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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - DMD2CURE (Correction of duplications in the DMD gene by a CRISPR/Cas9 approach)

Teaser

Summary

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 ~5â€“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 patientsâ€™ 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.

Work performed

The main objective of the DMD2CURE proposal was to develop a therapeutic strategy for DMD duplications. During the two years of the grant, Dr. Bovolenta published the proof of concept of the CRISPR/Cas9 strategy for DMD duplications in DMD patientsâ€™ cells and tuned the components and expression of the AAV vectors to deliver the gene editing systems. The challenges of this project were related to the creation of a new mouse model and the expression of a transgene (Cas9) larger than the AAV encapsidation limit.
Dr. Bovolenta improved the cloning processes for the gRNAs and the assembly of AAV vectors and exploited the more recent advances in the DMD and gene editing field to build AAV vectors containing a full functional CRISPR/Cas9 system. This allowed the comparison of different CRISPR/Cas systems and the selection of the more efficient ones.
The translation of this proof of concept to the clinic require the development of a new mice model that was not possible to create during the frame of the DMD2CURE. However, Dr. Bovolenta is still pursuing the objective and is now collaborating with a large consortium that has as well the aim to obtain the proof-of-principle for a CRISPR/Cas9 therapeutic approach for DMD in large animal models.
These two aspects constitute the core of the experimental activity of Dr. Bovolenta for the next years. Importantly, the improvement of the gene transfer efficacy and the targeting of muscles are primary goals in the AAV gene therapy field. More in general, innovations derived from the project of Dr. Bovolenta will contribute to the advancement of the AAV gene therapy for neuromuscular diseases.
Exploitation and dissemination:
Dr. Bovolenta participated in the preparation of 4 scientific publications. Of note, two publications focused on DMD and in one of them, Dr. Bovolenta was last author. One oral presentation and one poster about DMD was presented at international meetings. In addition to that, Dr. Bovolenta is member of the ARRIGE (Association for Responsible Research and Innovation in Genome Editing)
Finally, along the two years Dr. Bovolenta has been invited as speaker in patients advocacy/support meetings as the AFM-Telethon â€œJournÃ©es des Famillesâ€ and was interviewed by Telematin, a TV program aired on France 2.

Final results

Duchenne Muscular Dystrophy is still a disease without cure and in particular therapeutic options for duplications in DMD are limited. The main impact of the DMD2CURE was the demonstration of a proof-of-concept for a gene editing therapeutic strategy for the duplication of the dystrophin exon 2 in patientsâ€™ muscle cells and the development of AAV vectors for the in vivo delivery of the CRISPR/Cas9 system to the muscles.
The research activities performed in the frame of this project will be instrumental not only for duplications in DMD but also for other kind of mutations and other neuromuscular diseases.
Dr. Bovolenta is continuing the DMD2CURE project and pursuing the development of therapeutic approaches for DMD by participating in a consortium committed to the development of personalized medicine using CRISPR/Cas9 for DMD and other neuromuscular disorders.