Abstract
Duchenne muscular dystrophy (DMD) is a genetic disorder caused by mutations in the dystrophin-encoding DMD gene. The DMD gene, spanning over 2.4 megabases along the short arm of the X chromosome (Xp21.2), is the largest genetic locus known in the human genome. The size of DMD, combined with the complexity of the DMD phenotype and the extent of the affected tissues, begs for the development of novel, ideally complementary, therapeutic approaches. Genome editing based on the delivery of sequence-specific programmable nucleases into dystrophin-defective cells has recently enriched the portfolio of potential therapies under investigation. Experiments involving different programmable nuclease platforms and target cell types have established that the application of genome-editing principles to the targeted manipulation of defective DMD loci can result in the rescue of dystrophin protein synthesis in gene-edited cells. Looking towards translation into the clinic, these proof-of-principle experiments have been swiftly followed by the conversion of well-established viral vector systems into delivery agents for DMD editing. These gene-editing tools consist of zinc-finger nucleases (ZFNs), engineered homing endoculeases (HEs), transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases (RGNs) based on clustered, regularly interspaced, short palindromic repeats (CRISPR)–Cas9 systems. Here, we succinctly review these fast-paced developments and technologies, highlighting their relative merits and potential bottlenecks, when used as part of in vivo and ex vivo gene-editing strategies.
Highlights
Duchenne muscular dystrophy (DMD) is a lethal Xlinked genetic disorder [1] caused by mutations in the ~2.4-megabase DMD gene [2] which lead to irrevocable muscle wasting owing to the absence of dystrophin in the striated muscle cell lineage [3]
The very high amounts of dystrophin seen in activated Pax7-positive satellite cells are followed by very low and intermediate levels of the protein in myoblasts and differentiated muscle cells, respectively [56]. Such differentiation-stage-specific oscillations in dystrophin amounts strengthen the rationale for repairing the genetic defects by direct endogenous DMD editing, as this strategy is expected to restore proper regulation of dystrophin synthesis. These findings demonstrate that recombinant adeno-associated viral vector (rAAV) delivery of RNA-guided nuclease (RGN) complexes can result in the structural improvement of treated striated tissues and lead to the partial rescue of specific muscle functions in dystrophic mice
Provided that clinically applicable delivery vehicles of gene-editing tools become available, ex vivo DMD editing can naturally build upon the numerous investigations that are being conducted on the isolation, characterization, and testing of human myogenic cells isolated from different tissues for treating muscular dystrophies [69,70,71,72,73]
Summary
Duchenne muscular dystrophy (DMD) is a lethal Xlinked genetic disorder (affecting approximately 1 in 5000 boys) [1] caused by mutations in the ~2.4-megabase DMD gene [2] which lead to irrevocable muscle wasting owing to the absence of dystrophin in the striated muscle cell lineage [3]. In part related to this potential issue, T-cell immunity directed to epitopes corresponding to wild-type dystrophin sequences was detected in two patients undergoing a clinical trial based on recombinant adeno-associated viral vector (rAAV) delivery of a microdystrophin construct [31].
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