Monday, June 4, 2007

RNAi by another Means: Dicer-substrate RNAs

The most widely known and used triggers of RNAi are small interfering RNAs (siRNAs) and DNA-directed hairpin structures. Whereas siRNAs are channelled into the downstream steps of the RNAi pathway, the RiSC complex, hairpin RNAs in humans first have to undergo processing by the endogenous microRNA pathway to yield the active small silencing RNAs. There is, however, a third way to induce RNAi, and these are so called Dicer-substrate RNAs. Their potential utility for gene silencing was recognised following the finding by the Cleary/Hannon [Siolas et al. (2005) Nat. Biotechnol. 23:181] and Rossi [Kim et al. (2005) Nat. Biotechnol. 23: 222] groups that longer RNA duplexes of around 25-27 bp that undergo initial processing by the RNAi enzyme Dicer to yield the active small RNA are in some cases more efficient in gene than the equivalent siRNA. This is thought to be the consequence of Dicer actually forming part of the RiSC loading complex, thereby ensuring efficient hand-off of the small RNA product into the RiSC silencing complex.

There are a number of reasons why Dicer-substrate RNAs have not become a mainstream tool for inducing RNAi yet. Among these were the difficulty of manufacturing Dicer-substrate RNAs that would yield predictable small RNA effectors, non-specific perturbations of gene expression due to cytokine induction by the dsRNA, and the lack of reliable Dicer-substrate RNA design rules. Finally, RNAi inducers upstream of siRNAs may compete with more elements of the microRNA pathway than necessary and the longer length of RNAs will add to the cost and complexity of synthesis.

Some of these challenges, however, are being met mostly as a result of a collaboration between the Rossi lab of the City of Hope, California, and the nucleic acid synthesis company IDT which licensed Dicer-substrate RNAs for use in non-therapeutic applications. Creating dsRNA with one blunt end that contains DNA nucleotides on one strand and 2 nucleotide 3’ overhangs on the other end introduced directionality into Dicer processing. Moreover, it appears that the same modifications that can be introduced into standard siRNAs to avoid cytokine induction also work well for Dicer-substrate RNAs. One weakness that remains, however, is the lack of efficient Dicer-substrate RNA design rules. However, in collaboration with Bio-Rad, IDT is screening and developing sets of validated Dicer-substrate RNAs that have greater than 85% knockdown efficiencies.

It remains to be seen how well accepted Dicer-substrate RNAs will eventually become. With IDT, possibly the world’s largest synthetic nucleic acids supplier for research purposes, behind the technology, Dicer-substrate RNAs should be able to reach most researchers in the field. It will therefore be their hands-on experience, publications showing the benefits of Dicer-substrate RNAs and word-of-mouth that will determine the success of Dicer-substrate RNAs. As siRNAs have shown, a biotechnology that works predictably does not need much advertisement. Some interest meanwhile is demonstrated by the fact that Novartis is apparently testing a small library of Dicer-substrate RNAs for target validation purposes, and Nastech Pharmaceutical Company has obtained an exclusive license from the City of Hope for developing Dicer-substrate RNAs as therapeutics against a handful of gene targets.

With regards to IP issues, I would expect that their therapeutic use would require some kind of licensing agreement from the beneficiaries of the Tuschl I and II patents partly because of the 2nt 3’ overhang structures and the fact that Dicer-substrate RNAs are the immediate precursors of siRNAs. A precedent for this kind of licensing agreement has been set before by Benitec, which in 2005 has taken a license from Alnylam for the “targeted gene silencing mediated by short interfering RNAs (siRNAs) generated from DNA constructs introduced into cells”.

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By Dirk Haussecker. All rights reserved.

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