Allele-specific gene silencing in two mouse models of autosomal dominant skeletal myopathy

PLoS One. 2012;7(11):e49757. doi: 10.1371/journal.pone.0049757. Epub 2012 Nov 12.


We explored the potential of mutant allele-specific gene silencing (ASGS) in providing therapeutic benefit in two established mouse models of the autosomal dominantly-inherited muscle disorders, Malignant Hyperthermia (MH) and Central Core Disease (CCD). Candidate ASGS siRNAs were designed and validated for efficacy and specificity on ryanodine receptor (RyR1) cDNA mini-constructs expressed in HEK293 cells using RT-PCR- and confocal microscopy-based assays. In vivo delivery of the most efficacious identified siRNAs into flexor digitorum brevis (FDB) muscles was achieved by injection/electroporation of footpads of 4-6 month old heterozygous Ryr1(Y524S/+) (YS/+) and Ryr1(I4895T/+) (IT/+) knock-in mice, established mouse models of MH with cores and CCD, respectively. Treatment of IT/+ mice resulted in a modest rescue of deficits in the maximum rate (∼38% rescue) and magnitude (∼78%) of ligand-induced Ca(2+) release that occurred in the absence of a change in the magnitude of electrically-evoked Ca(2+) release. Compared to the difference between the caffeine sensitivity of Ca(2+) release in FDB fibers from YS/+ and WT mice treated with SCR siRNA (EC(50): 1.1 mM versus 4.4 mM, respectively), caffeine sensitivity was normalized in FDB fibers from YS/+ mice following 2 (EC(50): 2.8 mM) and 4 week (EC(50): 6.6 mM) treatment with YS allele-specific siRNA. Moreover, the temperature-dependent increase in resting Ca(2+) observed in FDB fibers from YS/+ mice was normalized to WT levels after 2 weeks of treatment with YS allele-specific siRNA. As determined by quantitative real time PCR, the degree of functional rescue in YS/+ and IT/+ mice correlated well with the relative increase in fractional WT allele expression.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles*
  • Animals
  • Disease Models, Animal
  • Gene Knock-In Techniques
  • Gene Knockdown Techniques
  • Gene Silencing*
  • Genes, Dominant / genetics*
  • Genetic Testing
  • HEK293 Cells
  • Humans
  • Mice
  • Muscles / pathology*
  • Muscular Diseases / genetics*
  • Muscular Diseases / pathology*
  • Mutation / genetics
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / metabolism
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction
  • Ryanodine Receptor Calcium Release Channel / metabolism


  • RNA, Messenger
  • RNA, Small Interfering
  • Ryanodine Receptor Calcium Release Channel