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 7

AON-mediated reading frame correction in human SGCG mutant cells.

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AON-mediated reading frame correction in human SGCG mutant cells. (A) Sh...
(A) Shown are the 2 different alleles from an individual with LGMD 2C affecting the SGCG locus, a 1.4 MB deletion encompassing multiple genes (blue shaded area, top) and a smaller deletion encompassing SGCG exon 6 in its entirety (blue shaded area, middle schematic). Numbers refer to genome position in hg19. The exon organization for exon 6 deletion (ex6del) mutant SGCG transcripts is shown in the lower schematic. Asterisk represents transcription start site. Red arrowhead indicates premature stop codon. (B) Skin fibroblasts from control and ex6del were obtained and reprogrammed to the myogenic lineage using a tamoxifen-inducible MyoD (31, 32). Desmin expression (red) and multinucleated myotube formation indicated myogenic reprogramming after 4OH-tamoxifen exposure (5 μM, 48 hours) and culture in differentiation media. Nuclei were marked with Hoechst 3342 (blue). Scale bars: 10 μM. (C) RT-PCR demonstrated SGCG transcripts from control and ex6del mutant in reprogrammed (right) fibroblasts after differentiation (5 μM 4OH-tamoxifen, 48 hours; 12-day differentiation). (D) MyoD-reprogrammed fibroblasts were treated with AONs targeting exons 4, 5, and 7 (100 nM/AON, 300 nM total). RT-PCR demonstrated the expected skipped products, including the smallest product representing exons 2, 3, and 8 and deleted for exons 4, 5, and 7 (red arrow). Results from 3 independent replicates are shown for AON treatment. NTC, no-template control. Black arrow indicates the single exon skipping of exon 7 in the ex6del SGCG transcript, which occurs in the absence of AON treatment.