ISIS Demonstrates Wider Utility of RNaseH ASOs for Nuclear Targets

While research astounds us on a daily basis with unexpected discoveries, sometimes it is what we don’t know and haven’t bothered to ask is what is astounding.  One example of the latter in the field of Oligonucleotide Therapeutics is the poor understanding of which tissues and cell types and therefore disease indications are most appropriate for a particular (delivery) approach based on the ability to engage targets there.   

Antisense Therapeutics, over 30 years in the making, has been the biggest violator of this principle.  Smug in the belief that delivery is not necessary, the approach has been to just apply the oligonucleotide and then pray that it will go to the right place and work its magic, especially in cancers.  Only after decades, the field through much clinical trial and error has come to the realization that the liver and kidney may be pharmacologically favored target organs.

In a long overdue tour-de-force, Hung and colleagues from ISIS Pharmaceuticals recently published in the journal Nucleic Acid Therapeutics a detailed investigation of the global biodistribution and RNaseH knockdown efficacy of phosphorotioate antisense chemistries (PS-ASO) following systemic application: Characterization of target mRNA reduction through in situ RNA hybridization in multiple organ systems following systemic antisense treatment in animals. This parallels a similar study for the direct application of this chemistry to the CNS presented at this year's OTS meeting which has yielded the surprising insight of the broad CNS distribution of PS-ASOs following focal administration. 

As a result of the latest research, a roadmap of target organs was created.  Importantly, through the application of newer RNA immunohistochemistry methods rather than the old harvesting and mashing up organs, the study looked at the specific cell types within an organ that were amenable to RNaseH knockdown.  This is important in at least two ways.  Firstly, it allows us to reject a potential target in organs where bulk knockdowns have shown a rather deep knockdown, but where the detailed organ analysis shows that the particular cell type in which one desired the knockdown does not show such a knockdown (e.g. kidney).  Secondly, it allows one to reconsider targets and cell types within organs for which bulk knockdowns have not been observed (e.g. the small intestines).

Another valuable piece of insight of the study was that it compared the old, second-generation 2’ MOE chemistry with the higher affinity locked nucleic acid chemistry version pioneered by Santaris (in this case the cET ISIS knock-off version of LNAs).  In addition to increasing the knockdown potency in traditional tissues such as liver, kidney, and adipose tissue, the chemistry allows for appreciable knockdowns in some less traditional tissues such as muscles.  Unfortunately, the direct comparison between 2’ MOE and LNAs was only performed in mice and at the very high 50mg/kg dose.  In the non-human primate study, also at a very high (35mg/kg) dose, no such direct comparison was  performed and from this, it seems that the new organs enabled by the higher-affinity chemistries were limited to the muscle and lung.

Why Marina Biotech could be the 2014 high-flyer

Regular readers will notice that I have shifted some of my investment attention to Marina Biotech.  The main reason for this is that this company which is considered by many to be dead, actually owns the rights to a high-affinity ASO chemistry (CRN) of a potency that is equivalent to Santaris’ LNAs and probably superior to ISIS’ cET while the market cap of Marina is just one-thousandth that of ISIS Pharmaceuticals.  Even when one considers that the in vivo safety (especially) and potency evaluations lag behind the others due to the budget constraints of Marina Bio, I believe it is a risk worth taking given the enormous valuation gap and the fact that CRN PS-ASO biodistributions and activities can be assumed to be similar to the competing chemistries.

What is more, Marina Bio is pursuing a program in type I myotonic dystrophy which represents the sweet spot of indications uniquely facilitated by these chemistries: muscle as a new druggable target organ and still shielded from superior RNAi competition; a rare, severe orphan disease; and a toxic nuclear RNA.

Largely depending on the recapitalization strategy (partnering first before capital raise or vice versa), this program together with SMARTICLE RNAi delivery and access to usiRNAi triggers, has made me accumulate 1.5% of the company with the intention of increasing my position.  Of course, financial success can only happen if other investors share my view that we should therefore give Marina Bio another chance.  As always, invest at your own risk and according to your unique financial circumstances.

