TY - JOUR
T1 - A gene-edited mouse model of limb-girdle muscular dystrophy 2C for testing exon skipping
AU - Demonbreun, Alexis R.
AU - Wyatt, Eugene J.
AU - Fallon, Katherine S.
AU - Oosterbaan, Claire C.
AU - Page, Patrick G.
AU - Hadhazy, Michele
AU - Quattrocelli, Mattia
AU - Barefield, David Y.
AU - McNally, Elizabeth M.
N1 - Funding Information:
This work was supported by the National Institutes of Health (HL61322, AR052646) and the Kurt+Peter Foundation.
Publisher Copyright:
© 2019 Published by The Company of Biologists Ltd.
PY - 2020
Y1 - 2020
N2 - Limb-girdle muscular dystrophy type 2C is caused by autosomal recessive mutations in the γ-sarcoglycan (SGCG) gene. The most common SGCG mutation is a single nucleotide deletion from a stretch of five thymine residues in SGCG exon 6 (521ΔT). This founder mutation disrupts the transcript reading frame, abolishing protein expression. An antisense oligonucleotide exon-skipping method to reframe the human 521ΔT transcript requires skipping four exons to generate a functional, internally truncated protein. In vivo evaluation of this multi-exon skipping, antisense-mediated therapy requires a genetically appropriate mouse model. The human and mouse γ-sarcoglycan genes are highly homologous in sequence and gene structure, including the exon 6 region harboring the founder mutation. Herein, we describe a new mouse model of this form of limb-girdle muscular dystrophy generated using CRISPR/Cas9-mediated gene editing to introduce a single thymine deletion in murine exon 6, recreating the 521ΔT point mutation in Sgcg. These mice express the 521ΔT transcript, lack γ-sarcoglycan protein and exhibit a severe dystrophic phenotype. Phenotypic characterization demonstrated reduced muscle mass, increased sarcolemmal leak and fragility, and decreased muscle function, consistent with the human pathological findings. Furthermore, we showed that intramuscular administration of a murine-specific multiple exon-directed antisense oligonucleotide cocktail effectively corrected the 521ΔT reading frame. These data demonstrate a molecularly and pathologically suitable model for in vivo testing of a multi-exon skipping strategy to advance preclinical development of this genetic correction approach.
AB - Limb-girdle muscular dystrophy type 2C is caused by autosomal recessive mutations in the γ-sarcoglycan (SGCG) gene. The most common SGCG mutation is a single nucleotide deletion from a stretch of five thymine residues in SGCG exon 6 (521ΔT). This founder mutation disrupts the transcript reading frame, abolishing protein expression. An antisense oligonucleotide exon-skipping method to reframe the human 521ΔT transcript requires skipping four exons to generate a functional, internally truncated protein. In vivo evaluation of this multi-exon skipping, antisense-mediated therapy requires a genetically appropriate mouse model. The human and mouse γ-sarcoglycan genes are highly homologous in sequence and gene structure, including the exon 6 region harboring the founder mutation. Herein, we describe a new mouse model of this form of limb-girdle muscular dystrophy generated using CRISPR/Cas9-mediated gene editing to introduce a single thymine deletion in murine exon 6, recreating the 521ΔT point mutation in Sgcg. These mice express the 521ΔT transcript, lack γ-sarcoglycan protein and exhibit a severe dystrophic phenotype. Phenotypic characterization demonstrated reduced muscle mass, increased sarcolemmal leak and fragility, and decreased muscle function, consistent with the human pathological findings. Furthermore, we showed that intramuscular administration of a murine-specific multiple exon-directed antisense oligonucleotide cocktail effectively corrected the 521ΔT reading frame. These data demonstrate a molecularly and pathologically suitable model for in vivo testing of a multi-exon skipping strategy to advance preclinical development of this genetic correction approach.
KW - Antisense oligonucleotide
KW - Dystrophin
KW - Gene correction
KW - LGMD 2C
KW - Mouse
KW - Sarcoglycan
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U2 - 10.1242/dmm.040832
DO - 10.1242/dmm.040832
M3 - Article
C2 - 31582396
AN - SCOPUS:85074552903
VL - 13
JO - DMM Disease Models and Mechanisms
JF - DMM Disease Models and Mechanisms
SN - 1754-8403
IS - 2
M1 - dmm040832
ER -