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
  • Concise Communication
  • Editorials
  • Viewpoint
  • Top read articles
  • About
  • Editors
  • Consulting Editors
  • For authors
  • Publication ethics
  • Transfers
  • Advertising
  • Job board
  • Contact
Base editing repairs an SGCA mutation in human primary muscle stem cells
Helena Escobar, … , Florian Heyd, Simone Spuler
Helena Escobar, … , Florian Heyd, Simone Spuler
Published April 13, 2021
Citation Information: JCI Insight. 2021;6(10):e145994. https://doi.org/10.1172/jci.insight.145994.
View: Text | PDF
Research Article Stem cells Therapeutics

Base editing repairs an SGCA mutation in human primary muscle stem cells

  • Text
  • PDF
Abstract

Skeletal muscle can regenerate from muscle stem cells and their myogenic precursor cell progeny, myoblasts. However, precise gene editing in human muscle stem cells for autologous cell replacement therapies of untreatable genetic muscle diseases has not yet been reported. Loss-of-function mutations in SGCA, encoding α-sarcoglycan, cause limb-girdle muscular dystrophy 2D/R3, an early-onset, severe, and rapidly progressive form of muscular dystrophy affecting both male and female patients. Patients suffer from muscle degeneration and atrophy affecting the limbs, respiratory muscles, and heart. We isolated human muscle stem cells from 2 donors, with the common SGCA c.157G>A mutation affecting the last coding nucleotide of exon 2. We found that c.157G>A is an exonic splicing mutation that induces skipping of 2 coregulated exons. Using adenine base editing, we corrected the mutation in the cells from both donors with > 90% efficiency, thereby rescuing the splicing defect and α-sarcoglycan expression. Base-edited patient cells regenerated muscle and contributed to the Pax7+ satellite cell compartment in vivo in mouse xenografts. Here, we provide the first evidence to our knowledge that autologous gene–repaired human muscle stem cells can be harnessed for cell replacement therapies of muscular dystrophies.

Authors

Helena Escobar, Anne Krause, Sandra Keiper, Janine Kieshauer, Stefanie Müthel, Manuel García de Paredes, Eric Metzler, Ralf Kühn, Florian Heyd, Simone Spuler

×

Figure 5

SGCA c.157Grep patient MuSC express α-sarcoglycan, and they are viable and myogenic.

Options: View larger image (or click on image) Download as PowerPoint

SGCA c.157Grep patient MuSC express α-sarcoglycan, and they are viable ...
(A) qPCR analysis of SGCA mRNA in SGCA c.157Grep compared with unedited (U) patient and carrier myotubes. Values are normalized to GAPDH and relativized to control 3. SGCA mRNA values were additionally normalized to MYH2 to correct for differences in the differentiation stage of the cells, which may affect promoter activity. Each data point represents the mean ± SD for the n = 3 technical replicates of each biological sample. (B) Western blot analysis of α-sarcoglycan protein in SGCA c.157Grep patient myotubes compared with unedited (U) patient and control 3 myotubes. Myosin heavy chain (MyHC) was used as a differentiation marker. Vinculin and GAPDH were used as loading controls. The intensity of α-sarcoglycan Western blot bands was quantified using ImageJ and normalized to Vinculin and MyHC. (C) SGCA c.157Grep patient MuSC were immunostained for Pax7, Desmin, MyoD, Myf5, and Ki-67. The analysis was performed for all SGCA c.157Grep patient cell populations, and a quantification is shown in Supplemental Table 1. (D) SGCA c.157Grep MuSC readily fused into multinucleated myotubes expressing MyHC. The analysis was performed for all SGCA c.157Grep patient cell populations. (E) α-Sarcoglycan immunostaining in control myotubes, as well as unedited and SGCA c.157Grep patient myotubes. The analysis was performed for all SGCA c.157Grep patient cell populations. MT, myotubes. Scale bars: 50 μm.

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

Sign up for email alerts