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

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 2

SGCA c.157G>A is an exonic splicing mutation.

Options: View larger image (or click on image) Download as PowerPoint
SGCA c.157G>A is an exonic splicing mutation. (A) RT-PCR analysis of...
(A) RT-PCR analysis of SGCA mRNA in muscle tissue from controls and the c.157G>A carrier. Primer binding sites and expected band sizes are displayed in the panel above. Control 3 is a heterozygous SGCA c.748-2A>G carrier with a WT SGCA exon 2 sequence. The RT-PCR was performed 3 times. (B) Sequencing of the bands from A shows skipping of exon 2 or exon 2+3 in the c.157G>A carrier. (C) qPCR analysis of SGCA mRNA in muscle tissue from controls and the c.157G>A carrier using primers against the exon 1–2 or exon 6–7 boundaries. Control 3 is represented by a green square. The qPCR was performed in technical triplicates. Values were normalized to GAPDH and relativized to the mean of controls. (D) Strength scores of the SGCA exon 2 splice donor for the WT and the mutant sequence predicted by MaxEntScan:score5ss. (E) Minigene construct schemes. FL, full-length SGCA exon 1–4 (blue boxes) with the intermediate introns (black lines); +498, a 498 bp-long low complexity intronic sequence from the human HBB gene (yellow) was inserted to extend intron 2. The size of each intron is indicated above in gray. For intron 2, the size before and after the branch point is indicated below in gray. UT, untransfected; EV, empty vector; WT, WT SGCA; G>A, c.157G>A mutation. (F) Minigene splicing patterns in HEK293T cells analyzed by low-cycle RT-PCR with a 32P-labeled forward primer; products were separated by denaturing PAGE. The splice isoforms identified are shown on the right (identity confirmed by Sanger sequencing). *, intron-retention isoforms; 2*, truncated exon 2 (40 nt); 3*, truncated exon 3 (–70 nt). (G) Splice isoform quantification from F using Phosphorimager analysis. Quantified values are presented as mean ± SD (n = 3). E, exon; I, intron.