Thursday, August 16, 2007

MicroRNA Sponges! A Gene Therapy Approach to Inhibiting MicroRNAs

A report from the lab of Phil Sharp, a scientific co-founder and advisor of Alnylam, in the August 12 online edition of Nature Methods demonstrates the inhibition of microRNA function in mammalian cells through the expression of so called “microRNA sponges” (Ebert et al. (2007): MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nature Methods, DOI: 10.1038/NMeth1079). This comes less than a month after the publication on a similar process in plants in which microRNA activity was shown to be inhibited by target mimicry (3 August 2007 Blog: “Journal Club: Target Mimicry as a New Way to Regulate microRNA Activity”).

MicroRNA sponges are expressed RNAs that function by competing with the natural targets of a given microRNA for its binding and which consequently de-represses these targets. They are therefore functionally equivalent to synthetic antisense inhibitors of microRNAs, sometimes referred to as “antagomirs”, and by the same token should facilitate the functional investigation of microRNAs. Equally exciting is the prospect of using microRNA sponges for the therapeutic inhibition of microRNAs in a gene therapy approach. This new development is analogous to the situation in the RNAi arena where synthetic and gene therapy approaches exist to bring about gene silencing.

Importantly, Ebert and colleagues demonstrate that microRNAs sharing the same seed sequence could be inhibited by the same sponge. This illustrates that sponges work by the normal microRNA-target recognition process and the inhibition is not simply due to sense-antisense interactions. Feeding into this endogenous pathway may also explain why at least in this study competition for microRNA binding with sponges was more potent than with antisense molecules, and why sponges with bulged target sites were more effective than those with perfectly complementary target sites, at least when expressed from Pol II promoters.

Practically, one could therefore imagine a sponge silencing whole families of functionally and sequence-related microRNA families, something that the authors show cannot easily be achieved with single antisense molecules. This is likely to be therapeutically relevant, as it is known for example that members of so called “oncomir” families redundantly contribute to cancer progression. Moreover, due to the modularity of the sponge system, different microRNA binding sites could be incorporated into a single sponge expression cassette to inhibit structurally unrelated microRNAs.

Next to therapeutic applications in microRNA targeting, this study is also of relevance for the RNAi Therapeutics field as this paper confirms now for human cells that siRNA off-targeting through a microRNA mechanism not only raises safety issues, but may also reduce on-target efficacy. I think it would be well worth revisiting whether some of the siRNA modifications shown to reduce off-target gene silencing also enhance on-target gene silencing. Such studies may eventually allow for the development of even more potent siRNAs that work at lower concentrations.

Finally, I would like to point out that RNAs could also function as sponges when expressed from the quite potent RNA polymerase III U6 promoter. Similar to the normal U6 snRNA product, these sponges can only be detected in the nucleus- yet they successfully compete for microRNA binding. As it is unlikely that these RNAs can inhibit the maturation of highly structured microRNA precursors which are present in the nucleus of a cell, this data adds to the growing body of evidence that microRNAs and RNAi-related processes occur in the nucleus. What could be the function of such nuclear small RNAs? Personally, my favourite hypothesis on how microRNAs act is that they bind their targets in the nucleus following transcription but before theire export into the cytoplasm. This is in contrast to most models that hold that microRNAs mature in the cytoplasm and recognise their targets there.

It will also be interesting to find out whether Pol III-driven sponge activity, as shown for Pol II-driven sponges, is seed dependent as this would suggest that transcripts other than those generated by Pol II may functionally associate with the microRNA/RNAi silencing apparatus.

While it has taken the microRNA sponge concept almost 6 years longer than for expressed RNAi to emerge, it will now be very exciting to follow up on the potential implications for basic RNAi/microRNA biology and microRNA-based therapies. And with regards to investment opportunities, it is anybody’s guess as to who will pick up the IP on microRNA sponges since so many different technologies and corporate strategies overlap with, but do not completely cover it.

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By Dirk Haussecker. All rights reserved.

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