Antisense suppression of donor splice site mutations in the dystrophin gene transcript

Abstract

We describe two donor splice site mutations, affecting dystrophin exons 16 and45 that led to Duchenne muscular dystrophy (DMD), through catastrophicinactivation of the mRNA. These gene lesions unexpectedly resulted in theretention of the downstream introns, thereby increasing the length of the dystrophinmRNA by 20.2 and 36 kb, respectively. Splice-switching antisense oligomerstargeted to exon 16 excised this in-frame exon and the following intronfrom the patient dystrophin transcript very efficiently in vitro, thereby restoringthe reading frame and allowing synthesis of near-normal levels of a putativelyfunctional dystrophin isoform. In contrast, targeting splice-switching oligomersto exon 45 in patient cells promoted only modest levels of an out-of-frame dystrophintranscript after transfection at high oligomer concentrations, whereasdual targeting of exons 44 and 45 or 45 and 46 resulted in more efficient exonskipping, with concomitant removal of intron 45. The splice site mutationsreported here appear highly amenable to antisense oligomer intervention. Wesuggest that other splice site mutations may need to be evaluated for oligomerinterventions on a case-by-case basis.