In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Evaluation of DNA segments in 2′-modified RNA sequences in designing efficient splice switching antisense oligonucleotides
Today’s pick is from RSV advances (royal chemistry society) by Le et al on using gapmers for Duchenne exon skipping. Early stage work from Rakesh Veedu. Doi 10.1039/d1ra00878a
Antisense oligonucleotides (ASOs) can modulate splicing or reduce the amount of transcripts. For the latter gapmers are used, pieces of PS-DNA in the center allow RNase H to bind and cleave the target transcripts. For splice modulation, no gaps are used as you do not want cleavage.
For splice modulations fully modified ASOs are used, PMOs (with poor bioavailability thus leading to high doses in clinical application) or 2O modified PS ASOs, with higher bioavailability but also more side effects (see drisapersen trial for Duchenne).
In order to try and get the best of both worlds, authors here try to introduce gaps into 2OMe and MOE PS oligos targeting mouse dystrophin exon 23. They used gaps at different positions, and kept them small so RNase H is not activated. Red is the gap, blue is MOE, black 2OMe.
Surprisingly the Tm was only mildly affected, except for ASO3. ASOs were transfected with lipofectamine and most ASOs induced exon skipping even at lower doses (some as low as 2.5 nM), while ASO3 was not effective. Authors suggest that ASOs with a gap may be safer.
They also stress this is early stage work. I agree. However, I do not know whether these gapmers are in fact safer. A lot of the side effects of PS modified ASOs are driven by the PS modification, and the 2O modifications protect to some extent.
So for in vivo treatment, the gaps may be less safe rather than more safe. Another comment is that now only lipofectamine transfection was used, which results in very efficient delivery of ASOs. I would be interested to see what happens after gymnotic delivery.
Finally, authors mention exon skipping percentages – however, we know this will be hugely overestimated, as a nested PCR was used to assess exon skipping. There is no other way for cultured cells due to low dystrophin expression levels, however, shorter fragment amplification bias will inflate the exon skipping levels a lot. 31 and 33 cycles were used even if the skipped fragment is amplified 1% more efficiently, after 64 cycles this adds up to 2 fold more skip. Note that this does not matter when checking which is the best ASO.
Provided it is tested in the same PCR the amplification bias applies to all samples. However, the percentages mentioned are not absolute amounts. Looking forward to more work from this group and hope I am wrong about the gapmers being less safe.