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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 3

2OMePS-mediated exon skipping of the SGCG transcript.

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2OMePS-mediated exon skipping of the SGCG transcript.
(A) Correction of ...
(A) Correction of the SGCG 521ΔT frameshift mutation (red triangle) requires a multi-AON exon-skipping strategy targeting exons 4, 5, 6, and 7 (yellow boxes) to generate the reading frame–corrected Mini-Gamma transcript encoded by exons 2, 3, and 8. Arrows denote the location of the RT-PCR primers, and the expected amplicon lengths are indicated. Myogenic cells were exposed to a multiexon–skipping cocktail of 2OMePS AONs targeting exons 4, 5, 6, and 7. RT-PCR analysis demonstrated the expression of the 521ΔT transcript (black arrowhead), along with PCR products representing the skipping of 1, 2, and 3 exons (white arrowheads). A PCR product representing the 4-AON–mediated generation of the Mini-Gamma was observed at the expected size (red arrowhead). (B) Treatment of reprogrammed normal control fibroblasts with 2OMePS AONs targeting exons 4, 5, 6, and 7 also induced exon skipping. RT-PCR analysis demonstrated the expression of the SGCG transcript (black arrowhead), along with PCR products representing the skipping of 1, 2, and 3 exons (white arrowheads). No PCR product representing the 4-AON–mediated generation of the Mini-Gamma was observed at the expected size (red arrowhead).

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