Duchenne muscular dystrophy (DMD) is a severe hereditary disorder caused by a loss of dystrophin, an essential musculoskeletal protein. Decades of promising research have yielded only modest gains in survival and quality of life for these patients and there have been no approved gene therapies for DMD to date. There are two significant hurdles to creating effective gene therapies for DMD;​ it is difficult to deliver a replacement dystrophin gene due to its large size and current strategies to restore the native dystrophin gene likely require life-long administration of a gene-modifying drug. This thesis presents a novel method to address these challenges through restoring dystrophin expression by genetically correcting the native dystrophin gene using engineered nucleases that target one or more exons in a mutational hotspot in exons 45- 55 of the dystrophin gene. Importantly, this hotspot mutational region collectively represents approximately 62% of all DMD mutations. In this work, we utilize various engineered nuclease platforms to create genetic modifications that can correct a variety of DMD patient mutations.

Initially, we demonstrate that genome editing can efficiently correct the dystrophin reading frame and restore protein expression by introducing micro-frameshifts in exon 51, which is adjacent to a hotspot mutational region in the dystrophin gene. Transcription activator-like effector nucleases (TALENs) were engineered to mediate highly efficient gene editing after introducing a single TALEN pair targeted to exon 51 of the dystrophin gene. This led to restoration of dystrophin protein expression in cells from DMD patients, including skeletal myoblasts and dermal fibroblasts that were reprogrammed to the myogenic lineage by MyoD. We show that our engineered TALENs have minimal cytotoxicity and exome sequencing of cells with targeted modifications of the dystrophin locus showed no TALEN-mediated off-target changes to the protein coding regions of the genome, as predicted by in silico target site analysis.

In an alternative approach, we capitalized on the recent advances in genome editing to generate permanent exclusion of exons by using zinc-finger nucleases (ZFNs) to selectively remove sequences important in specific exon recognition. This strategy has the advantage of creating predictable frame restoration and protein expression, although it relies on simultaneous nuclease activity to generate genomic deletions. ZFNs were designed to remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript, a method that can potentially restore the dystrophin reading frame in up to 13% of DMD patients. Nucleases were assembled by extended modular assembly and context-dependent assembly methods and screened for activity in human cells. Selected ZFNs had moderate observable cytotoxicity and one ZFN showed off-target activity at two chromosomal loci. Two active ZFN pairs flanking the exon 51 splice acceptor site were transfected into DMD patient cells and a clonal population was isolated with this region deleted from the genome. Deletion of the genomic sequence containing the splice acceptor resulted in the loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin expression in vitro. Furthermore, transplantation of corrected cells into the hind limb of immunodeficient mice resulted in efficient human dystrophin expression localized to the sarcolemma.

Finally, we exploited the increased versatility, efficiency, and multiplexing capabilities of the CRISPR/Cas9 system to enable a variety of otherwise challenging gene correction strategies for DMD. Single or multiplexed sgRNAs were designed to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45-55 and introducing either intraexonic small insertions and deletions, or large deletions of one or more exons. Significantly, we generated a large deletion of 336 kb across the entire exon 45-55 region that is applicable to correction of approximately 62% of DMD patient mutations. We show that, for selected sgRNAs, CRISPR/Cas9 gene editing displays minimal cytotoxicity and limited aberrant mutagenesis at off-target chromosomal loci. Following treatment with Cas9 nuclease and one or more sgRNAs, dystrophin expression was restored in Duchenne patient muscle cells in vitro. Human dystrophin was detected in vivo following transplantation of genetically corrected patient cells into immunodeficient mice.

In summary, the objective of this work was to develop methods to genetically correct the native dystrophin as a potential therapy for DMD. These studies integrate the rapid advances in gene editing technologies to create targeted frameshifts that restore the dystrophin gene around patient mutations in non-essential coding regions. Collectively, this thesis presents several gene editing methods that can correct patient mutations by modification of specific exons or by deletion of one or more exons that results in restoration of the dystrophin reading frame. Importantly, the gene correction methods described here are compatible with leading cell-based therapies and in vivo gene delivery strategies for DMD, providing an avenue towards a cure for this devastating disease.