Allele specific silencing by RNAi of R92Q and R173W mutations in cardiac troponin T

Many familial forms of hypertrophic and dilated cardiomyopathy (HCM and DCM) are linked to autosomal dominant mutations in genes encoding sarcomeric proteins. Several efforts have been done to develop therapeutic approaches for these patients, including the design of Allele-Specific Silencing approaches by RNA interference (ASP-RNAi). ASP-RNAi represents a powerful and promising strategy to counteract genetic defects by using duplex small interfering RNAs (siRNAs) to target mutant alleles with minimal suppression of the corresponding wild-type allele. We focused on the R92Q and R173W missense mutations in the cardiac Troponin T gene (cTnT) associated to HCM and DCM, respectively and developed an ASP-RNAi strategy to specifically knock-down the mutant alleles. Following siRNA design and generation, the specific silencing of the mutant allele was tested by a luciferase reporter gene assay and further confirmed on HEK293T cells expressing either the wild type or mutant cTnT alleles. siRNAs fully complementary to the target sequence or containing single-base mismatches downstream the targeted mutations were analyzed and evaluated. Results obtained showed that optimal allele discrimination was obtained with the use of siRNA containing a single-base mismatch both for the R92Q and the R173W mutations.