Roche Buys Santaris (Because It Could Not Buy ISIS)

Minutes ago, Roche announced the acquisition of Danish antisense company Santaris for $250M in upfront cash and $200M in contingent payment.  Given that Santaris only has a microRNA inhibitor for HCV in the clinical pipeline which many in the industry believe is outdated given recent successes in the treatment of HCV, this purchase is a big vote of confidence by Roche in the RNA Therapeutics platform.

This latest development comes after Roche struck a Huntington’s-based deal with ISIS Pharmaceuticals in April 2013 resulting in a development candidate as revealed today by ISIS in its earnings report.  Roche was fortunate to get that deal, because ISIS was about to strike a muchbroader neurology deal with BiogenIdec, leaving it hungry for all those other targets.  Apparently, Roche was happy with what it saw, also following a smaller deal with Santaris just in January this year on undisclosed targets.

This deal flow is reminiscent of what Roche did in RNAi Therapeutics in 2006/7.  After Merck took out Sirna Therapeutics, Roche saw a need to partner with Alnylam instead, at least this is how the narrative goes. 

You can bet your house that Roche at one point or another wanted to buy ISIS, but given its varied partnerships and general unwillingness to sell itself had to settle with archrival Santaris (--> ongoing litigation which perhaps Roche is more able to settle on ISIS' terms).  But, hey, maybe history is repeating itself and ISIS already signed itself away to AstraZeneca and we will hear about it soon.

PS: for the stock junkies (I know there are many here), this news also bodes well for Marina Biotech given its highly similar chemistry to that of Santaris: LNA and CRN.

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.   

Regulus Borrows from RNAi Delivery Technologies…5 Years Late

With the gift of birth of having access to all the technology toys from parent companies Alnylam and ISIS, Regulus Therapeutics in 2007 was poised to speed into the clinic a new class of RNA Therapeutics: microRNA Therapeutics.  Today, Regulus has yet to put a single compound into clinical development.  This not only reflects the increased biological complexity of microRNAs as opposed to for example single gene knockdown approaches such as RNAi Therapeutics, it also reflects a fear of breaking with the dogma that for microRNA inhibition ‘naked’ antisense technologies should be used.

It turns out that only now, Regulus finds that in order to optimize the product profile for their lead program, anti-miR122 for the pan-genotypic treatment of HCV infection, they really ought to borrow from RNAi delivery, in this case GalNAc-conjugates from Alnylam.  With this, they will achieve more potent miR-122 inhibition as well as increase the therapeutic window through the targeted delivery.  This is opposed to saturating the target organ along with the rest of the body with ‘naked’ phosphorothioate molecules, a concept I'm struggling to get comfortable with. 

The case for such a facilitated delivery strategy is even stronger for an antiviral anti-miR candidate such as this one because hitting the virus instantly instead of only slowly reaching effective drug concentrations in the target organ reduces the risk of drug resistance.

As a result, Regulus will enter the clinic with RG-101 at a time when the first all-oral HCV therapeutics with high cure rates will have hit the market.  This is a commercial catastrophe for an otherwise scientifically sound target.

One already has to be thankful that a small organization such as Regulus is able to show such flexibility at all (I don't even want to know in which antiquated terms Big Pharma is thinking about microRNA Therapeutics).  This achievement almost makes me afraid to ask why they have not thought it through to the logical end and adopted more structured, and much more potent anti-miR structures along with these delivery technologies?  This armchair CEO would have had a SNALP/structured anti-miR strategy tested in 2008.

However, Regulus Therapeutics has a chance to redeem itself- at least partly.  As it evaluates GalNAcs and LNPs (SNALP?) for an anti-miR program in liver cancer (HCC), it could for example help the RNAi Therapeutics industry understand whether the GalNac-receptor (ASGPR) is suitable for RNAi Therapeutics candidates for the attractive liver cancer indication.  So as Tekmira has followed Arrowhead Research into HBV, maybe Arrowhead Research may want to follow Tekmira (TKM-PLK1) into HCC.

Disclosure: The idea for this blog entry came after I started to look into RGLS ahead of the AASLD meeting (Nov1-5).  I concluded that there would be a good chance that with presenting a promising profile for RG-101 at AASLD and maybe some new data for the Alport Syndrome at the Kidney Week also in early November (Nov5-10), we could see RGLS bounce back into the $9-10 range by mid-November.

