Pini, Veronica; (2019) Development of a CRISPR-based gene therapy approach to correct duplications causing Duchenne Muscular Dystrophy. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Duchenne Muscular Dystrophy is a severe neurodegenerative disorder caused by deletions, duplications or point mutations in the DMD gene, which encodes dystrophin. In absence of dystrophin, muscle fibres degenerate and patients become wheelchair dependent by their early teens. Cardiac and respiratory muscles are also affected, causing premature death by the third decade of life. Among the approaches currently being tested in clinical trials to treat this disease, none is suitable to permanently restore dystrophin by removing either small or large multi-exon dystrophin duplications, which account for 10-15% of DMD cases. In this thesis, I designed a genome editing approach to correct duplications in the DMD gene by using a single CRISPR/Cas9 target site. First, I identified a CRISPR/Cas9 nuclease able to efficiently target DMD intron 9, which would be suitable for gene editing in patients harbouring DMD duplications in the mutational hotspot 2-201. Then, I tested both integrating lentiviral particles and nuclear electroporation as tools to deliver and express CRISPR/Cas9 in patient-derived cells carrying different dystrophin duplications. Patient-derived myoblasts allowed me to assess dystrophin restoration at the genomic, transcriptional and protein level by means of the T7 assay, quantitative-PCR and western blot, respectively. I confirmed dystrophin correction in transduced as well as electroporated cells expressing CRISPR/Cas9, and I demonstrated that both a constitutive and a transient nuclease expression led to a similar extent of protein restoration of around 50%. These outcomes allowed me to conclude that CRISPR/Cas9 editing tool is a suitable approach to remove large genomic duplications in vitro. Furthermore, the data presented in this thesis provides the basis for the design of new therapeutic approaches to be tested in vivo in Duchenne Muscular Dystrophy animal models. These include both in vivo CRISPR/Cas9-mediated gene therapy and cell-therapy based on transplantation of ex vivo corrected myoblasts expressing corrected wild-type dystrophin.

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