Thursday, March 27, 2008
Day 2 and 3 of Keystone RNAi Conference
David Bartel (MIT) started off the second day of the conference. Well known for his groundbreaking work on microRNA discovery and microRNA target prediction work, he usually has two or three biological short stories to share that are well done and insightful. Same this time. One had to do with the technical challenge of assessing the genome-wide impact on protein expression by a given microRNA. While transcriptional profiling is a well-established and affordable method to measure changes in mRNA levels (RNA that gets translated into proteins), because microRNAs were initially believed to regulate gene expression largely by inhibiting translation without changing RNA levels, a lot of the real targets (according to Bartel’s own studies about 100-200 per microRNA) may have been missed by just looking at transcript levels (note: siRNAs largely regulate their genes by destroying the mRNA, so mRNA quantitation is a reasonable way of measuring target knockdown).
Like Klaus Rajewski (Berlin) who presented tonight, Bartel’s group compared the transcriptional impact of a microRNA by gene expression microarray and compared that to a less well known method of quantitatively measuring genome-wide changes in protein levels, SILAC. SILAC involves the incubation of cells in the presence of light (e.g. control) versus heavy (e.g. treatment) isotope media. When added at the time of the microRNA addition, newly synthesized proteins will incorporate these isotopes. If an mRNA for a given protein had been targeted by a microRNA less of that protein would be made. Cell lysates are then prepared and mixed (control and experiment) and the entire protein content analyzed by mass-spectrometry. Although the peptide signature of the mass-spec for a given protein is essentially unchanged, the incorporation of the heavy isotope would cause the profile to slightly shift for the sample grown in the heavy media, so both treatments can be compared with the relative intensity of the corresponding peptide signals indicative of microRNA regulation (say for a microRNA target the peak would be only 60% of control, meaning a 40% knockdown).
After showing that most of the changes can be bioinformatically accounted for by microRNA regulation, thus validating the technique, both Bartel and Rajewski essentially came to the same conclusions. One is that most regulated genes change both in mRNA and protein content, with the translational inhibition only accounting for a small part of microRNA regulation. This is important since this adds to the validity of assessing off-targeting by transcriptional profiling. The other good news is that most of the changes by microRNAs, the mechanism that largely accounts for RNAi Therapeutic off-targeting, are rather subtle, less than 2-fold in most cases, thereby lowering the side-effect risk through off-targeting.
Unlike previous RNAi Keystone conferences that placed more emphasis on basic principles and mechanisms, the first 3 days were dominated by high-throughput technologies. Even more so than SILAC, deep-sequencing of small RNAs cannot be avoided. It is now possible to obtain millions of sequence reads from a single sequencing reaction, mostly to discover new populations of small RNAs, but also with a trend towards quantitating them by counting the sequence reads.
Other presentations made use of more ancient transcript analysis method, the microarray, but that does not mean with less biological insight. Tom Gingeras from Affymetrix, e.g. looked at the complexity of RNAs found in human cells by microarray and cloning, and one of his observations was that a given gene locus, including those coding for proteins, often harbor multiple overlapping transcripts. That poses the challenge that even though a gene may have been well validated as an RNAi Therapeutic target, one has to make sure that the right mRNA(s) is targeted to avoid unpleasant surprises. This may be even more so important when screening the genome with siRNAs for gene discovery purposes.
Dinshaw Patel (New York) reported on the molecular structure of an Argonaute protein in archaebacteria. This work shows how the guide strand nucleic acid is bound and also suggests how it may recognize and bind its targets. This may add to our understanding of what makes for an efficient siRNA and offers the prospect for thermodynamically optimized siRNA designs. It may also inform us what types of siRNA modifications may be optimal. For example, many of the protein-guide strand interactions are mediated by Argonaute amino acid side-chains making contact in a non-sequence specific manner to the phosphate backbone of the guide, and one would probably want to avoid disturbing this interaction too much. The structure also suggests that there is quite some spatial flexibility in the 3’ end of the guide RNA which may explain why microRNAs normally tolerate 3’ mismatches with their target RNA. How about then siRNAs which are bulky at their 3’ ends such that the spatial flexibility was reduced to enforce target specificity? It certainly will be exciting to watch the impact of RNAi structural biology on the design of future siRNAs.
