Gene Correction of a Duchenne Muscular Dystrophy Mutation by Meganuclease-Enhanced Exon Knock-In

Duchenne muscular dystrophy (DMD) is a severe inherited, muscle-wasting disorder caused by mutations in the DMD gene. Gene therapy development for DMD has concentrated on vector-based DMD minigene transfer, cell-based gene therapy using genetically modified adult muscle stem cells or healthy wild-type donor cells, and antisense oligonucleotide-induced exon-skipping therapy to restore the reading frame of the mutated DMD gene. This study is an investigation into DMD gene targeting-mediated correction of deletions in human patient myoblasts using a target-specific meganuclease (MN) and a homologous recombination repair matrix. The MN was designed to cleave within DMD intron 44, upstream of a deletion hotspot, and integration-competent lentiviral vectors expressing the nuclease (LVcMN) were generated. MN western blotting and deep gene sequencing for LVcMN-induced non-homologous end-joining InDels (microdeletions or microinsertions) confirmed efficient MN expression and activity in transduced DMD myoblasts. A homologous repair matrix carrying exons 45–52 (RM45–52) was designed and packaged into integration-deficient lentiviral vectors (IDLVs; LVdRM45–52). After cotransduction of DMD myoblasts harboring a deletion of exons 45 to 52 with LVcMN and LVdRM45–52 vectors, targeted knock-in of the RM45–52 region in the correct location in DMD intron 44, and expression of full-length, correctly spliced wild-type dystrophin mRNA containing exons 45–52 were observed. This work demonstrates that genome surgery on human DMD gene mutations can be achieved by MN-induced locus-specific genome cleavage and homologous recombination knock-in of deleted exons. The feasibility of human DMD gene repair in patient myoblasts has exciting therapeutic potential. Popplewell and colleagues develop a meganuclease that targets an intron upstream of many common mutations in the Duchenne muscular dystrophy (DMD) gene. They evaluate the efficiency of cleavage at this site following lentivirus vector–mediated expression of the meganuclease in DMD patient myoblasts, as well as the capacity of the meganuclease to induce recombination with a second lentivirus vector bearing the downstream DMD exons flanked by arms of homology. Using this approach, they are able to detect full-length DMD mRNA in patient myoblasts following cotransduction with the meganuclease and template-bearing vectors.