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 models. In detail, we will: 1) assemble CRISPR/Cas9 systems to target the DMD gene exon 2 duplication: the system will target an intronic portion of the duplicated region so that it will create two double strand breaks and stimulate the repair machinery to recombine these sites leading to the exclusion of the duplication; 2) deliver CRISPR/Cas9 systems in HEK293 and myogenic cells to identify the more efficient one; 3) produce immortalized myogenic cells from fibroblasts of patients with exon 2 duplications as cellular models for testing our therapeutic approach; 4) develop Adeno-Associated Virus (AAV) vectors for the delivery of the selected CRISPR/Cas9 systems in the myogenic cells derived from fibroblasts of patients with exon 2 duplication and characterize the resulting effect. 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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