Go to The Journal of Clinical Investigation
  • About
  • Editors
  • Consulting Editors
  • For authors
  • Publication ethics
  • Transfers
  • Advertising
  • Job board
  • Contact
  • Current issue
  • Past issues
  • By specialty
    • COVID-19
    • Cardiology
    • Immunology
    • Metabolism
    • Nephrology
    • Oncology
    • Pulmonology
    • All ...
  • Videos
  • Collections
    • Resource and Technical Advances
    • Clinical Medicine
    • Reviews
    • Editorials
    • Perspectives
    • Top read articles
  • JCI This Month
    • Current issue
    • Past issues

  • Current issue
  • Past issues
  • Specialties
  • In-Press Preview
  • Editorials
  • Viewpoint
  • Top read articles
  • About
  • Editors
  • Consulting Editors
  • For authors
  • Publication ethics
  • Transfers
  • Advertising
  • Job board
  • Contact
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.
View: Text | PDF
Research Article Genetics Muscle biology

Efficient exon skipping of SGCG mutations mediated by phosphorodiamidate morpholino oligomers

  • Text
  • PDF
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

×

Figure 6

Restoration of SGCG expression as Mini-Gamma protein.

Options: View larger image (or click on image) Download as PowerPoint
Restoration of SGCG expression as Mini-Gamma protein.
Reprogrammed fibro...
Reprogrammed fibroblasts were treated for 3 days with either a nontargeting control vivo-PMO (nontarget) or a mutation-specific vivo-PMO exon skipping cocktail (Mini-Gamma). (A) Representative IFM images of reprogrammed 521ΔT cells that were treated as indicated. Treatment with reading frame–correcting vivo-PMOs resulted in the expression of the Mini-Gamma protein (green). Myotubes were labeled with α-actinin (red), and nuclei were labeled with Hoechst 3342 (blue). (B) Image analysis demonstrated a significant increase in Mini-Gamma fluorescence after treatment with vivo-PMOs that corrected the transcript reading frame (n = 6) as compared with nontargeting controls (n = 5). (C and D) Reprogrammed cells harboring a deletion of SGCG exons 5 and 6 (ex5/6del) were treated as indicated. IFM image analysis demonstrated a significant increase in Mini-Gamma fluorescence after treatment with vivo-PMOs that corrected the reading transcript reading frame (n = 4) as compared with nontargeting controls (n = 3). (E and F) Reprogrammed cells harboring a deletion of SGCG exons 6 (ex6del) were treated as indicated. IFM image analysis demonstrated a significant increase in Mini-Gamma fluorescence after treatment with vivo-PMOs that corrected the transcript reading frame (n = 5) as compared with nontargeting controls (n = 3). Data represent the mean γ-sarcoglycan fluorescence per α-actinin–positive area normalized to the mean of the untreated group. A minimum of 3 independent fields were analyzed for each sample. *P < 0.05 as determined by 2-tailed Student’s t test. Data represent the mean ± SEM. Scale bars: 50 μM.

Copyright © 2023 American Society for Clinical Investigation
ISSN 2379-3708

Sign up for email alerts