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 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 2

Mini-Gamma interacts with β- and δ-sarcoglycans.

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Mini-Gamma interacts with β- and δ-sarcoglycans. Plasmids encoding mamma...
Plasmids encoding mammalian sarcoglycans were expressed in HEK293T cells. (A) Expression of either Mini-Gamma or full-length γ-sarcoglycan (GSG) alone resulted in cytoplasmic and perinuclear accumulation. This observation is consistent with previous reports that association with the β/δ-sarcoglycan core is required for membrane targeting (26). Arrows in left panels indicate little to no plasma membrane trafficking. Coexpression of full-length γ-sarcoglycan with β- and δ-sarcoglycan (BSG and DSG) resulted in plasma membrane translocation of γ-sarcoglycan (arrow in top right panel). Similarly, expression of Mini-Gamma with β- and δ-sarcoglycan resulted in plasma membrane translocation of Mini-Gamma (arrow in bottom right panel). Scale bars: 5 μm. (B) Co-IP was performed to examine sarcoglycan-complex formation from HEK293T heterologous cell–expression experiments. After IP with an anti–β-sarcoglycan antibody, a complex containing β-, δ-, and γ-sarcoglycan was detected in β/δ/γ coexpressing cells (upper panels). Likewise, IP with the same anti–β-sarcoglycan antibody demonstrated an interaction among β- and δ-sarcoglycan and Mini-Gamma (lower panels). IP for Mini-Gamma using an antibody against the Xpress tag also detected β- and δ-sarcoglycan. MG, Mini-Gamma.