Antisense suppression of donor splice site mutations in the dystrophin gene transcript.
We describe two donor splice site mutations, affecting dystrophin exons 16 and 45 that led to Duchenne muscular dystrophy (DMD), through catastrophic inactivation of the mRNA. These gene lesions unexpectedly resulted in the retention of the downstream introns, thereby increasing the length of the dystrophin mRNA by 20.2 and 36 kb, respectively. Splice-switching antisense oligomers targeted to exon 16 excised this in-frame exon and the following intron from the patient dystrophin transcript very efficiently in vitro, thereby restoring the reading frame and allowing synthesis of near-normal levels of a putatively functional dystrophin isoform. In contrast, targeting splice-switching oligomers to exon 45 in patient cells promoted only modest levels of an out-of-frame dystrophin transcript after transfection at high oligomer concentrations, whereas dual targeting of exons 44 and 45 or 45 and 46 resulted in more efficient exon skipping, with concomitant removal of intron 45. The splice site mutations reported here appear highly amenable to antisense oligomer intervention. We suggest that other splice site mutations may need to be evaluated for oligomer interventions on a case-by-case basis.
Centre for Comparative Genomics, Murdoch University South St, 6150, Perth, Western Australia, Australia ; Centre for Neuromuscular and Neurological Disorders, University of Western Australia Perth 6009, Western Australia, Australia.
Sue
S
Fletcher
Centre for Neuromuscular and Neurological Disorders, University of Western Australia Perth 6009, Western Australia, Australia.
Penny L
PL
Meloni
Centre for Neuromuscular and Neurological Disorders, University of Western Australia Perth 6009, Western Australia, Australia.
Russell D
RD
Johnsen
Department of Pediatrics, Cincinnati Children's Hospital Medical Centre and University of Cincinnati College of Medicine Cincinnati, 45229-3039, Ohio.
Brenda L
BL
Wong
The Dubowitz Neuromuscular Centre, University College London Institute of Child Health London London, WC1N 1EH, United Kingdom.
Francesco
F
Muntoni
Centre for Comparative Genomics, Murdoch University South St, 6150, Perth, Western Australia, Australia ; Centre for Neuromuscular and Neurological Disorders, University of Western Australia Perth 6009, Western Australia, Australia.
Stephen D
SD
Wilton
Duchenne muscular dystrophy; exon skipping; splice mutation; splice-switching oligomer