Heemskerk HA, de Winter CL, de Kimpe SJ, van Kuik-Romeijn P, Heuvelmans N, Platenburg GJ, van Ommen GJ, van Deutekom JC, Aartsma-Rus A. In vivo comparison of 2’-O-methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping. J Gene Med. 2009 Jan 12. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/19140108

Now for some opinions, based on the paper’s abstract and my understanding of these oligo types (later note: reading the text has not dissuaded me from these views). I’m not surprised by the experimental outcome described in their abstract. In terms of efficacy and sequence specificity, 2’-O-methyl RNA is the best competitor for Morpholinos as pharmaceutical antisense. However, toxicity could be the deciding factor.

Phosphorothioate intersubunit linkages were introduced into nucleic acid oligos to slow their degradation. Natural nucleic acids have a phosphate intersubunit linkage which degrades rapidly in cells. Replacing the phosphate with a sulfur-substituted phosphorothioate makes the oligos resist degradation by nuclease enzymes. However, degradation still occurs, albeit more slowly, and the monomers that are released bear a free phosphorothioate group — and that group is toxic. This has been shown extensively in the literature describing the phosphorothioate antisense structural type – in particular, see the work of Cy Stein. Phosphorothioate groups are used primarily in two applications: antisense and pesticides (try a Google search of “phosphorothioate”).

The phosphorothioate linkage is used in 2’-O-methyl RNA oligos to resist degradation. Often just a few phosphorothioate linkages are used, producing a “chimeric” oligo with phosphate-linked subunits in the middle and a few phosphorothioate-linked subunits at the ends, a strategy that slows degradation from the ends while minimizing the toxins released by the eventual degradation of the oligos.

Morpholinos don’t degrade and they are far less toxic — the toxicity associated with Morpholinos comes from interactions of the oligo base sequence with RNA, a form of toxicity unavoidable with any kind of antisense. A well-chosen sequence can be administered at high doses without toxicity. In contrast, the toxicity of phosphorothioates is a combination of sequence-specific toxicity (like Morpholinos or any other antisense) and the chemical toxicity of the free phosphorothioate monomers.

Now I hope both camps and some independent groups quickly get on with comparative toxicity studies. If I am proven wrong, great — get a phosphorothioate exon skipper for DMD to market ASAP. An exon-skipping drug for DMD is desperately needed.