can bypass mutations and restore reading frame disruption by generating internally truncated, functional proteins to rescue the loss of native gene expression. Limb-girdle muscular dystrophy type 2C is caused by autosomal recessive mutations in the SGCG gene, which encodes the dystrophin-associated protein γ-sarcoglycan. The most common SGCG mutations disrupt the transcript reading frame abrogating γ-sarcoglycan protein expression. In order to treat most SGCG gene mutations, it is necessary to skip 4 exons in order to restore the SGCG transcript reading frame, creating an internally truncated protein referred to as Mini-Gamma. Using direct reprogramming of human cells with MyoD, myogenic cells were tested with 2 antisense oligonucleotide chemistries, 2’-O-methyl phosphorothioate oligonucleotides and vivo–phosphorodiamidate morpholino oligomers, to induce exon skipping. Treatment with vivo–phosphorodiamidate morpholino oligomers demonstrated efficient skipping of the targeted exons and corrected the mutant reading frame, resulting in the expression of a functional Mini-Gamma protein. Antisense-induced exon skipping of SGCG occurred in normal cells and those with multiple distinct SGCG mutations, including the most common 521ΔT mutation. These findings demonstrate a multiexon-skipping strategy applicable to the majority of limb-girdle muscular dystrophy 2C patients.

Authors

Eugene J. Wyatt, Alexis R. Demonbreun, Ellis Y. Kim, Megan J. Puckelwartz, Andy H. Vo, Lisa M. Dellefave-Castillo, Quan Q. Gao, Mariz Vainzof, Rita C. M. Pavanello, Mayana Zatz, Elizabeth M. McNally

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Figure 2

SGCG mRNA and protein expression in fibroblasts reprogrammed into the myogenic lineage.

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SGCG mRNA and protein expression in fibroblasts reprogrammed into the m...
(A) Schematic of the SGCG pre-mRNA transcript. The most common LGMD 2C mutation is a single thymine (T) deletion from a string of 5Ts in SGCG exon 6. This mutation, designated 521ΔT, results in a transcript frame shift, which generates a premature stop codon (red octagon). Asterisks represent the transcription start sites, and the numbers denote the mRNA region encoded by each exon. Fibroblasts from a normal control subject (SGCG intact) and a LGMD 2C individual with the 521ΔT mutation were directly reprogrammed into the myogenic lineage using a tamoxifen (Tam) inducible MyoD lentiviral construct (iMyoD) (32). (B) RT-PCR analysis demonstrated expression of the SGCG mRNA in normal control and 521ΔT cells after tamoxifen induction and culture in differentiation media (5 μM 4OH-tamoxifen, 48 hours, 9 days differentiation). (C) Immunofluorescence microscopy (IFM) demonstrated the efficient transduction and myogenic reprogramming of both the control and 521ΔT cell lines (5 μM 4OH-tamoxifen, 48 hours, 12 days differentiation). Nuclear expression of MyoD (red) is observed in the iMyoD-transduced cells. Reprogrammed cells expressed the myogenic marker desmin (green) and formed multinucleated myotubes. Nuclei were labeled with Hoechst 3342 (blue). Scale bar: 50 μM. (D) γ-Sarcoglycan protein (green) was readily detected in the reprogrammed control cells, but not in reprogrammed 521ΔT cells, in accordance with their mutation status. Expression of α-actinin (red) indicated the myogenic reprogramming of each cell line. Scale bar: 50 μM.

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