A shameful title

If you re-read the title of the paper and even the entire publication, you may be forgiven for going away with the impression that it is open season for RNaseH knockdown in muscles and other tissues and organs.  This is far from the truth as the ‘exemplary’ target chosen in the study was the nuclear non-coding RNA MALAT.   This is because a high-profile Nature publication by ISIS Pharmaceuticals itself (Wheeler et al. 2012) has shown that whereas largely cytoplasmic m-e-s-s-e-n-g-e-r RNAs (i.e. RNAs encoding for proteins as even a decent high-school kid will know) expressed in muscles were entirely recalcitrant to RNase H knockdown, the mutated nuclear retained DMPK underlying myotonic dystrophy was susceptible to such action.  Curiously, while Wheeler et al. was cited in the Hung paper, the authors failed to point out this important and very obvious caveat.

This can be no innocuous omission as ISIS Pharmaceuticals in one of their patent applications has expressed the striking difference between mRNA and nuclear RNA druggability by PS-ASOs as follows (highlights are mine):

Reduction of Nuclear-Retained RNA

Data provided herein demonstrates that sensitivity to cleavage by ASOs is dramatically increased for a nuclear-retained RNA making it possible to reduce nuclear-retained targets in tissue that has low uptake of oligonucleotide by a pharmacologically relevant amount. For example, out of the more than 4,000 transcripts that Isis has targeted by antisense, MALAT1, a non-coding, nuclear-retained RNA, is demonstrated to be one of the most sensitive targets for antisense oligonucleotide/RNase H inhibition. The data demonstrate a great number of oligonucleotides targeting over the majority of the transcript that inhibit by more than 50% in vitro. The data also demonstrates very low IC50 values in multiple cell types. Half-life studies have also shown that the MALAT1 is stable over a period of at least 10 hours. Subcutaneous administration of oligonucleotide targeting MALAT1 at doses commensurate with other oligonucleotide drugs (e.g., liver targeting drugs) achieved pharmacologically relevant reduction of MALAT1 in skeletal and cardiac muscle. Dosing at 50 mg/kg biweekly for 3.5 weeks achieved a 89% and 85% reduction in gastrocnemius and quadriceps, respectively, and 54% reduction in heart (as compared to 95% reduction in liver). Pharmacologically relevant reduction of MALAT1 has also been achieved in tumor xenograft models.

As a member of the Oligonucleotide Therapeutics Society, it greatly saddens me that the related journal is letting ISIS Pharmaceuticals get away with the highly misleading, and simply wrong title.  There is no arguing around it.  Followers of the competitive oligonucleotide therapeutics investment arena know that the game here is to make RNaseH antisense appear much more widely applicable than it actually is.  What is more, it was at the 2011 OTS meeting in Boston where the ISIS CEO Stan Crooke in his keynote made the ignonimous statement that ‘mipomersen has no side effects’.  

I strongly suggest to the society and the journal Nucleic Acid Therapeutics which are supposed to foster the development of the technology broadly to keep a watchful eye on the growing corporate influence, especially by 'generous sponsors' ISIS and Alnylam Pharmaceuticals.   

ISIS Pharmaceuticals Next-Gen cET Technology on Shaky Grounds

As I was listening to a presentation by ISIS satellite company Regulus Therapeutics at the Future Leaders in the Biotech Industry Conference last week, I noticed that the fight between ISIS/Regulus and Santaris over intellectual property has increased in bitterness. On the same day, Santaris announced that the USPTO had rebuffed the second patent challenge concerning its conformationally constrained nucleotide technology (LNA).  As ISIS and Regulus have decided on making the LNA-derived cET technology their next-generation antisense technology, the increased aggression (including an ongoing gapmer patent infringement suit brought by ISIS against Santaris) may indicate that also ISIS sees cET technology on shaky IP grounds.

Thanks for the Validation!

Even during the hard-fought Tekmira vs Alnylam battle, the participants largely refrained from making direct snarky comments.  This, however, cannot be said of last week’s presentation by Regulus, when it sarcastically thanked Santaris for being years ahead in their miR-122 HCV program and doing all the clinical validation- for them.  The comment obviously refers to the fact that Stanford University had licensed the seminal Sarnow patent related to miR-122 inhibition for HCV therapy to Regulus, not Santaris.  It is also what made GSK dump Santaris and partner with Regulus on the program instead.  I would agree that Regulus has a strong case in this matter, but the unmitigated sarcasm was remarkable.

Is it all about cET?

