In vivo comparison of 2'-O-methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping.
BACKGROUND
Antisense-mediated exon skipping is a putative treatment for Duchenne muscular dystrophy (DMD). Using antisense oligonucleotides (AONs), the disrupted DMD reading frame is restored, allowing generation of partially functional dystrophin and conversion of a severe Duchenne into a milder Becker muscular dystrophy phenotype. In vivo studies are mainly performed using 2'-O-methyl phosphorothioate (2OMePS) or morpholino (PMO) AONs. These compounds were never directly compared.


METHODS
mdx and humanized (h)DMD mice were injected intramuscularly and intravenously with short versus long 2OMePS and PMO for mouse exon 23 and human exons 44, 45, 46 and 51.


RESULTS
Intramuscular injection showed that increasing the length of 2OMePS AONs enhanced skipping efficiencies of human exon 45, but decreased efficiency for mouse exon 23. Although PMO induced more mouse exon 23 skipping, PMO and 2OMePS were more comparable for human exons. After intravenous administration, exon skipping and novel protein was shown in the heart with both chemistries. Furthermore, PMO showed lower intramuscular concentrations with higher exon 23 skipping levels compared to 2OMePS, which may be due to sequestration in the extracellular matrix. Finally, two mismatches rendered 2OMePS but not PMO AONs nearly ineffective.


CONCLUSIONS
The results obtained in the present study indicate that increasing AON length improves skipping efficiency in some but not all cases. It is feasible to induce exon skipping and dystrophin restoration in the heart after injection of 2OMePS and unconjugated PMO. Furthermore, differences in efficiency between PMO and 2OMePS appear to be sequence and not chemistry dependent. Finally, the results indicate that PMOs may be less sequence specific than 2OMePS.
Heemskerk
Hans A
HA
DMD Genetic Therapy Group, Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
de Winter
Christa L
CL
de Kimpe
Sjef J
SJ
van Kuik-Romeijn
Petra
P
Heuvelmans
Niki
N
Platenburg
Gerard J
GJ
van Ommen
Gert-Jan B
GJ
van Deutekom
Judith C T
JC
Aartsma-Rus
Annemieke
A
D000818:Animals; D001483:Base Sequence; D005091:Exons / Q000235:genetics*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D051379:Mice; D018101:Mice, Inbred mdx; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000628:therapy; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000235:genetics; D054735:Phosphorothioate Oligonucleotides / Q000008:administration & dosage / Q000235:genetics
D016376:Oligonucleotides, Antisense; D054735:Phosphorothioate Oligonucleotides