ISIS/GSK and Tekmira Come Out with HBV Knockdown Plans

If you did not appreciate the value the pharmaceutical industry has come to place on the HBsAg knockdown concept for achieving a functional cure for chronic Hepatitis B (HBV) infection, the last two days will have woken you up. 

Yesterday, ISIS Pharmaceuticals reported that it had received a $7M milestone payment related to the development of an antiviral RNaseH development candidate (ISIS-GSK3Rx, aka ISIS-HBVRx) which, although undisclosed for competitive reasons, hasgot to be for HBV.  And today, Tekmira publicly announced that they will file an IND for an HBV-RNAi candidate in 2014 while hinting at the partnering potential of such a treatment candidate.

Arrowhead Research is thus not alone in their efforts any more.  Coincidentally, Arrowhead reported today the completion of their enrollment of the phase I single-dose, healthy volunteer study with ARC520, their DPC-delivered candidate for chronic HBV.  Accordingly, the dose escalation was able to run through all the pre-planned 6 dose cohorts up to the top dose of 2.0mg/kg. 

Apparently, there were no signs of significant dose-related toxicities.  The only finding of concern among the 36 volunteers, 24 of which received drug, was 2 cases of lightheadedness of uncertain clinical relevance.  As these occurred at the highest dose, it seems that the company suspects that it could have been drug-related although the study remains blinded for follow-up.

A dose of 2mg/kg without any serious adverse events or dose-limiting toxicities is a great start for DPC delivery technology.  This is especially the case when one considers that the single-molecule subQ version of DPC that I hope will form the basis for the upcoming pipeline candidates, except for the next one perhaps, will be much more potent than the two-molecule version of intravenously delivered ARC520 based on the non-human primate data presented at last year's OTS meeting.

With 2mg/kg of ARC520, I further believe that HBsAg knockdowns of over 90% are likely.  The biggest challenge going forward with this program will be setting a knockdown goal and getting the dose and dose frequency right.

For more about increasingly lively HBsAg knockdown for the treatment of chronic HBV, please follow my HBV Knockdown Blog.

Also today: Tekmira and Arrowhead Research Rapidly Filling Pipeline

In addition to chronic HBV, Tekmira further disclosed development plans for candidates addressing Marburg infection, alcoholism, hypertriglyceridemia and severe orphan glycogen disorders.  The presentation made clear that the company has not sat on its hands since the settlement with Alnylam and is close to having at least 4 drug candidates in active clinical development by early 2015 (TKM-PLK1, TKM-EBOLA, TKM-ALDH2, TKM-HBV).

Meanwhile, previously $200M market cap Arrowhead Research has succeeded with a $60M private placement without having had to offer a discount.  In my opinion, this reflects the broadened investor interest in the space and makes Arrowhead an even better investment as it can now, freed from financial constraints, immediately pursue some of the attractive liver targets with best-in-class solutions and beyond.

Wondering whether we are in an RNA Therapeutics bubble?  Then I suggest you get my OTS 2013 meeting report.  Dare I say that oligonucleotide therapeutics is a more capable and exciting technology than monoclonal antibodies already?  At least if you believe orphan diseases are the way to go in today's pharmaceutical drug development, there is no doubt about it coming out of the meeting. 

ISIS-CRP Phase I Results: The Decreasing Competitiveness of RNaseH Antisense for Liver Gene Knockdown

To maintain is positive trajectory of the recent comeback, it is important for RNAi Therapeutics to firmly claim gene knockdown in the liver for itself.  This is because liver gene knockdown is the lowest hanging fruit for the technology, yet RNaseH antisense has proven to be capable of gene knockdown there as well.   If RNaseH were to crystallize as the preferred technology, it would jeopardize a considerable portion of the near to midterm market potential of RNAi Therapeutics.  Just think of Alnylam's 5x15(TM) portfolio of drug candidates.

As I have explained before, dose/tissue oligonucleotide concentrations and, related to that, safety/tolerability will determine which technology will win the competition.  Although the odds seem in favor of RNAi Therapeutics, ISIS Pharmaceuticals keeps promising that improved chemistry and screening methods will improve upon the modest potency (~30-35% ApoB knockdown) of and the safety concerns with mipomersen.  Due to the importance of liver gene knockdown to RNAi Therapeutics, this blog will continue to comment on the respective data points as they come in.

