Reengineering a transmembrane protein to treat muscular dystrophy using exon skipping
Quan Q. Gao, … , Matthew Wolf, Elizabeth M. McNally
Quan Q. Gao, … , Matthew Wolf, Elizabeth M. McNally
Published November 2, 2015; First published October 12, 2015
Citation Information: J Clin Invest. 2015;125(11):4186-4195. https://doi.org/10.1172/JCI82768.
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Research Article Genetics

Reengineering a transmembrane protein to treat muscular dystrophy using exon skipping

Abstract

Exon skipping uses antisense oligonucleotides as a treatment for genetic diseases. The antisense oligonucleotides used for exon skipping are designed to bypass premature stop codons in the target RNA and restore reading frame disruption. Exon skipping is currently being tested in humans with dystrophin gene mutations who have Duchenne muscular dystrophy. For Duchenne muscular dystrophy, the rationale for exon skipping derived from observations in patients with naturally occurring dystrophin gene mutations that generated internally deleted but partially functional dystrophin proteins. We have now expanded the potential for exon skipping by testing whether an internal, in-frame truncation of a transmembrane protein γ-sarcoglycan is functional. We generated an internally truncated γ-sarcoglycan protein that we have termed Mini-Gamma by deleting a large portion of the extracellular domain. Mini-Gamma provided functional and pathological benefits to correct the loss of γ-sarcoglycan in a Drosophila model, in heterologous cell expression studies, and in transgenic mice lacking γ-sarcoglycan. We generated a cellular model of human muscle disease and showed that multiple exon skipping could be induced in RNA that encodes a mutant human γ-sarcoglycan. Since Mini-Gamma represents removal of 4 of the 7 coding exons in γ-sarcoglycan, this approach provides a viable strategy to treat the majority of patients with γ-sarcoglycan gene mutations.

Authors

Quan Q. Gao, Eugene Wyatt, Jeff A. Goldstein, Peter LoPresti, Lisa M. Castillo, Alec Gazda, Natalie Petrossian, Judy U. Earley, Michele Hadhazy, David Y. Barefield, Alexis R. Demonbreun, Carsten Bönnemann, Matthew Wolf, Elizabeth M. McNally

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Figure 1

Mini-Gamma rescues Drosophila muscular dystrophy.

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Mini-Gamma rescues Drosophila muscular dystrophy. (A) γ-Sarcoglycan (GSG...
(A) γ-Sarcoglycan (GSG) is a type II transmembrane protein with a cytoplasmic amino-terminus and an extracellular carboxy-terminus. The SGCG gene encoding γ-sarcoglycan is composed of 8 exons, and the most common mutation falls within exon 6 and disrupts the reading frame (15). To restore the reading frame, skipping exon 4–7 is required. This approach removes a portion of the extracellular domain, producing an internally truncated protein, referred to as Mini-Gamma. (B) The GAL4/UAS system was used to express full-length mGSG and Mini-Gamma as transgenes in Sgcd840 flies, a sarcoglycan-deficient model of muscular dystrophy. Mini-Gamma protein localized to the plasma membrane in Sgcd840 fly skeletal muscle (Mef2-Gal4, UAS-Mini-Gamma), similar to full-length mGSG (Mhc-Gal4, UAS-mGSG). In fly heart tube, Mini-Gamma also showed a plasma membrane staining (TinΔC-Gal4, UAS-Mini-Gamma). Scale bars: 20 μm. (C) OCT was used to measure fly heart function (24). Sgcd840 flies had dilated heart tubes with increased ESD compared with WT flies. Expression of Mini-Gamma in the Sgcd840 heart tube restored ESD to WT level (Mef-Gal4, UAS-Mini-Gamma) (n = 10~12 flies per genotype). (D) The MB5 monitor was used to record fly spontaneous activity. Nocturnal activity is shown (12 am–8 am). Expression of Mini-Gamma improved nocturnal activity of Sgcd840. The degree of rescue was similar between full-length mGSG and Mini-Gamma (n = 20~35 flies per genotype). Student’s t test was used to compare results between 2 groups.