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

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.

SUBMITTER: Zaw K

PROVIDER: S-EPMC6951410 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Zaw Khine K Greer Kane K Aung-Htut May Thandar MT Mitrpant Chalermchai C Veedu Rakesh N RN Fletcher Sue S Wilton Steve D SD

Frontiers in genetics 20191220

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 ...[more]

PMID: 31956327

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Project description:BackgroundAntisense oligonucleotides (AOs) can interfere with exon recognition and intron removal during pre-mRNA processing, and induce excision of a targeted exon from the mature gene transcript. AOs have been used in vitro and in vivo to redirect dystrophin pre-mRNA processing in human and animal cells. Targeted exon skipping of selected exons in the dystrophin gene transcript can remove nonsense or frame-shifting mutations that would otherwise have lead to Duchenne Muscular Dystrophy, the most common childhood form of muscle wasting.ResultsAlthough many dystrophin exons can be excised using a single AO, several exons require two motifs to be masked for efficient or specific exon skipping. Some AOs were inactive when applied individually, yet pronounced exon excision was induced in transfected cells when the AOs were used in select combinations, clearly indicating synergistic rather than cumulative effects on splicing. The necessity for AO cocktails to induce efficient exon removal was observed with 2 different chemistries, 2'-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers. Similarly, other trends in exon skipping, as a consequence of 2'-O-methyl AO action, such as removal of additional flanking exons or variations in exon skipping efficiency with overlapping AOs, were also seen when the corresponding sequences were prepared as phosphorodiamidate morpholino oligomers.ConclusionThe combination of 2 AOs, directed at appropriate motifs in target exons was found to induce very efficient targeted exon skipping during processing of the dystrophin pre-mRNA. This combinatorial effect is clearly synergistic and is not influenced by the chemistry of the AOs used to induce exon excision. A hierarchy in exon skipping efficiency, observed with overlapping AOs composed of 2'-O-methyl modified bases, was also observed when these same sequences were evaluated as phosphorodiamidate morpholino oligomers, indicating design parameters established with one chemistry may be applied to the other.

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

Publications

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

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