Similar improvements in efficacy and specificity may come from studying the function of Argonautes in humans (Argonautes are the effector proteins of RNAi). There are 4 human Argonaute proteins, with Ago-2 being the one that efficiently cleaves target mRNAs using siRNAs, whereas the function of Ago1, 3, and 4 is less well known. Some believe that all four Agos may get loaded with a given siRNA and this may very well affect knockdown efficiency and influence off-targeting. Joel Belasco e.g. proposed that the differential expression pattern of the 4 Agos in the various tissues may affect just that. Consistent with that, Dirk Grimm (Heidelberg) found that the Argonautes compete with each other for hairpin-derived small RNAs with the relative level of Ago2 determining knockdown efficiency. One idea therefore would be to design siRNAs that specifically get incorporated into Ago2 while avoiding the other Agos. The molecular structure of all four human Agos would certainly be invaluable in that effort.
Some of you may be wondering whether transcriptional gene silencing (TGS, adding an siRNA to change the chromatin structure so that the resident gene is silenced at the transcriptional level, that is less RNA produced rather than destroying it by RNAi once produced) occurs in mammals and whether it may have therapeutic utility. One problem this aspect of RNAi has suffered from was poor reproducibility and controversy around some of the studies. A particularly interesting talk was therefore given by Danesh Moazed from Harvard as his studies offered the possibility that these discrepancies may have been due to differences in the chromatin context of the targeted genes, and some human gene promoters may therefore be more amenable to TGS than others. I probably would not rush investing into therapeutic TGS just yet (there is no company based on TGS that I am aware of), but I would certainly not want to write it off either. Adding to the credibility of siRNA-induced TGS in humans, a poster by New England Biolabs, a well respected vendor of reliable molecular biology reagents, reported on the identification of efficient promoter-silencing dsRNAs.
The program of the next two days of the meeting will have more on RNAi Therapeutics, but there have been some abstracts readers here may find of interest (I had to miss those, so just the highlights here from the abstract book). One was a study by Alnylam and USAMRIID where they targeted the Ebola virus (Protiva had a similar publication with the US Army in 2006) using liposomally (probably SNALP) formulated siRNAs. Encouragingly, this formulation was able to protect both mice and guinea pigs from an otherwise lethal dose of virus- in an apparently target sequence-specific manner. Moreover, viral titers were reduced when RNAi was administered not only before viral challenge, but also after. Of course, I would have loved to ask about the toxicity profile of these treatments, but hopefully this will be addressed in one of the upcoming oral presentations of this meeting.
I haven’t had a chance to read the latest miR-122 inhibition study in Nature using Santaris’ LNA technology, but a poster abstract by Santaris described the long-lasting (up to 90 days) inhibition of miR-122 with the expected LDL cholesterol lowering effects, all of this at reasonable doses of around 3mg/kg. LNAs are certainly a promising microRNA-inhibition technology and an IPO should not be too far off. Not to be outdone, Regulus Therapeutics described an improvement of their original antisense chemistry for miR-122 inhibition with an apparently 8-fold increase in potency. I assume this must have been through a phenotypic read-out (e.g. cholesterol lowering) since they now find that this chemistry inhibits, but does not destroy the microRNA.
A lesser known method of inducing RNAi developed by Cequent Pharmaceuticals in Boston, hairpins expressed in E. coli (transkingdom RNAi/tkRNAi), is slowly getting ready to enter the clinic within the next year. In their initial program, they will orally administer tkRNAi bacteria targeting beta-catenin for the treatment of familial adenomatous polyposis. IND-enabling studies are under way.
Lastly, a poster from Rosetta Genomics illustrated the potential of microRNA diagnostics beyond cancer by detecting changes of microRNAs in the body fluid that reflected a disease state.
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