We developed a gene editing system to correct a subset of mutations in the DMD gene using homology-independent targeted integration (HITI).

Duchenne muscular dystrophy (DMD) is a progressive muscle disorder affecting 1 in 5000 male newborns. In order to restore expression of full-length dystrophin, we designed gene editing systems targeting exons 1-19 and exon 45, two of the common mutational hotspots. We had previously demonstrated that the former system can restore 11% of normal dystrophin levels in the heart in a mouse model of DMD.

HITI allows insertion of large donor fragments into the genome without relying on homology-directed repair pathways. Mutations in exons 1-19 can be corrected through insertion of a new copy of these exons into intron 19, along with a new promoter, effectively bypassing transcription from the native promoter. Deletion of exon 45 can be corrected by insertion of a new copy of this single exon.

The choices of target, spacer length, and scaffold were optimized through in vitro experiments. For in vivo experiments, the editing systems were each delivered using a pair of AAV vectors administered intramuscularly or systemically. Higher biodistribution was achieved using the MyoAAV3A serotype than AAV9. The highest editing levels were seen in the heart, with lower efficiency in skeletal muscles. Efficiency in skeletal muscles was hampered by a loss of both vector genomes and edited genomes between 1 and 3 months post-treatment. Long-read sequencing identified knock-in of fragmentary and recombined AAV-derived sequences at the target site, which reduced the proportion of therapeutic edits. 

HITI can be used to correct mutations in the DMD gene, particularly when combined with knock-in of a highly active promoter. Key obstacles to efficient gene editing in muscle included recombination between AAV vectors and turnover of dystrophic muscle.