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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 4

Vivo-PMO–mediated exon skipping of the SGCG transcript.

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Vivo-PMO–mediated exon skipping of the SGCG transcript.
(A) A multi-AON ...
(A) A multi-AON exon-skipping cocktail using vivo-PMO chemistry was employed to correct the SGCG 521ΔT reading frame in myogenic cells. RT-PCR analysis showed treatment with vivo-PMOs targeting exons 4, 5, 6, and 7 generated the Mini-Gamma reading frame–corrected transcript (red arrowhead) with minimal residual expression of the mutant 521ΔT transcript (black arrowhead). Arrows in schematic denote the location of the RT-PCR primers, and the expected amplicon lengths are indicated. (B) Normal control myogenic cells were treated with the same multiexon-skipping vivo-PMO cocktail. RT-PCR analysis showed expression of the Mini-Gamma transcript (red arrowhead), indicating that normal SGCG transcript could be skipped using vivo-PMOs. Expression of the SGCG transcript in untreated cells is indicated (black arrowhead). (C and D) Sequence analysis of PCR products confirmed the generation of the Mini-Gamma transcripts in each of the mutant cell lines, with the splicing of SGCG exons 3 and 8 demonstrating the removal of exons 4, 5, 6, and 7 from the mature transcripts.

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ISSN 2379-3708

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