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

Hum Gene Ther. 2013 Jul;24(7):692-701. doi: 10.1089/hum.2013.081.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • DNA Repair / genetics
  • Deoxyribonucleases / metabolism
  • Exons / genetics
  • Gene Knock-In Techniques / methods
  • Genetic Therapy / methods*
  • Genetic Vectors / genetics*
  • High-Throughput Nucleotide Sequencing
  • Humans
  • INDEL Mutation / genetics
  • Lentivirus
  • Muscular Dystrophy, Duchenne / genetics*
  • Muscular Dystrophy, Duchenne / therapy*
  • Mutation / genetics*
  • Myoblasts / metabolism
  • Oligonucleotides, Antisense / genetics
  • Targeted Gene Repair / methods*

Substances

  • Oligonucleotides, Antisense
  • Deoxyribonucleases