There were quite a number of RNAi Therapeutics related talks on the last day of the conference. Muthiah Manoharan from Alnylam Pharmaceuticals gave an overview of some of the important chemistries and formulations the company is developing.
The liposomal delivery section of his presentation, Alnylam develops liposomes both for dsRNA as well as single-stranded antagomir delivery, proved a bit hard to follow (or maybe it was just me having difficulties concentrating towards the end of the conference) since some data related to Protiva SNALP technology, then Tekmira developed novel cationic liposome formulations, and then the next moment to lipid-like particles. It is befitting that given the overlap and complementarity in terms of biodistribution and pharmacokinetics of these technologies much of this has now been consolidated into the new Tekmira. If I have it right, the previously shown successful repeat-administration data for sustained gene knockdown in the liver made use of MIT’s lipidoid technology, while the impressive mouse liver cancer data were the result of a Protiva collaboration.
Conjugation of siRNAs to cholesterol is well known for the systemic delivery to the liver and jejunum. Less well known in the context of siRNAs is the PEG-conjugation. Since according to Dr. Manoharan this conjugation facilitates equally efficient delivery to the jejunum as cholesterol does, but largely avoids the liver, PEGylation strategies may lessen the risk for liver toxities in targeted therapies of the jejunum.
With respect to modification technology, in contrast to Merck’s (notably absent from the speaker roster) emphasis on siRNA modification (‘siNAs’), Alnylam follows the natural trend and applies modifications as little as possible. It is interesting that they are still using the classical Tuschl dTdT 3’ overhang structure, albeit substituting a thioate for a phosphate in the last linkage. Their favorite modification, however, appears to be the 2’-fluoro modification, long a workhorse for many RNA technology platforms before RNAi, which has now also proven to be very promising for RNAi Therapeutics. This modification imparts not only stability on the siRNA, but also helps in avoiding immune recognition and favors a molecular conformation that may aid in target recognition. In addition to the associated higher lipophilicity, all of this appears to result in significantly improved knockdown efficiencies.
Next on the list was the session chair of the afternoon RNAi Therapeutics workshop, Frank Bennett from the antisense company ISIS Pharmaceuticals. ISIS is developing single-stranded RNAs for the induction of therapeutic RNAi. This may be due to their IP estate in single-stranded antisense as well as the notion that single-stranded molecules may have cost and delivery advantages. The flexibility of single-stranded molecules plus its amphipathic nature, particularly when extensive phosphortioate backbone modifications are applied, should make it easier for ssRNAs to cross the cell membrane (note, however, that naked antisense delivery may not always be sufficient as their own Regulus venture is looking at systemically formulated, conjugated or liposomal, antisense for the inhibition of microRNAs). Maybe not surprising, ISIS, too, has found 2’fluoro modifications to be “magic” as this has significantly improved single-strand RNAi potency, with the IC50s in a number of cases in the low or even subnanomolar range. But his own comparisons show that since 2’fluoro also improved the potency of dsRNAi, the fold-difference in potency between single-stranded vs dsRNA RNAi has not really changed, with ssRNAi being 10-fold less efficient at best (please correct me if I am wrong here, but numbers probably based on lipoplexed oligo delivery in tissue culture). For example, the most potent ssRNAi shown molecule had an IC50 of 0.43nM, while the corresponding 2’fluoro siRNA came in at 0.01nM.
Part in me believes that ISIS has a sincere interest in developing ssRNAi for their future pipeline, but another part makes me believe that ISIS’ work is supposed to widen their claim onto IP in RNAi. Accordingly, Bennett noted that in was in 1998 that ISIS evaluated the use of single-stranded RNAs to modulate gene regulation, and as it turns out, these RNAs worked via RNAi. In any case, this was a fact-based presentation and not meant as an advertisement of single-strand RNAi or antisense technology and it will be interesting to follow just how efficiently single-stranded RNAs can be designed so that they are recognized by RNAi.
David Lewis (Mirus Bio) then gave us an update on his company’s progress with Dynamic Polyconjugates (DPC), a non-liposomal technology for the systemic delivery of RNAi to the liver. As I had
noted here before , DPCs may be particularly promising in terms of safety, as depending on the attached sugar, Kupffer cells and hepatocytes can be selectively targeted. This was corroborated by the presentation of extensive safety data, a topic that unfortunately was not really addressed in the previous two speakers’ presentations. Knockdown was remarkably sustained, and particles repeat-administrable in mice for over 100 days without loss of silencing activity, at relatively low 1.25mg/kg doses. Moreover, sustained silencing was also demonstrated for non-human primates. It is notable that by targeting ApoB in monkeys, they, like many other groups, also observe the fatty liver phenotype. The small size of DPCs should make the technology also a candidate for more difficult-to-penetrate tissues.
