Researchers have developed homology-independent targeted integration (HITI)-mediated gene editing to correct full-length human dystrophin in Duchenne muscular dystrophy (DMD).

The authors of this new method observed full-length dystrophin restoration in skeletal and cardiac muscles of a humanized mouse model of DMD, the hDMDΔ52/mdx model, recently published in Molecular Therapy. The mice contained a full-length human DMD gene with a frame-disrupting deletion of exon 52, which is within the DMD patient mutational hotspot (exons 45-55).

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In addition to the targeted integration of the coding sequence of the missing exon 52, the researchers engineered a superexon encoding the last 28 exons of the DMD gene. They observed full-length dystrophin restoration following this approach.

Pickar-Oliver et al used a dual adeno-associated virus (AAV) delivery system to express the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9). In addition, they used guide RNAs (gRNAs) to generate and target genomic double-strand breaks to deliver donor templates for integration at the cut site.

Site-specific integration was mediated by an error-prone nonhomologous end-joining-based method.

“These HITI-based CRISPR gene editing strategies could potentially be effective therapies to restore full-length dystrophin for >20% of the DMD patient population and could be extended to an even broader population,” the authors said.

“Future efforts can expand this approach to all patients with mutations within and downstream the exon 45-55 hotspot (>50% of all patients) by developing a dual AAV-based system with one AAV that encodes SaCas9 [Staphylococcus aureus Cas9] and a gRNA targeting intron 44, and a second donor AAV vector that contains the human dystrophin cDNA coding sequence downstream of exon 44 (exons 45-79).”


Pickar-Oliver A, Gough V, Bohning JD, et al. Full-length dystrophin restoration via targeted genomic integration by AAV-CRISPR in a humanized mouse model of Duchenne muscular dystrophyMol Ther. Published online September 9, 2021. doi:10.1016/j.ymthe.2021.09.003