Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle-wasting disease, characterized by progressive weakening of skeletal, respiratory, and cardiac muscle followed by necrosis and fibrosis. DMD affects ~1:5000 live male births and is associated with delayed motor milestones. DMD occurs as a result of mutations within the DMD gene that lead to premature termination of translation.
At present, despite the several therapeutic and pharmacological approaches under development, no cure exists for DMD.
Duplications in DMD account for ~5–10% of all reported mutations in DMD, although the incidence may be higher. Despite the limited number in DMD, duplications are widespread in almost all diseases and are generally neglected by therapeutic approaches. New molecular tools, now represented by genome-editing technologies that use synthetic nucleases in order to introduce targeted alterations at specific sites in the genome, hold great promises for revolutionizing the gene therapy strategies. According to these premises, we conceived and validated a strategy based on the CRISPR/Cas9 system to repair tandem duplications by removing the mutation: compared to the exon deletions approach which uses two guideRNA (gRNA) targeting two unique regions defining the sequence to be deleted, our approach employs only one gRNA against a unique intronic sequence within the tandem duplication. This strategy exploits the characteristic of tandem duplication (two identical contiguous sequences) and results in the removal of the mutation restoring the wild-type dystrophin expression.
DMD2CURE had the objectives to test and select the best gene editing systems for the creation an adeno-associated virus (AAV) vector-mediated gene therapy for duplications in DMD, the creation of a new mouse model with DMD caused by a duplication and to obtain the proof-of concept of a CRISPR/Cas AAV vector-mediated gene editing therapy for the treatment of DMD.
In conclusion data obtained in the frame of the DMD2CURE project, allowed the validation of the CRISPR/Cas9 strategy for duplications in DMD patients’ cells with duplications of the exon 2, the selection of muscle specific promoters for the expression of Cas9 and the construction of AAV vectors for the delivery of a full CRISPR/Cas9 system in muscles. Despite the intense research activity, the creation new mouse model bearing the duplication of the DMD exon 2 was not achieved during the frame of the project and the test of the developed vectors in this model delayed.