Beverly Davidson (Iowa) summarized her group’s experience with AAV-shRNAs for the treatment of Huntington’s Disease. Like many other laboratories have observed, they also find toxicity following U6-driven shRNA expression. Albeit very potent, it is probably recommended to stay away from the U6 promoter for future development programs, especially when high copy-numbers of expression cassettes may get into individual cells, and there are more than enough alternative potent hairpin expression technologies that do not cause toxicity. According to Davidson, incorporating the RNAi trigger within a microRNA context for example was safe. AAV delivery to the striatum, the anatomical site that matters most in HD, was “phenomenal”, particularly with a viral preparation from their collaborators at Targeted Genetics (Seattle). Before RNAi gene therapy for HD can progress into the clinic though, it will be important to determine whether knocking down both the wild-type and mutant huntingtin alleles is safe or whether allele-specific knockdown is required. Studies that address this issue are now ongoing. Overall, I am quite optimistic about the future of AAV- and lentivirally mediated RNAi gene therapy.
Judy Lieberman (Harvard), together with Phil Sharp a co-organizer of the conference, presented more data on targeting cancer stem cells by RNAi, the underlying rationale being that in order to get rid of a tumour, you really need to get rid of the cancer stem cells from which the tumour mass derives. While previous work centered on antibody-protamine siRNAs targeted to stem cell-specific surface receptors, her latest work makes use of targeted liposomes, which can be potentially more consistently manufactured as well as have the added advantage that many siRNAs can be delivered with just one liposome. Using this to deliver let-7 mimics, a microRNA that is becoming more and more clear to serve as a key differentiation factor, her group showed that breast cancer stem cells dramatically lose their ability to self-renew as well as metastasize to the lung and liver.
Equally interesting was Markus Stoffel’s presentation (ETH, Zurich) on the mechanism of cholesterol-conjugated siRNA uptake. In a study previously published in Nature Biotech and discussed here , Stoffel and his collaborators at Alnylam found that the favorable pharmacokinetics and liver uptake of cholesterol-conjugates is due to their association into lipoprotein particles of the blood, and that it is cholesterol-siRNA bound by LDL that carries the RNAi to the LDL-receptor on the hepatocytes, thereby causing gene silencing in the liver. Moreover, cholesterol-siRNA pre-formulated with LDL in vitro and then administered into mice, proved useful in enhancing the hepatic delivery of RNAi.
Extending this work, his group has now found that cholesterol-conjugated siRNAs can also be consistently formulated with Intralipid , a clinically tested fat emulsion used for nutritional purposes as well as a drug carrier for certain anaesthetics, especially for indications of the heart. Quite excitingly, and in retrospect maybe not that surprising, it turns out that cholesterol-siRNAs are not delivered to the liver (a potential safety advantage when targeting other tissues), but instead go to the lung and even better, the muscle, including heart. At reasonable 10mg/kg, they observe an 80% ApoB knockdown in the lung, but this needs to be repeated now targeting genes more highly expressed in muscle/lung. Developments such as this are all the more important, since the ability to target new tissues opens up RNAi Therapeutics to a whole new spectrum of disease (note that this is an siRNA method and gene therapy methods such as AAV-mediated RNAi may also be useful for the systemic treatment of the heart).
Represented in the poster session was work by Nastech on meroduplexes. Maybe I don’t get it, but the suggested advantages of meroduplexes are so tenuous while creating all the additional challenges in developing tri-partite siRNAs. If Nastech was really making progress on delivery as has been suggested, it would make more sense focusing on those efforts which should be sufficient to create partnering interest and financing at favorable terms. I’m sure a company like Alnylam would be more than happy to then cross-license siRNA and delivery IP.
Finally, a poster by Takanori Yokota (Tokyo) reported on the delivery of alpha-tocopherol conjugated Dicer-substrate siRNAs to the liver. Effective silencing of ApoB in mice was achieved at low mg/kg dosages and was apparently well tolerated, except for the- you hear it once again- ApoB knockdown-specific fatty liver phenotype.
Overall, this year’s Keystone has proven a must-go for those that can only go to only a limited number of scientific meetings, giving them a chance to catch up on the important developments in the field over the past year and getting a glimpse into emerging trends. Next year, the RNAi community will be split between three RNAi-related Keystone meetings, one on the basic mechanisms of RNAi, another one centered on RNAi Therapeutics, and finally one on microRNAs and cancer just illustrating how much this field has grown and diversified.
Showing posts with label Keystone RNAi. Show all posts
Showing posts with label Keystone RNAi. Show all posts
Tuesday, April 1, 2008
Monday, March 31, 2008
Day 4 of the RNAi Keystone Conference
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.
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.
Friday, March 28, 2008
Day 2 and 3 of Keystone RNAi Conference
The Keystone conference kept me kind of busy, so I did not find the time to summarize what’s been happening here in the mountains up Vancouver, so here only a general impression on where the field seems to be heading and some highlights from the abstracts.
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.
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.
Subscribe to:
Posts (Atom)