As I alluded to already in my previous post on the conference, the issue of transcriptional gene silencing (TGS) mediated by RNAi-related mechanisms in mammals has been somewhat clouded. If it existed and the rules for inducing them could be clearly laid out, it may represent an alternative means of harnessing RNAi for therapy. Presentations on the (at least in mammals) germline-specific Piwi-associated small RNAs (piRNAs; by Alexei Aravin, Cold Spring Harbor) in mice and the small RNA profile from human embryonic stem cells showing that many of them correspond to transposable elements, often regarded as the parasites of our genomes and therefore have to be contained (e.g. by RNAi), the nuclear localizations of Dicer (Witold Filipowicz, Basel) and Argonautes (poster by the Meister group in Munich), and various data on the nuclear localization, and possibly processing, of microRNA precursors into mature microRNAs, are all very suggestive that promoter-targeted TGS may be part of biologically-relevant gene regulation and have promise as a therapeutic technology platform.
In addition to the nuclear localization of Dicer, there was additional data on this central RNAi enzyme. What is often so amazing about science and was also demonstrated a number of times at this conference, different groups working on apparently different phenomena converge on the same results. John Rossi (City of Hope) for example had been studying whether DNA-directed long shRNAs could be processed such that HIV would be targeted by more than a single small RNA, thereby minimizing the likelihood of escape mutants. Like the structural biologist Jennifer Doudna (Berkeley), he finds that mutations in the helicase domain converts human Dicer from an enzyme that does not like to processively chop long dsRNAs into a string of short RNAs into one that does it with reasonable efficacy. Of course, we always hope here that findings like these may allow us to adjust therapeutic designs such that in this case long DNA-directed shRNAs may be processed more efficiently by Dicer. From a basic biology perspective, studies such as Doudna’s on the structural requirements of Dicer should also encourage us to consider endogenous RNAs as Dicer substrates even if they do not result in small RNAs.
Somewhat surprising to me, Thomas Tuschl’s (Rockefeller) presentation focused on the use of microRNAs as diagnostics. It is worth remembering that his group discovered and patented many of the first human microRNAs and that IP may have most immediate value in diagnostics. Specifically, his group is refining technologies for detecting microRNAs in situ (using LNA-spiked probes) and the quantitative profiling of microRNAs by counting. It appears to me that while first-generation microRNA diagnostics in the clinic will involve interrogating a limited number of microRNAs by qRT-PCR (one of Rosetta’s first diagnostics for squamous versus non-squamous lung cancer appears to be based on the PCR quantitation of a single microRNA), while in another 10-20 years quantitative sequencing technologies take over, possibly by-passing array-based detection methods for clinical use altogether.
Phil Sharp (MIT) reminded us that the complexity of the transcriptome may have important implications for our understanding of microRNA regulation. 3’ UTRs are the main site of microRNA action (although there were some bioinformatics talks at the meeting that strongly supported functional microRNA target sites in the open-reading frames of genes as well, though to a much lesser extent than in 3’ UTRs) and may be dynamically regulated according to the state of the cell, e.g. proliferation. Since the extent of these 3’ UTR changes (due to alternative splicing or polyadenylation) may be quite considerable, this complicates efforts to unravel the functional microRNA interactions for the identification, including safety, of candidate microRNA therapeutics.
High-throughput RNAi library screening for drug target identification (commercial examples are Cenix Biosciences, Galapagos Genomics), using synthetic siRNAs as well as plasmid and viral-based shRNA, is now well established. According to Ed Harlow (Harvard), this really has opened up mammalian biology for the type of genetic experimentation long reserved to model organisms. Focusing on the human kinome and cancer (all the predicted human kinases), he made the observation that the functional outcomes of suppressing a given gene may differ considerably according to the experimental system (e.g. depending on the cell lines). This should serve to caution us that context such as the presence of an oncogene or tumor-suppressor gene or not and redundancy can have a profound impact on the importance of a kinase in cell signaling. Another observation from his screens was that the resulting drug target candidates coming out of these screens were not biased towards the kinases that have been already subject of the majority of kinase-related studies, illustrating how RNAi may also benefit small molecule drug development. While in vitro RNAi screens are common, the costs associated with high-throughput in vivo screens would seem prohibitive. It is therefore notable that Michael Hemann’s (MIT) approach to studying the role of genes in cancer drug resistance involves the transduction of a library of shRNAs into cancer cells of the blood and administering them to mice. Changes in the relative abundance of a given shRNA prior to and after drug treatment are then monitored as a measure of the importance of the targeted gene in drug response and resistance. I could imagine that similar approaches to extend the power of RNAi screens may have near-term potential not only for cancer, but also other settings where it is possible to select for relative cell viability (viral infection, degenerative diseases etc).
