Allele specific silencing as an innovative approach for the treatment of autosomal dominant Catecholaminergic Polymorphic Ventricular Tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease characterized by adrenergically mediated bidirectional or polymorphic ventricular tachycardia leading to syncope and/or sudden cardiac death in individuals without structural heart defects. The autosomal dominant form of CPVT is caused by mutations in the gene encoding the cardiac Sarcoplasmic Reticulum calcium release channel, Ryanodine Receptor 2 (RyR2). The aim of our work is to explore the feasibility of allele-specific silencing by RNAi (ASP- RNAi) by selectively reducing the expression of the mutated allele encoding the mutated protein. This would be a potential approach to correct calcium handling dysfunctions characteristic of autosomal dominant CPVT. In order to achieve the optimal result the interfering molecules were designed to be able to knockdown one allele at the transcript level, leaving unaltered the mRNA from the second allele. Since on the RYR2 gene more than 150 CPVT causative mutations are present, generating silencing molecules able to specifically target each different mutation is not feasible. In this scenario, the idea of targeting common SNPs would allow to treating several patients with different genetic condition. The SNPs have to be present on only one allele, guaranteeing the possibility to discriminate it over the other, and have to be present in population mostly in heterozygosis so with a Minor Allele Frequency (MAF%) around 50%. The SNPs that we found to meet this criteria were: rs376509 (c.1359C>T; S453S), rs684923 (c.7806C>T; H2602H) and rs34967813 (c.8873A>G; Q2958R) and were chosen since they had a Minor Allele Frequency (MAF%) of 48.8%, 45% and 22.5% respectively. In the present study, following the work previously done on the hRYR2-S453S SNP an in vitro system to screen a set of siRNA duplex and miRNA molecules was developed, in order to determine those exhibiting an higher efficiency and specificity in silencing the target mRNA. An artificial heterozygous condition in human cultured cells was established with by the use of two plasmids that simulate the WT and the SNP- containing allele, both containing different reporter genes. Subsequently, human cultured cells were co-transfected with the interfering molecules and the two reporter alleles. The effects of the silencing molecules on suppressing target allele transcripts, as well as off-target effect, were investigated by mRNA and protein based assays. The screening was performed taking in consideration the case on which the SNP could be either in cis or in trans with a specific CPVT causative mutation. The study led to the identification of selective and powerful molecules that are being developed for the creation of an artificial viral miRNA expressing vector for further in vitro and in vivo analysis.