In the first two years of the Eurhythmy project, we developed a gene therapy strategy able to use RNA-interference to reduce the production of the mutant protein, thus improving the RyR2-mediated calcium movements in cardiac cells. The molecules of RNA that can improve the function of the RyR2 protein are delivered to the heart using a virus called AAV, modified so as to be unable to replicate inside the cells and damage them. Rather, it is able to deliver in the nucleus of the cells the curative RNA molecules. We have shown that when mice are treated at birth, they grow without manifesting the disease while if they are treated in adulthood, within 8 weeks from the treatment they stop manifesting arrhythmias.
We also reasoned that our approach might not be ideal for clinical application since it would require developing a silencing molecule for each pathogenic mutation. We, therefore, opted to target highly prevalent single nucleotide polymorphisms (SNPs) present in the coding region of the RYR2 gene. Considering that each SNPs of RYR2 may co-segregate with the mutations in the same allele (in cis) or in the opposite (in trans). we designed six therapeutic molecules: three targeting the SNPs in cis with the mutation, and three that are effective when the SNPs are in trans with the mutation thus developing a kit of molecules to silence the mutant allele irrespective of the disease-causing mutation. The above data reported for the first time the efficacy of RNAi strategy to treat a cardiac channelopathy and data were published in Circulation Research 2017 Aug 18;121(5):525-536. This discovery was presented over the years in several conferences held by Prof Priori and by the first author of the Circulation Research paper by Mrs. Rossana Bongianino, Ph.D. Additionally, a patent (WO-PTC) has been filed and is currently under evaluation WO2017141157A1WIPO (PCT) and subsequently patents were filed in Europe (EP3417063A1), United States (US20210189401A1); Canada (CA3014550A1); Australia (AU2017220774A1).
In the meantime, we have also advanced the attempt to develop a pig model of Timothy Syndrome (TS), since rodent model is often flawed with issues of anatomy and physiology, as in cardiac diseases. Yet, a pig model requires the sort of clinical assistance that is required for a human patient: thanks to the competence of our partner AVANTEA, a company with a solid background in the development of animal models for scientific biomedical research, we embarked in this effort driven by the motivation to understand mechanisms causing arrhythmias in Timothy syndrome and to develop gene therapy for the disease. The relevant mutation has been thus inserted in fibroblasts from pigs using state-of-the-art genome editing techniques; subsequently, the nucleus of fibroblasts is inserted in an oocyte of a sow (somatic cell nuclear transfer, SCNT) to create an embryo implanted in the uterus of a sow serving as a surrogate mother. The success of this part, which represents the very high-risk component of the project, has been conducted by our partner Avantea that in only 30 months has induced the first pregnancies leading to the delivery of several litters of pigs carrying the causative point mutation that causes TS.