John Rossi (City of Hope) reported on progress with the first DNA-directed RNAi gene therapy trial (corporate sponsor: Benitec) involving a triple RNA therapeutic against HIV, including a U6-driven shRNA, lentivirally delivered to bone marrow transplant cells in AIDS lymphoma patients that are in need of a BMT anyway. This is a limited trial with an ultimate enrolment goal of 6 patients. Given the complexity of the therapeutic and trial designs it has taken considerable efforts before the first patient could be dosed, but happily the first patient has now been treated a couple of weeks ago. It will be exciting to follow the progress of this patient. Since he/she also received untreated BMT cells for safety reasons, it should be possible to monitor the enrichment/non-enrichment of treated vs untreated T-cells as an indicator of treatment success. Since lentiviral vectors have not been associated with oncogene activation, probably my main safety concern for this trial is the use of a U6-driven shRNA. Not only did having two U6 promoters in one vector cause excision of the U6-shRNA cassette due to a recombination event in some of the genomically integrated vectors, it is known that U6-shRNAs have a tendency to be toxic due to sheer promoter strength as well as possibly its particular processing. For future DNA-directed RNAi development programs, the use of H1 promoters may therefore be even more promising.
A word here on viral escape mutants to RNAi Therapeutics as antivirals. The triple construct in the HIV trial was based on the notion that hitting the virus through three different mechanisms should limit viral escape. As such, it was reported in studies leading up to this trial that HIV likes to mutate around shRNA target sites. To my knowledge, this had not been reported for the HIV sequences targeted by the ribozyme and decoy components in this vector. Contrary to popular opinion, I am actually quite pleased to see such mutations being selected for since this is a clear indication of the efficacy of the shRNA. Moreover, with RNAi the target site for the shRNA could be chosen such that largely unfit viruses were selected for.
Although all three components of the triple construct are RNA-based therapeutics, it illustrates the potential of combining RNAi Therapeutics with other mechanisms of actions into one drug. Similarly, Rossi’s group is now developing an aptamer designed to both neutralize circulating HIV virions as well as target attached anti-HIV siRNAs to HIV infected cells. Another example may be liposomally delivered siRNAs, which in the case of a cancer therapy could be designed such that not an oncogene would be targeted, but also such that the RNA would stimulate the immune response against the cancer. Interestingly, a poster presented by Nigel McMillan (Brisbane, Australia) reported on the maybe somewhat surprising finding that the targeting of an mRNA led to the production of a truncated protein (presumably by translation from the cleaved mRNA) which stimulated an immune response against the oncogene with, if it holds up, some interesting implications for microRNA evolution (cleavage vs non-cleavage) and RNAi Therapeutics.
The aptamer-siRNA combination involved the Dicer-substrate design. The rationale being that such a combination necessitates the covalent attachment of the RNAi trigger to the carrier so that Dicer cleavage has to liberate the active small RNA from the carrier. This is reasonable, but one should note that smaller siRNAs covalently attached to cholesterol on the passenger strand, and probably also the 3’ end of the guide, are also known to be bioactive (although I am not sure whether some efficacy was compromised by such designs). Also, I would be curious to see whether attachment via disulfide bonds that would break in the reductive cytoplasm would liberate even larger amounts of bioactive dsRNAs, both long and small, thus further illustrating that covalent attachment to the delivery vehicle does not represent a fundamental disadvantage for the delivery of short siRNAs.
Of course, this gets us right into the IP issue. A poster by RXi was instructive of how companies aim to get around Alnylam’s perceived dominance in this arena (almost like watching a virus mutate around an shRNA target site). As many of you know already, the focus is on the use of dsRNAs longer than 23bp as well as blunt-end versus overhang dsRNAs. Without discussing the relative scientific merits of the different approaches, it is notable that RXi’s poster put great emphasis that their 25bp Stealth siRNAs were not processed by Dicer into smaller siRNAs as would have been expected for equally long unmodified dsRNAs, yet were still active in gene silencing. Unlike Dicer-substrate oriented approaches, RXi’s intention is to circumvent any claims by Alnylam that Dicer-substrates are nothing more than pro-drugs to the classical Tuschl design, certainly novel and non-obvious from an argumentation point of view. Scientifically, I would be interested in whether these dsRNAs work as efficiently in Dicer knockdown or knockout cells, in other words whether Dicer would still recognize these particular dsRNAs and facilitate RNAi by handing them over to RiSC, independent of whether Dicer had processed or not, and whether this rather than dicing is how Dicer facilitates gene silencing for a range of non-diceable RNAi triggers. In other words, since Dicer is known to aid in loading the RNAi effector complex, RiSC, aren’t all dsRNAs, including siRNAs, Dicer substrates and would the size of microRNAs/siRNAs be a consequence of Dicer processing, but not necessarily a requirement for RNAi activity? Tuschl’s early work on single-stranded RNAi, showing that ssRNAs of various length, i.e. not only 19-21mers, but also 29mers and others, were equally efficient in triggering RNAi suggests just that [Martinez et al. (2002). Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110:563].
Local Tekmira had a poster on the 10-fold improvement in efficacy of some of the new liposomal nanoparticles down to 0.1mg/kg, similar to what had been reported on Protiva's website and at Alnylam's R&D day. But rather than discussing here more about SNALP-like technology which I have done extensively in the past, and before I will summarize the conference’s last day, I will have to first read up on the Tekmira news that came out today, an apparent victory of reason and impressive management skill.
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