A study by the Zamore group published in Science (Ameres et al.: Target RNA-directed Trimming and Tailing of Small Silencing RNAs) provides intriguing insights into an important mechanism impacting siRNA stability by showing that guide strands that are perfectly complementary to their target mRNAs are subject to tailing and subsequent degradation. By better understanding siRNA stability, such work has the potential to decrease the frequency and dose with which synthetic siRNAs have to be administered, thus improving the overall clinical and commercial profile of this class of RNAi Therapeutics.
Actually, siRNA Therapeutics turned out to have a better pharmacokinetic profile than I had thought when I first started to work with siRNAs in late 2002. Most experiments in these early days for mammalian RNAi were conducted in rapidly dividing tissue culture systems where, somewhat depending on the natural turnover of the target mRNA/protein, silencing would be maximal between day 1-3 after siRNA administration and then quickly subside by ~day 5. Luckily, it was then found that it is cell division that is responsible for the relatively rapidly diminishing silencing effect observed in many tissue culture systems by diluting the siRNA-loaded RISC silencing complex between daughter cells. By contrast, silencing in non-dividing tissues, particularly in whole organisms typically persists for about 2-4 weeks after single administration- and on top of it is btw also typically more potent there on a per molecule basis as well!
Especially since many of the first batch of RNAi Therapeutics are likely to be intravenously and intravitreously (needle injection into eye) administered, the longer the interval between drug administrations, the broader the adoption of the technology should be, particularly for non-lethal, chronic diseases. 2-4 weeks is at the lower-to-mid-range for what is practiced for many monoclonal antibodies (subQ and IV) and in my opinion quite acceptable for most diseases where it is worth going to seek treatment for. On the other hand, extending this to something like 6-10 weeks, could in some cases quite significantly impact the adoption of RNAi Therapeutics, especially in situations where the administration procedure itself is associated with cumulative risk e.g. as is the case for the intravitreous route. This realization should also be the motivation for the recently formed collaboration between ophthalmic drug company Surmodics and Egen for ocular controlled-release siRNA formulations.
What Ameres and colleagues show in their paper is that small RNAs such as microRNAs and siRNAs are destabilized in the presence of target mRNAs with perfect complementarity, especially with regard to the 3’ end of the guide strand. For reasons that remain to be determined, the cell somehow recognizes this configuration triggering a polymerase to add a few nucleotides to the guide strand 3’ end which in turn is recruits the ubiquitous RNA degradation complex, the Exosome.
This finding has direct practical implications for the design of siRNAs. Because RNAi gene silencing molecules tolerate mismatches towards their target mRNAs without losing efficacy, especially at the 3’ end, it should be possible to increase siRNA stability by introducing such mismatches. Incidentally, such mismatches should have the added benefit of increasing RISC turnover rates, a finding made by the same group earlier and covered by the first Zamore siRNA design rule patent issued in the US a few months ago and exclusively licensed to Silence Therapeutics.
It is, however, also likely that instead of introducing 3’ mismatches, (blocking) nucleotide modifications at the guide RNA 3’ end would have the same effect by not being suitable substrates for the polymerase.
It will now be important to test how these findings translate to silencing in large animals and develop in vitro assay systems that faithfully recapitulate such small RNA destabilization. If the stability of the guide strand within the RISC complex is indeed a major determinant of the gene silencing longevity in mammals (rather than stability of Argonautes e.g., in which case slow-release strategies would gain in relative attractiveness), then this line of work should have significant potential for improving the eventual clinical and commercial profile of RNAi Therapeutics.