title: Exon-skipped dystrophins for treatment of Duchenne muscular dystrophy: mass spectrometry mapping of most exons and cooperative domain designs based on single molecule mechanics.
abstract: Force-bearing linkages between the cytoskeleton and extracellular matrix are clearly important to normal cell viability-as is evident in a disease such as Duchenne muscular dystrophy (DMD) which arises in the absence of the linkage protein dystrophin. Therapeutic approaches to DMD include antisense-mediated skipping of exons to delete nonsense mutations while maintaining reading frame, but the structure and stability of the resulting proteins are generally unclear. Here we use mass spectrometry to detect most dystrophin exons, and we express and physically characterize dystrophin "nano"-constructs based on multiexon deletions that might find use in a large percentage of DMD patients. The primary structure challenge is addressed first with liquid chromatography tandem mass spectrometry (LC-MS/MS) which can detect tryptic peptides from 53 of dystrophin's 79 exons; equivalent information from immunodetection would require 53 different high-specificity antibodies. Folding predictions for the nano-constructs reveal novel helical bundle domains that arise out of exon-deleted "linkers," while secondary structure studies confirm high helicity and also melting temperatures well above physiological. Extensional forces with an atomic force microscope nonetheless unfold the constructs, and the ensemble of unfolding trajectories reveal the number of folded domains, proving consistent with structure predictions. A mechanical cooperativity parameter for unfolding of tandem domains is also introduced as the best predictor of a multiexon deletion that is asymptomatic in humans. The results thereby provide insight and confidence in exon-skipped designs.
authors.affiliation: Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, USA.
authors.forename: Christine Carag
authors.initials: CC
authors.lastname: Krieger
authors.forename: Nishant
authors.initials: N
authors.lastname: Bhasin
authors.forename: Manorama
authors.initials: M
authors.lastname: Tewari
authors.forename: Andre E X
authors.initials: AE
authors.lastname: Brown
authors.forename: Daniel
authors.initials: D
authors.lastname: Safer
authors.forename: H Lee
authors.initials: HL
authors.lastname: Sweeney
authors.forename: Dennis E
authors.initials: DE
authors.lastname: Discher
mesh_terms: D002470:Cell Survival / Q000235:genetics; D002853:Chromatography, Liquid; D002874:Chromosome Mapping; D002942:Circular Dichroism; D016189:Dystrophin / Q000235:genetics*; D005091:Exons; D020440:Gene Duplication; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics* / Q000628:therapy*; D017434:Protein Structure, Tertiary
chemical_list: D016189:Dystrophin