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

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 7

SGCG reading frame correction in urine-derived cells (UDCs).

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SGCG reading frame correction in urine-derived cells (UDCs). UDCs from a...
UDCs from a normal control subject and an LGMD 2C patient with a deletion of exon 6 (ex6del) were reprogrammed into a myogenic lineage. (A) γ-Sarcoglycan protein (green) was detected by IFM in the reprogrammed normal control myotubes but not in reprogrammed ex6del cells. α-Actinin, red; nuclei, blue. (B) RT-PCR analysis demonstrated reading frame–corrected Mini-Gamma transcript expression (red arrowhead). (C) Representative IFM images showed the restoration of γ-sarcoglycan protein expression in cells treated with Mini-Gamma vivo-PMOs. (D) Significant increase in γ-sarcoglycan protein fluorescence was observed after treatment with vivo-PMOs (n = 5) as compared with nontargeting control vivo-PMOs (n = 5). A minimum of 3 independent fields were analyzed for each sample. (E) To assess membrane stability in response to vivo-PMO treatment, reprogrammed cells were challenged with hypo-osmotic shock and membrane leak was monitored by release of creatine kinase (CK). Vivo-PMO treatment significantly decreased the relative amount of CK release consistent with increased membrane stability. Data represent the percent of CK released relative to the total CK from 4 independent experiments (n = 3–4, for each). Data are presented as the mean CK released in cells treated with exon-skipping vivo-PMOs relative to the mean in cells treated with a nontargeting vivo-PMO. (F) Model depicting the increased membrane stability that resulted from vivo-PMO–mediated reading frame correction of an SGCG frameshift mutation. *P < 0.05 as determined by 2-tailed Student’s t test. Data represent the mean ± SEM. Scale bars: 50 μM.