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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 1

An exon-skipping strategy to treat LGMD 2C.

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An exon-skipping strategy to treat LGMD 2C.
(A) The SGCG gene, which enc...
(A) The SGCG gene, which encodes γ-sarcoglycan, is comprised of 8 exons, and many SGCG mutations disrupt the transcript reading frame, causing Limb-girdle muscular dystrophy (LGMD) 2C. (B) Correction of the reading frame requires the skipping of SGCG exons 4, 5, 6, and 7 at the pre-mRNA level. Four antisense oligonucleotides (AON) were designed to exclude these exons from the mature mRNA transcript. The bypass of exons 4–7 generates an internally truncated product termed Mini-Gamma encoded by exons 2, 3, and 8. (C) Mini-Gamma protein lacks a portion of the extracellular domain encoded by exons 4–7 (red box) and retains the essential cytosolic, transmembrane, and extracellular domains required for functionality (30). (D) Exon skipping AONs are chemically modified to avoid nuclease degradation and destruction of the mRNA transcript. Shown are the chemical structures of 2 types of AONs used for exon skipping. 2’-O-methyl phosphorothioates (2OMePS) include a methyl group on the ribose ring and substitution of sulfur for oxygen to create a phosphothioate bond (red). Phosphorodiamidate morpholino oligomers (PMOs) incorporate morpholino rings rather than ribose rings and substitute nitrogen for oxygen to create a phosphorodiamidate bond (blue) (18). Vivo-PMOs are synthesized with a covalently linked 3′ octa-guanidine delivery moiety for enhanced cell delivery (31).

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