title: Allele-specific gene silencing in two mouse models of autosomal dominant skeletal myopathy.
abstract: 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.
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester, Rochester, New York, United States of America.
authors.forename: Ryan E
authors.initials: RE
authors.lastname: Loy
authors.forename: John D
authors.initials: JD
authors.lastname: Lueck
authors.forename: Mohammed A
authors.initials: MA
authors.lastname: Mostajo-Radji
authors.forename: Ellie M
authors.initials: EM
authors.lastname: Carrell
authors.forename: Robert T
authors.initials: RT
authors.lastname: Dirksen
mesh_terms: D000483:Alleles; D000818:Animals; D004195:Disease Models, Animal; D055879:Gene Knock-In Techniques; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D005799:Genes, Dominant / Q000235:genetics*; D005820:Genetic Testing; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D009132:Muscles / Q000473:pathology*; D009135:Muscular Diseases / Q000235:genetics* / Q000473:pathology*; D009154:Mutation / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D019837:Ryanodine Receptor Calcium Release Channel