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Molecular strategies to treat inherited arrhythmias

Periodic Reporting for period 3 - EU-rhythmy (Molecular strategies to treat inherited arrhythmias)

Reporting period: 2018-11-01 to 2020-04-30

"What is the problem being addressed by the EUrhythmy project? EUrythmy is dedicated to the problem of sudden cardiac death in young people affected by genetic diseases that increase the propensity of the heart to develop arrhythmias.
These paitents may have an apparently normal life, sometime they also are eganged in competitive sports and perform well being considered healthy and perfectly trained, the underlying genetic disease may in fact remain silent for many years and even medical assessmente may fail to indetify majora abnormalties in the electrocardiogram and cardiac arrest may be the first manifestation of the disease. Electrocardiographic screening performed in young asymptomatic individuals has shown some benefit facilitating the identification of affected individuals. Similarly the availabibility of genetic screeing have advanced the ability of clinicians to identify affected individuals before they start manifesting symptoms that frequently are syncopal episodes during stress and emotions.
Why is this problem important for society? The young age of victims of inherited arrhythmias and the evidence that if cardiac arrest can be prevented they may enjoy a full life with the same expectation of long term survival as observed in the general population. For these reason several goals remain important targets for reasearch and for improvement of clinical pracrice. Some advancement has been made in establishing electrocardiographic screenings in chidlren and adolescence that can allow to establish the diagnosis before symptoms manifests and direct patients to molecular diagnosis that may allow to further identify affected family members. What still remain unsolved is how to advance the management of the disease from ""treatment"" to ""cure"". Treatment is what is available today, basically for each disease the first line treatmente is made of life style adjustment and pharmachological therapy: despite this approache has been shown to reduce mortality, it is still limited by the fact that even skipping a single tablet may be sufficient to precipitate malignant arrhythmias in patients: this is what we define the limitation of ""treatment"". Accordingly we believe that it is now time to move to the development of ""cure"" for these diseases modifying the genetic substrate that leads to arrhythmias.
What are the overall objectives of EUrhythmy? EUrhythmy intends to develop a gene therapy appraoch to cure two of the inhertied arrhythmias in which available ""treatment"" is unsufficient to protect young patients from adverse otucome and to lift the spell from their life that are often overwhelmed by the concern of dying from an arrhythmic episode that may unexpectedly occur. We aim at developing a gene therapy for a disease called catecholaminergic polymorphic ventricular tachycardia a name abbreviated as CPVT that is caused by a genetic defect in a gene called RyR2 that is the gene that produced a protein that controls calcium movements in the heart. Patients suffer from arrhythmias because this protein releases too much calcium thus inducing arrhythmias: we discovered that the RyR2 gene is responsible for CPVT in 2000 and subsequently we have developed a Knock in mouse model of CPVT that mimics the clinical profile of patients in the affected mice.
The second disease for which we will try to develop a curative gene therapy strategy is called Long QT Syndrome 8 or Timothy Syndrome. The disease is a complex syndrome that beside leading to arrhythmias also causes behavioral abnormalities such as autism, syndactyly, i.e. a malformation of hands and feet in which fingers are attached to each other, hypoglycemia, i.e dramatic and unexplained drop of sugar levels that are not responsive to glucose administration, hyptohermia i.e. lower than normal body temperature. Unfortunately an animal model that replicate the disease and presents the same genetic abnormality that causes the disease in humans does not exist and therefore we are lacking an appropriate model to test new therapeutic strategies. We have therefore planned that the EU-rhythmy project will fill this gap and develop a model of Timothy sydnrome that not only will be useful to us for the development of gene therapy but may also be used in collaboration with other teams to devolop innovative small molecules and drugs to improve the differenty symptoms of the disease."
In the first two years of the Eurhythmy project we have developed a gene therapy strategy that is able to use a method called RNA-interference to reduce the production of the mutant protein thus improving the RyR2-medated calcium movements in cardiac cells and abolish . The molecules of RNA that can improve the function of the RyR2 protein are delivered to the heart using a virus called AAV) that has been modified to be unable to replicate inside the cells and to cause damage and rather 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 and if they are treated in adulthood, after they already manifest the arrhythmias, whithin 8 weeks from treatment they stop manifesting arrhythmias.

In the first 30 moths of the Eurhythmy project, we have not only successfully developed the gene therapy strategy for CPVT, but we have also advanced the attempt to develop a pig model of Timothy Syndrome (TS). Pig models of human diseases are becoming more popular in the circumstances where the rodent model is inadequate because they do not reproduce the phenotype or there are issues of anatomy and physiology like in cardiac diseases. Making a pig model however it is a challenge both from the economic and practical point of view as it will require 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 on the development of animal models for scientific biomedical research, we have embarked in this effort driven by the motivation to impact understanding of mechanisms that cause arrhythmias in Timothy syndrome as well as to develop a gene therapy for the disease. The technology used to achieve this goal is based on the insertion of the mutation of interest in fibroblasts from pigs using state of the art genome editing techniques, once this goal is achieved the nucleus of fibroblasts is inserted in a oocyte of a sow (somatic cell nuclear transfer, SCNT) to create an embryo that is implanted in the uterus of a sow that serves as surrogate mother. We consider highly remarkable that in 30 months we have been able to induce the first pregnancies that has lead to the delivery of several litters of pigs carrying one of the required precise point mutation of the TS. It is a great success to observe that in the litter we have some piglets carrier of the mutations, as well as age matched controls, that causes the disease in humans. We will study the pigs in the next few months to establish which clinical profile they manifest and hopefully, if they present cardiac phenotype of TS we will be able to start designing molecular strategies to cure this severe disease.
"The results obtained in the WP1 have really moved the field beyond the state of the heart. we have demonstrated that RNA-interference is an appropriate gene therapy strategy able to abolish arrhythmias. The radical effect of the RNA molecule that we have developed to reduce the transcripts of mutant RyR2 and its ability to prevent arrhythmias development in mice infected in the early days of life combined with the ability to suppress arrhythmias in mice infected during adulthood have provided a solid demonstration of a new paradigm in the field. It is infact important to consider the widespread skepticism against the concept that gene therapy could be effective to cure arrhythmia because of the concern that the inhomogeneous infection of cardiac cells would increase, rather than cure, arrhythmias. Our hypothesis, at variance with the prevailing attitude, was that even if a fraction of all cardiac cells would be 2cured"" by our gene therapy approach, it would have been suffcient to block propagation of arrhythmais thus benefitting patients: data on the CPVT mice confirmed our hypothesis. We are therefore very satisfied to realize that we have now opened a new field for treatment of cardiac arrhythmias with gene therapy.
The most challenging part of the EUrythmy project will be conducted in the next 30 months when we will face a double challenge and we will have to test in our LQT8 pigs the efficacy of gene therapy. These experiments will really raise the bar of complexity becuase the gene therapy strategy that we will use in LQT8 is complex and different and it will require expressing the wild type channel with a methodology never applied in the heart befoer. The next challenge is related to the difficulty to infect an adequate number of cells in a large heart as that of siwnes and finally since the mechanism of arrhythmias initiation in lQT8 is differen than that present in CPVT, we may encounter proarrhythmias or lack of efficacy. We are however confident that we will overcome the complexity of the challenge and be able to achieve two remarkable results : 1) develop the first knock in animal model of LQT8 that will substantially advance the field not only with this project but also through collaborations that we will establish in the future to understand mechanisms of extracardiac manifestations of the disease and 2) we will develop the first gene therapy approach for Long QT syndrome

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