The long term goal of this project is to generate the first therapeutic approach for tandem exon duplications. The selected target will be the most frequent duplication in the dystrophin gene (DMD): exon 2 duplication. In order to open the way to a therapy for this class of neglected variations we will develop viral vectors to test the efficacy of the approach in in vitro and in vivo models. In detail, we have assembled CRISPR/Cas9 systems to target the DMD gene exon 2 duplication with the idea to target intronic portions of the duplicated region creating two double strand breaks and stimulate the repair machinery to recombine these sites leading to the exclusion of the duplication. The aim of the project are: 1) testing of CRISPR/Cas9 activity in myogenic immortalized cells; 2) creation of a hDMD mdx mouse model bearing exon 2 duplications; 3) AAV-CRISPR/Cas9 vector creation for the treatment of relevant DMD mouse models; 4) assessment of the induced modifications and of rescue of dystrophin synthesis, 5) assessment of the off-target cleavage by specific Cas9-ChIP protocol. This proposal will evaluate the feasibility of a gene targeting approach to rescue the exon 2 duplication which results in a DMD phenotype. Overall these objectives will provide the proof-of-principle for a therapeutic approach for duplications paving the way for the treatment of several other diseases.
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