The goal of exon skipping to treat DMD is to induce retention of out-of-frame exons and to create an intact reading frame. In DMD, internally deleted forms of dystrophin are known to be functional and result in a milder form of disease known as Becker muscular dystrophy (BMD) ( Koenig et al., 1989 Monaco et al., 1988). Chemically modified AONs can modulate pre-mRNA splicing, bypassing mutations and generating an internally truncated protein that is able to partially rescue the loss of the full-length protein ( Kole et al., 2012). 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.Įxon skipping relies on chemically modified antisense oligonucleotides (AONs) to modulate gene expression, including correction of an aberrant reading frame which abrogates protein expression ( Chan et al., 2006).
Furthermore, we showed that intramuscular administration of a murine-specific multiple exon-directed antisense oligonucleotide cocktail effectively corrected the 521ΔT reading frame. Phenotypic characterization demonstrated reduced muscle mass, increased sarcolemmal leak and fragility, and decreased muscle function, consistent with the human pathological findings. These mice express the 521ΔT transcript, lack γ-sarcoglycan protein and exhibit a severe dystrophic phenotype. 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. The human and mouse γ-sarcoglycan genes are highly homologous in sequence and gene structure, including the exon 6 region harboring the founder mutation. In vivo evaluation of this multi-exon skipping, antisense-mediated therapy requires a genetically appropriate mouse model. An antisense oligonucleotide exon-skipping method to reframe the human 521ΔT transcript requires skipping four exons to generate a functional, internally truncated protein.
This founder mutation disrupts the transcript reading frame, abolishing protein expression. The most common SGCG mutation is a single nucleotide deletion from a stretch of five thymine residues in SGCG exon 6 (521ΔT). Limb-girdle muscular dystrophy type 2C is caused by autosomal recessive mutations in the γ-sarcoglycan ( SGCG) gene.
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