When ISISfirst reported on their next-generation (gen 2.5 that is) nucleotide modification, it (cET) was explicitly derived from LNAs.  The rationale was that although LNA is the most potent antisense technology around, it suffered from poor safety (as supported by the premature termination of the PCSK9 and ApoB programs by Santaris).  Consequently, additional moieties were incorporated into the bridge that fixes the conformation of the backbone ribose in place.  As a result, the high affinity of the parent LNA was retained, but the safety issues were apparently overcome.

As a LNA-derived technology, the obvious question is whether Santaris, which controls fundamental LNA patents, has IP rights in cET.  Given the importance of cET to ISIS Pharmaceuticals and Regulus such uncertainty is intolerable.  Hence, it would make sense for ISIS and Regulus to attack Santaris and its LNA patents, which is exactly what ISIS has been doing.  Two such pre-emptive strikes, however, have failed according to last week's news.  And as a pre-emptive reminder on my part, a patent only gives you the right to exclude, not freedom-to-operate.

The gapmer patent infringement suit against Santaris may thus be viewed as an effort to get Santaris to the negotiating table.  The miR-122 situation, albeit unrelated to the LNA issue, would be further ammunition to ISIS and Regulus in that effort.

Expect the confrontation to get heatier still, as Santaris should strike back as the overall strategy by ISIS Pharmaceuticals, which is continuing to closeone deal after the other, seems to be working despite the lost patent re-examination battles last week, and is taking away the financial oxygen from Santaris.

Register for the GTC RNAi Research and Therapeutics meeting in San Francisco today (June 20-21).  Get a free RNAi Therapeutics blog T-shirt and 20% discount on registration by entering discount code 'RNABLG13'.

Santaris Initiates First Clinical Trial for a MicroRNA Therapeutic

Another notable milestone has been reached in the history of small RNA-based therapeutics. Yesterday, Santaris announced that it had commenced a clinical trial for SPC3649, an LNA-based antisense molecule against miR-122 for the treatment of Hepatitis C. The phase I trial will be a double-blind, randomized dose-escalation study involving 48 healthy male volunteers.

MiR-122 has been shown to facilitate HCV replication as well as to have a role in cholesterol synthesis. Because of these potential applications and its expression in the liver, the tissue that can be best addressed with today’s systemic delivery technologies for nucleic acid-based therapeutics, miR-122 has become the favorite target for first generation microRNA-based development programs.

Santaris has made rapid progress and appears to be leading anti-miR-122 efforts, at least based on the published literature, with most prominently a recent Nature study demonstrating miR-122 antagonism in non-human primates, but also a nice transcriptomic study looking at changes in gene expression following miR-122 inhibition in mice. Santaris is using so-called LNA-“mixmers” which consist of a combination of interspersed DNA and LNA nucleotides and a phosphorothioate backbone and appear to act by sequestering the microRNA in the cytoplasm.

In mice, this LNA-antimiR-122 was quite a bit more potent than a competing technology developed originally by Alnylam and now owned by Regulus Therapeutics (‘antagomirs’) which were thought to induce the degradation of miR-122, possibly by RNase H. While these differences may also relate to the tissue concentrations and tightness of hybridization achieved by the various chemistries, my impression is that sequestration rather than RNaseH is the more promising approach to antagonizing microRNAs while for mRNA inhibition by antisense the opposite may be the case (e.g. morpholino versus gapmers). Moreover, the exact mechanism of action of the antagomir is still somewhat controversial, and I am curious to learn about the potency of antagomirs in vivo when formulated into SNALP-like particles.

A remarkable aspect of the LNA-antimiR-122 studies in non-human primates was the long duration of activity, up to 100 days after 3 loading doses of the antisense as judged by the lowering of plasma cholesterol. However, at this time point, the shift in the Northern blots which was taken to directly reflect microRNA sequestration, was not observed at this point. This could either mean that the Northern blot shift may represent an experimental artifact due to hybridization of antisense to miR-122 following sample preparation, and/or that the cholesterol lowering had persisted after LNA-antimiR-122 has ceased to sequester miR-122.

Of course, while the speedy technological progress is encouraging, there are a number of risks associated with this program. One is that it is unclear whether successfully inhibiting miR-122 in a chronic hepatitis C infection in man will have a meaningful effecgt on HCV burden. Compared to targets in RNAi Therapeutics trials, little is known about the actual role of miR-122 in HCV replication. Unlike in tissue culture liver cell lines which generally express less miR-122, miR-122 is a very abundant microRNA in the liver and may not be as rate limiting for HCV repliation in vivo. The argument, however, that this drug candidate lacks in vivo validation could be applied to a range of other HCV therapeutic programs, some of which have proven to be successful. The development of more convenient animal models for HCV infection over the next 5 years should change this and benefit not only the development of miR-122-based HCV therapeutics, but particularly RNAi-based HCV antivirals.

