Antisense PMO found in dystrophic dog model was effective in cells from exon 7-deleted DMD patient.
BACKGROUND
Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD). We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO) targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD(J)) lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient's cells.


RESULTS
We converted fibroblasts of CXMD(J) and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD(J) and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species.


CONCLUSIONS
Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.
Saito
Takashi
T
Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan.
Nakamura
Akinori
A
Aoki
Yoshitsugu
Y
Yokota
Toshifumi
T
Okada
Takashi
T
Osawa
Makiko
M
Takeda
Shin'ichi
S
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D004252:DNA Mutational Analysis; D004195:Disease Models, Animal; D004285:Dogs; D005091:Exons / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008297:Male; D009025:Morpholines / Q000378:metabolism*; D060172:Morpholinos; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics* / Q000473:pathology*; D017570:MyoD Protein / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D017384:Sequence Deletion; D014161:Transduction, Genetic; D055815:Young Adult
D009025:Morpholines; D060172:Morpholinos; D017570:MyoD Protein
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