Estudo da instabilidade somática associada a expansões de tripletos CAG e de proteínas relacionadas com a reparação do DNA como alvos terapêuticos para a ataxia espinocerebelosa do tipo 3

This project aims to study novel and previously unexplored therapeutic targets for SCA3 - a currently untreatable disorder-focusing on the stabilization of the disease-causing expanded CAG repeats at the somatic level. Our experimental plan should allow us, if our working hypothesis holds true, to provide mechanistic proof of concept for the targeting of the MutS beta mismatch repair complex (comprising MSH enzymes) as a therapeutic approach for this disease. Specific aims and underlying hypotheses: 1. Hypothesis: Somatic instability is a driver of disease severity/progression in SCA3 patients and is modulated by variation in DNA repair genes AIM: to explore the correlation of somatic expansion (SE) of the CAG repeat with clinical parameters and the role of DNA repair genes (including FAN1 and MSH3) as modifiers of SE and of disease presentation. Blood samples of a large and multicentric cohort of SCA3 mutation carriers will be used, as well as samples from post-mortem brains of patients. SE will be determined using an innovative MiSeq technology and a previously validated analysis pipeline; allelic variants of DNA repair genes, as well as their transcriptomic levels will be correlated with clinical parameters (age at onset, ataxia severity and rate of disease progression) and with the SE ratio. 2. Hypothesis: Fan1 and Msh3 are modifiers of somatic CAG repeat instability in SCA3 mice AIM: To understand the role of Fan1 and Msh3 in SE of the CAG repeats within the human ATXN3 gene expressed in SCA3 mice, as a proof-of-concept for the relevance of the DNA repair pathway regulated by these genes in SCA3. For this, we will use gene editing approaches in a well-established model of disease. After crossing the transgenic CMVMJD135 SCA3 (Q135+/-) with a constitutive Cas9-expressing mouse, an adeno-associated virus (AAV)-based system will be used to deliver targeted guide RNAs and specifically silence Fan1 and Msh3. We will collect brain, spinal cord and peripheral tissue samples of the Q135+/- mice, confirm deletion of Fan1 and Msh3, and analyse the CAG SE pattern in several affected and non-affected brain regions, by PCR amplification and capillary electrophoresis, comparing to Q135+/- controls. The construct generation, production and delivery of these molecules was already optimized by Dr. Mouro Pinto, a consultant in this project (see letter of support). This will allow us to determine if the ablation of Fan1 or Msh3 modifies the CAG repeat instability in the SCA3 mouse tissues, and to verify whether the changes are in the same direction or different than seen for HD. 3. Hypothesis: The mismatch repair enzyme Msh3 contributes to SCA3 pathogenesis and its inactivation is neuroprotective AIM: To evaluate the therapeutic effect of inactivating Msh3 in SCA3 mice, validating the involvement of this enzyme and the role of somatic instability in neurotoxicity. Similarly to the approach used in AIM2, the Q135+/-;Cas9+/- animals will be used, followed by the systemic treatment of the mice with an AAV as a delivery system for sgRNAs against Msh3. After viral transduction and confirmation of Msh3 ablation, we will conduct a preclinical trial to evaluate the impact of this modulation on disease severity at the behavioural and neuropathological levels. Preclinical trials using this SCA3 mouse model have been our main research focus in the last decade, therefore we have a completely optimized setup and expertise to perform this task.