The Nature studies have also shown that anti-miR-122 may have limited utility for the treatment of hypercholesterolemia as its inhibition downregulated the “good cholesterol” even more than it did the “bad cholesterol”. On the other hand, given its abundance in the liver, it is likely that other indications for miR-122 antagonism will emerge and for which the Santaris phase I trial could be leveraged.

As far as the business strategy is concerned, maybe Santaris ought to solve the IP issues surrounding miR-122 for HCV once they initiate HCV-specific studies, as related IP fundamental to such an application has been exclusively licensed to Regulus and may thus limit the partnership value of this promising program.

Regulus Deal Makes GSK Clear Force in MicroRNA Therapeutics

Only months after closing a similar agreement for the development of antiviral microRNA therapeutics with LNA company Santaris (Santaris PR), GSK today agreed to a co-development partnership with the Alnylam-ISIS microRNA therapeutics joint venture Regulus Therapeutics in the inflammatory disease area (Regulus PR).

In both deals, GSK has the option to select up to 4 microRNA therapeutics candidates developed by Santaris or Regulus in the respective disease areas for comparable financial terms that gives the microRNA therapeutics companies important and largely undilutive upfront cash, development milestones, and royalties (for exact terms, please refer to the Santaris and Reguls PRs).

It is also a vote of confidence by Big Pharma that it sees microRNA therapeutics as a significant new emerging treatment modality. While the antiviral programs with Santaris should be based on the straight-forward targeting of either viral or host microRNAs directly implicated in viral infection, the anti-inflammatory programs with Regulus may be based on more complex microRNA pathway biology.

Interestingly, in both cases the emphasis is on microRNAs as targets for antisense antagonists and much of the discussion in today’s conference call on Regulus’ general microRNA therapeutics activities centered on such antagonist approaches, although I previously was under the impression that microRNA agonist approaches would also fall within the scope of Regulus.

Actually, when I woke up this morning and saw the headlines, I thought that the GSK-Regulus alliance would relate to a mystery surrounding microRNA-122 in HCV replication that is currently being developed by both Santaris and Regulus. Since miR-122 is by far the most advanced antiviral microRNA therapeutics candidate program, it is no stretch of the imagination that GSK will exercise their rights to Santaris’ HCV program that, in collaboration with the Sarnow lab, was subject to a recent high-profile publication in Nature. The issue here is that the Sarnow patents for targeting miR-122 in HCV from Stanford were exclusively licensed to Alnylam (now part of Regulus IP) and GSK-Santaris would therefore ultimately need access to this important patent. A Regulus-Santaris collaboration, however, is complicated by the fact that this program pits ISIS’ against Santaris’ LNA antisense chemistries. It is hoped that miR-122 will eventually aid in synergizing microRNA therapeutics efforts rather than leading to a Merck-like situation where GSK would end up in mutually exclusive collaborations. This issue probably should be addressed early on before it may cause harm down the line.

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.

Impressions from the 3rd Annual Meeting of the Oligonucleotide Therapeutics Society: Day 1

The 3rd Annual Meeting of the Oligonucleotide Society (OTS) got under way in the stylish Langenbeck-Virchow Auditorium situated in the centre of a culturally vibrant Berlin. The OTS brings together the many disciplines in the rapidly expanding field of oligonucleotide-based drug development, ranging from aptamers, ribozymes, antisense, immunostimulatory nucleic acids to RNAi. Reports from clinical studies of these treatment modalities and oligonucleotide chemistries were at the centre of today’s lecture sessions.

Brett Monia from ISIS Pharmaceuticals chaired the first session and set the stage by reporting that while there are only four approved oligo-based therapies, more than 50 are currently in clinical development. Many of these are designed for cancer applications, but infectious, metabolic, and inflammatory disease applications are emerging as major new focus areas. This may not only be due to considerable unmet medical needs, but also because these may offer better success rates for clinical development. A few talks on cancer therapies with ASOs (antisense oligos) documented the challenges of designing smooth clinical trials for cancer therapies. One problem is the heterogeneity of cancers and matching the gene target with the right cancer. Early trials on a given compound typically involve small patient numbers with a wide spectrum of cancer manifestations. Although safety trials, almost anecdotal evidence of biological activity in a few patients then often guide the design of later phases of clinical development. Clearly, for cancer, there is a need for the development of more reliable biomarkers as early indicators of therapeutic efficacy and for patient stratification, which may be something that Rosetta Genomics’ microRNA diagnostics may be able to facilitate.

