Consequences of Making the Inactive Active Through Changes in Antisense Oligonucleotide Chemistries

Khine Zaw; Kane Greer; May Thandar Aung-Htut; Chalermchai Mitrpant; Rakesh N Veedu; Sue Fletcher; Steve D Wilton

doi:10.3389/fgene.2019.01249

Consequences of Making the Inactive Active Through Changes in Antisense Oligonucleotide Chemistries

Front Genet. 2019 Dec 20:10:1249. doi: 10.3389/fgene.2019.01249. eCollection 2019.

Authors

Khine Zaw^{1

2}, Kane Greer^{1

3}, May Thandar Aung-Htut^{1

3}, Chalermchai Mitrpant^{2

3}, Rakesh N Veedu^{1

3}, Sue Fletcher^{1

3}, Steve D Wilton^{1

3}

Affiliations

¹ Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth, WA, Australia.
² Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
³ Perron Institute for Neurological and Translational Science and Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, Perth, WA, Australia.

Abstract

Antisense oligonucleotides are short, single-stranded nucleic acid analogues that can interfere with pre-messenger RNA (pre-mRNA) processing and induce excision of a targeted exon from the mature transcript. When developing a panel of antisense oligonucleotides to skip every dystrophin exon, we found great variation in splice switching efficiencies, with some antisense oligonucleotides ineffective, even when directed to canonical splice sites and transfected into cells at high concentrations. In this study, we re-evaluated some of these ineffective antisense oligonucleotide sequences after incorporation of locked nucleic acid residues to increase annealing potential. Antisense oligonucleotides targeting exons 16, 23, and 51 of human DMD transcripts were synthesized as two different chemistries, 2'-O-methyl modified bases on a phosphorothioate backbone or mixmers containing several locked nucleic acid residues, which were then transfected into primary human myotubes, and DMD transcripts were analyzed for exon skipping. The ineffective 2'-O-methyl modified antisense oligonucleotides induced no detectable exon skipping, while all corresponding mixmers did induce excision of the targeted exons. Interestingly, the mixmer targeting exon 51 induced two unexpected transcripts arising from partial skipping of exon 51 with retention of 95 or 188 bases from the 5' region of exon 51. These results indicated that locked nucleic acid/2'-O-methyl mixmers are more effective at inducing exon skipping, however, this improvement may come at the cost of activating alternative cryptic splice sites and off-target effects on gene expression.

Keywords: DMD; antisense oligonucleotide; cryptic splice site; locked nucleic acid; locked nucleic acid/2′-O-methyl mixmer.