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 Ryr1Y524S/+ (YS/+) and Ryr1I4895T/+ (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 Ca2+ release that occurred in the absence of a change in the magnitude of electrically-evoked Ca2+ release. Compared to the difference between the caffeine sensitivity of Ca2+ release in FDB fibers from YS/+ and WT mice treated with SCR siRNA (EC50: 1.1 mM versus 4.4 mM, respectively), caffeine sensitivity was normalized in FDB fibers from YS/+ mice following 2 (EC50: 2.8 mM) and 4 week (EC50: 6.6 mM) treatment with YS allele-specific siRNA. Moreover, the temperature-dependent increase in resting Ca2+ 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.
Highlights
With the advent of high throughput sequencing and the completion of the human genome project, the implications of genetics on individualized patient health care are increasing exponentially
This name ends with the relative position of the mismatch: c, for center mismatch or s, for shifted mismatch. small interference RNAs (siRNAs) designed with additional mismatches were tested to determine if the changes increased mutant allele specificity by introducing additional nucleotide differences from the WT sequence in cases in which specificity was low [1]
We developed in vitro mRNA- and protein-based assays to screen multiple siRNAs for efficient and allelespecific silencing of RyR1 mini-constructs containing previously identified autosomal dominant human disease mutations
Summary
With the advent of high throughput sequencing and the completion of the human genome project, the implications of genetics on individualized patient health care are increasing exponentially These advances open the door for a genetic basis of personalized medicine, enabling the possibility of tailoring individualized therapy based on the underlying genetics of the patient in addition to their particular symptomatic presentation. In this context, allele-specific gene silencing (ASGS), a strategy designed to selectively target and silence one allele at the mRNA level using RNA interference (RNAi), represents an attractive approach to correct the fundamental genetic defect in individuals suffering from autosomal dominant disorders [1]. While RNAi knockdown strategies have shown promise for some dominantly inherited non-muscle disorders [1], this therapeutic approach has not yet been developed or tested for the treatment of autosomal dominant muscle diseases
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