Focusing on diseases like viral infection and metabolic disease where efficacy can be routinely evaluated in early stages of clinical development (viral titers, LDL-cholesterol, glucose) should therefore be lower hanging fruits for the next wave of oligonucleotide-based therapeutics. This transformation in the industry is illustrated by ISIS’ new focus on metabolic disease and its out-licensing of the more challenging ASO applications. Rosanne Crooke recounted the clinical experience with 301012, ISIS’ lead compound for the treatment of hypercholesterolemia. As I wrote earlier, one major concern with targeting ApoB100 is the risk of steatosis of the liver due to inhibition of a protein required for the export of triglycerides and which has indeed been observed by a number of groups targeting ApoB100 by RNAi. Possibly motivated by the existence of individuals with null mutations for PCSK9, the new target of choice for hypercholesterolemia, with no evidence for adverse effects due to lack of the LDL-receptor processing protein, a conference participants asked about the existence of ApoB100 knockout mice. Interestingly, no such mice exist, because ApoB100 would have additional roles during development.

Despite reports of steatosis due to knockdown of ApoB100 with RNAi, Sirna Therapeutics is yet another biotech company interested in targeting it for lowering cholesterol. In a talk about siRNA delivery to the liver, Barry Polisky, CSO of Sirna Therapeutics/Merck also noted that in Sirna’s experience, there are genes that can be knocked down extremely well (e.g. ApoB100) and some that are relatively refractory to knockdown by RNAi. Although anecdotal, given the extensive experience of Sirna Therapeutics with screening for effective siRNAs by tiling them across entire genes, and similar findings by Qiagen and others, it will be interesting to determine what makes a gene a good target for RNAi, particularly whether it involves its normal regulation by microRNAs.

Polisky further focused on the need for siRNA modifications to make RNAi therapeutics a reality. Currently, Sirna’s standard siRNA design seems to involve si(R)NAs that lack essentially all 2’OH groups, except for the three 5’ nucleotides of the guide strand, while the passenger strand has 5’ and 3’ modifications that avoid it to be loaded into RiSC to reduce off-targeting. Other speakers and posters also noted that since most nucleotide modifications had been developed with antisense therapies in mind, new modifications may exist for optimal RNAi efficacy, particularly in vivo. Indeed, new synthesis strategies are being developed that will allow this to be tested, such as the 6-membered carbohydrate substitutions of ribose reported by Piet Herdewijn from the University of Leuven, Belgium.

Polisky further characterized siRNA delivery to the liver as efficient, albeit not optimal. Progress is clearly illustrated by the fact that >30% of siRNA formulated into liposomal nanoparticles now end up in the liver with first-pass kinetics with no evidence for toxicity in the 1-9mg/kg range (mostly rodent studies), whereas >99% of unformulated siRNAs are rapidly filtered out by the kidney.

The liver is also the organ where the first microRNA-based therapies are likely to be targeted. Like Regulus and Rosetta, I was amazed, or maybe not, at how many groups are currently working at abrogating miR-122 function for treating either hypercholesterolemia or HCV infection. LNAs, owned by the privately held Danish company Santaris, appear to be a particularly promising oligonucleotide class for achieving potent microRNA down-regulation in vivo. Similarly, antisense strategies based on LNA appear to rival siRNA potency in a number of settings, and it will be interesting to learn more about the toxicologies of these compounds, which would certainly be facilitated if Santaris decided to enable to field by allowing better access to their technology. It also highlights the need for specialized chemistries for therapeutic targeting of microRNAs which in my mind is something that Regulus ought to consider.

With regard to delivery outside the liver following systemic administration, PEI-formulated siRNAs (see recent PolyPlus-Alnylam deal) are now being used successfully by a number of groups for lung delivery. Other interesting delivery data was presented by a Portuguese group in a poster on RNAi in the brain. By non-covalently adding transferrin protein to cationic liposome-siRNA particles, they were able to efficiently target neuronal cells both in vitro and in vivo which, similar to cancer cells (see Calando’s cancer cell targeting with transferrin), are studded with transferrin receptors.

