Periodic Reporting for period 4 - EU-rhythmy (Molecular strategies to treat inherited arrhythmias)
Période du rapport: 2020-05-01 au 2021-10-31
EU-rythmy looks into the sudden cardiac death of young people suffering from genetic heart diseases leading to develop arrhythmias. These patients live an apparently normal life, often engaging in competitive sports. The underlying genetic disease may in fact remain silent for years, as medical assessments may fail to identify major abnormalities in the electrocardiogram. Cardiac arrest may be the first manifestation of the disease. Electrocardiographic screening has facilitated the identification of affected individuals, while the availability of genetic screening has allowed clinicians to identify affected individuals before they start manifesting symptoms such as syncopal episodes during stress and emotions.
The young age of victims of inherited arrhythmias is a major health and social challenges, and there is strong evidence that cardiac arrest can be prevented so to allow these individuals to enjoy a full life with the same long-term survival rate as the general population. Clinical research and practice is thus faced with important targets, particularly in advancing the management of the disease from treatment – i.e. what is available today (starting from lifestyle adjustments to pharmacological therapy) – to cure. Treatment has been shown to reduce mortality, but it is still limited by the fact that even skipping a single tablet may be sufficient to precipitate malignant arrhythmias in patients. Accordingly, it is time to move to the development of a cure for these diseases by modifying the genetic substrate that leads to arrhythmias.
EU-rhythmy intends to develop a gene therapy approach aimed at curing two inherited arrhythmias in which current treatment is insufficient to protect young patients from adverse outcomes. The first disease is called catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1): as we discovered in 2000, it is
caused by a genetic defect in the gene RyR2, which produces a protein controlling calcium movements in the heart. Subsequently, we have developed a Knock in a mouse model of CPVT that mimics the clinical profile of patients. The second disease for which we will try to develop a curative gene therapy is called Long QT Syndrome 8 or Timothy Syndrome, a complex syndrome which – besides leading to arrhythmias – also causes behavioural abnormalities such as autism, syndactyly (i.e. a malformation of hands and feet whose fingers are attached to each other), dangerous hypoglycemia not responsive to glucose administration, hypothermia. Unfortunately – since an animal model that replicates the disease and presents the same genetic abnormalities caused in humans does not exist – we planned to develop a model of Timothy syndrome to be used for the development of gene therapy but also in collaboration with other teams to develop innovative small molecules and drugs to improve the different symptoms of the disease.
The young age of victims of inherited arrhythmias is a major health and social challenges, and there is strong evidence that cardiac arrest can be prevented so to allow these individuals to enjoy a full life with the same long-term survival rate as the general population. Clinical research and practice is thus faced with important targets, particularly in advancing the management of the disease from treatment – i.e. what is available today (starting from lifestyle adjustments to pharmacological therapy) – to cure. Treatment has been shown to reduce mortality, but it is still limited by the fact that even skipping a single tablet may be sufficient to precipitate malignant arrhythmias in patients. Accordingly, it is time to move to the development of a cure for these diseases by modifying the genetic substrate that leads to arrhythmias.
EU-rhythmy intends to develop a gene therapy approach aimed at curing two inherited arrhythmias in which current treatment is insufficient to protect young patients from adverse outcomes. The first disease is called catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1): as we discovered in 2000, it is
caused by a genetic defect in the gene RyR2, which produces a protein controlling calcium movements in the heart. Subsequently, we have developed a Knock in a mouse model of CPVT that mimics the clinical profile of patients. The second disease for which we will try to develop a curative gene therapy is called Long QT Syndrome 8 or Timothy Syndrome, a complex syndrome which – besides leading to arrhythmias – also causes behavioural abnormalities such as autism, syndactyly (i.e. a malformation of hands and feet whose fingers are attached to each other), dangerous hypoglycemia not responsive to glucose administration, hypothermia. Unfortunately – since an animal model that replicates the disease and presents the same genetic abnormalities caused in humans does not exist – we planned to develop a model of Timothy syndrome to be used for the development of gene therapy but also in collaboration with other teams to develop innovative small molecules and drugs to improve the different symptoms of the disease.
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.
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.
The availability of the knock-in pig model has allowed our team to map for the first time the heart of a large mammal model of TS1, discovering that the consequences of the G406R mutation extend far beyond the findings reported in mice models. The study of the hearts of our swine model has highlighted that the calcium overload present in isolated cardiac cells from TS1-affected pigs, leads to novel arrhythmogenic consequences in the pattern of activation of the heart that opens new avenues for therapy.
The model has also been exploited to test the efficacy of an RNA-interfering molecule that has demonstrated in vitro ability to specifically silence the G406R mutant allele. During the development of the project, we have solved major challenging aspects of the gene-therapy delivery to the heart, and we have devised a novel protocol that eliminates the Anti-AAV antibodies and leads to infection of cardiomyocytes in both the right and left ventricles, confirming the ability of the gene therapy to successfully reach its target. Thanks to the results obtained thanks to ERC, we have now been funded by the Italian government (PNRR funding Scheme: National Champions 3) to advance the use of RNA interference gene therapy in the field of inherited arrhythmias and amyloidosis in collaborations with partners at UNIPV.
The model has also been exploited to test the efficacy of an RNA-interfering molecule that has demonstrated in vitro ability to specifically silence the G406R mutant allele. During the development of the project, we have solved major challenging aspects of the gene-therapy delivery to the heart, and we have devised a novel protocol that eliminates the Anti-AAV antibodies and leads to infection of cardiomyocytes in both the right and left ventricles, confirming the ability of the gene therapy to successfully reach its target. Thanks to the results obtained thanks to ERC, we have now been funded by the Italian government (PNRR funding Scheme: National Champions 3) to advance the use of RNA interference gene therapy in the field of inherited arrhythmias and amyloidosis in collaborations with partners at UNIPV.