Use of homology-independent targeted integration gene editing to correct proximal hotspot DMD gene mutations


Pre-Clinical Research

Poster Number: 137


Anthony Stephenson, Center for Gene Therapy, Nationwide Children's Hospital, Stefan Nicolau, MD, Nationwide Children's Hospital, Tatyana Vetter, PhD, Nationwide Children's Hospital, Kevin Flanigan, MD, Nationwide Children's Hospital

Duchenne muscular dystrophy (DMD) is a progressive muscle disease which manifests as difficulty walking in childhood, loss of ambulation by the teens, and death early in adulthood. DMD is caused by loss of the muscle-specific structural protein dystrophin resulting in poor muscle cell integrity. Chronic cycles of muscle degeneration in individuals with DMD causes a decline in muscle mass, strength, cardiac performance, and ventilation over time. Adeno-associated virus (AAV) vectorized gene editing with CRISPR-Cas9 has recently emerged and shown great potential for restoring dystrophin protein expression in animal models of DMD. However, these approaches most often generate shortened mutant dystrophin isoforms with clinical benefits highly dependent on patients' specific DMD gene mutation. Importantly, as most patients exhibit single or multiple exon deletions, knock-in gene editing to restore full-length dystrophin expression is highly desirable. Progress in knock-in gene editing for DMD is stifled by the low efficiency of homology-mediated knock-in within non-dividing cells. However, a recently described CRISPR-Cas9-based technology called homology-independent targeted integration (HITI) allows knock-in with the ubiquitous and efficient non-homologous end joining DNA repair pathway. Here, we designed a HITI gene editing approach to replace exons 1 – 19 with a single “mega-exon” of ~2.5 kb comprised of the coding sequence of exons 1 – 19 without the intervening introns and driven by a muscle-specific promoter. Using a CRISPR guide RNA (gRNA) that targets DMD intron 19, we tested HITI-mediated replacement of exons 1 – 19 in patient-derived cells and a DMD mouse model carrying an exon 2 duplication (dup2 mice). Two AAV vectors were used together to deliver CRISPR-Cas9 and the HITI donor DNA encoding DMD exons 1-19. We found that our two-AAV system induced significant levels of knock-in of the ~2.5 kb mega-exon into intron 19 in patient cells and dup2 mice. This resulted in meaningful expression of full-length dystrophin in hearts and skeletal muscles of dup2 mice and provided proof-of-concept data to support further development of HITI gene editing to replace exons 1 – 19, a strategy that could benefit DMD patients with mutations upstream of intron 19 (~25% of patients).