PS: A nuisance to most scientists in the field, immunostimulation by oligonucleotides is exploited by Dynavax as an adjuvant for vaccines. Poor adjuvants activity lead to insufficient immune responses and the need for multiple booster injections affecting compliance. HBV vaccination is a case in point, and as a consequence around 50% of patients in the US fail to successfully complete vaccination. Very high HBsAg antibody titers after only two injections of recombinant HBsAg with an immunostimulatory oligo from Dynavax as adjuvant, instead of alum, suggests that they may be able to greatly improve on those numbers.

3-stranded siRNAs

The detailed knowledge about the steps that occur during the natural process of RNAi has inspired scientists to come up with new ways to harness this pathway through the use of novel trigger RNAs. DNA-directed sh-miRs and sh-RNAs, Dicer-substrate RNAs, siRNAs, and single-stranded RNAs can all be used with the aim of obtaining activated RiSC effector complexes containing the guide RNA to seek and destroy target mRNAs.

Now, Bramsen and colleagues report in an Advance Access publication in Nucleic Acid Research on August 28, 2007 on the use of modified 3-stranded siRNAs. The fundamental siRNA design comprises a guide RNA paired with a passenger RNA to form a double-stranded RNA, 21-23 base pairs in length with optimally 2 nucleotide 3’ overhangs. In the case of 3-stranded siRNAs, the passenger strand is split into two separate strands.

3-stranded siRNAs have their roots in stunningly beautiful crystal structures of the Argonaute protein, the Slicer endonuclease of RiSC that cuts the target mRNA, bound to the guide RNA and a complementary RNA (Barford and Patel groups, 2005). In that work, the complementary RNA could have been the target mRNA, but just as well the passenger RNA in an siRNA. Not long after these structures were published, a number of groups reported almost simultaneously that, indeed, the passenger strand of an siRNA is also cleaved. While passenger strand cleavage is not essential for RNAi to work, due to the existence of a bypass mechanism, the Martinez group (Vienna) demonstrated that it greatly stimulates target mRNA cleavage by RiSC. As pre-cleaved, that is 3-stranded siRNAs, could also trigger efficient RNAi, it was concluded that the disruption of the passenger strand allows RiSC to quickly free itself from the passenger RNA for it to act on its real mRNA targets.

The paper by Bramsen et al. now demonstrates and discusses some of the advantages that the use of 3-stranded siRNAs may have. Surprisingly, 3-stranded siRNAs appear to be very forgiving to nucleic acid modifications. RNA modification is considered important to endow siRNAs with the proper drug-like properties, particularly stability, but also reduced off-targeting. Importantly, the authors find that modifications in the guide RNA strand, which is typically more sensitive, tolerated modifications that would abrogate gene silencing within the context of an uninterrupted siRNA. This is especially interesting when it comes to minimising off-targeting by the guide RNA. Small modifications to the second nucleotide from the 5’ end have already been shown to greatly diminish most of the microRNA seed-dependent off-targeting, and the increased flexibility that the 3-stranded siRNA design allows promises further improvements with regard to specificity and also efficacy. Being Danish, the authors suggest the use of locked nucleic acids (LNAs) to achieve this goal. In addition, there is absolutely no off-targeting by the passenger strand as it cannot function any more as a RiSC effector RNA.

You may wonder about the commercial implications of 3-stranded siRNAs. I could imagine the Danish biotech company Santaris to become involved, an oligonucleotides therapeutics company specialising in the use of LNAs. LNAs are highly potent in binding complementary sequences and show great potential for the therapeutic inhibition of microRNAs. Santaris’ patent policy, however, has led to a situation where LNAs are still not very widely used due to their often prohibitively high cost, and for unknown reasons you can almost be assured that when there is an LNA-based publication it involves a group from Denmark.

I would further be interested in whether any of the other academic groups that have contributed to the development of 3-stranded siRNAs claim any IP rights. One of these could be the Zamore group from the University of Massachusetts with ties to CytRx and Alnylam. Martinez on the other hand came out of the Tuschl lab, a co-founder of Alnylam. Lastly, Nastech, a company apparently keen in using anything but the fundamental siRNA design, has applied for a patent involving 3-stranded siRNAs (WO2006US42978 20061103). As often in science, meaningful progress is often made simultaneously by a number of groups and it is almost impossible to determine who deserves ultimate credit.