RNaseH Antisense CRP Phase I Data

Today, ISIS Pharmaceuticals announced phase I knockdown data from its c-reactive protein (CRP) program, ISIS-CRPRx.   CRP, a gene expressed by the liver, is believed (by some, not all) to act as a central player in a host of inflammatory diseases such as atrial fibrillation and rheumatoid arthritis.  Phase II studies for these indications are ongoing.

The phase I study tested whether ISIS-CRPRx can blunt CRP induction upon inflammatory stress.  Accordingly, healthy volunteers were dosed 6 times over 3 weeks with either 400mg or 600mg of the phosphorothioate antisense molecule against CRP.  Following that, subjects received endotoxin, a common laboratory reagent that is interpreted by the body as an infection.

Subjects treated with placebo saw an approximately 30-fold increase in CRP levels due to endotoxin.  When pre-treated with 400mg and 600mg, however, the induction was reduced by 36% and 63%, respectively.

 

Although the study succeeded in showing that ISIS-CRPRx could blunt CRP induction, it is much less clear whether ISIS-CRPRx makes for a viable therapeutic.  A 63% knockdown knockdown may be OK for some indications, but intuitively one has to wonder whether to stop an acute inflammation in its tracks you need to step much harder on brakes like CRP.  Indeed, the press release proclaimed, as a testament to the specificity of the drug candidate, that ‘other important immune modulators’ were not changed- somewhat puzzling given that CRP is thought to play such a central role in inflammation.

Be that as it may, what the press release did not mention was the side effect profile seen in the ISIS-CRPRx phase I study; to wit, safety and tolerability is the primary objective of any phase I study.  What concerns me is that mipomersen is 200mg weekly and causes injection side reactions, flu-like symptoms, and indications of chronic inflammation which are likely the result of the elevated steady-state tissue concentrations required for RNaseH antisense efficacy.  In the CRP study, 2- to 3-times more phosphorothioate antisense molecules were given.  Although the side effect profile of an RNaseH antisense molecule is also sequence-dependent, doses of 400mg and higher have historically been associated with toxicity/tolerability considered unacceptable outside of cancer.   

My prediction is that due to the poor efficacy-dose relationship, ISIS-CRPRx will not go into phase III.  We have to see whether the gen2.5 cET chemistry brings any significant increases in clinical potencies as inherent molecular potencies as measured in cell culture studies may not take into consideration other pharmacological rate-limiting factors.  If gen2.5 can lower liver tissue concentrations required to obtain 50% gene silencing into the low single-digit microgram/g range, RNaseH would become more interesting again for the liver.  Otherwise, it seems that more fertile hunting grounds for antisense technologies may be found in the areas of noncoding RNAs (e.g. microRNAs and lincRNAs) and therapeutic splice regulation, especially when the target molecules have a long nuclear residence time.  With ISIS about to present phase I data for its spinal muscular atrophy splice modulation drug candidate at next week’s AAN meeting, a next step into that strategic re-direction may be taken.

Simply SNALP: RNAi TTR Knockdown Data Impress

Clearly, RNAi Therapeutics is back in fashion.  Well, almost.

Alnylam’s announcement of profound gene knockdown in the ALN-TTR02 phase I study for the treatment of TTR amyloidosis was greeted with an over 50% jump in the stock price on very high volume.  Various observers pointed out that this might have been an over-reaction given the early nature of the data.  On the other hand, considering that the over 80% peak knockdowns achieved with only a single dose of 0.15mg/kg are representative of a widely applicable delivery strategy, namely Tekmira’s SNALP technology, and considering that an accelerated approval path  should be possible for this orphan disease, determining the fair value of a SNALP-based company like Alnylam becomes difficult.

Yes, this was a study that enrolled just 17 subjects, healthy volunteers at that.  But it may be this that makes the tight variability in the pharmacodynamic response (=TTR knockdown) even more remarkable so: less than 5% relative standard deviations in the 0.15mg/kg and 0.30 mg/kg 3-person dose cohorts, the relevant groups for this analysis.  

When I had mentally listed the technical risks of SNALP-mediated delivery several years ago when I did my fundamental research on SNALP delivery, intersubject knockdown variability was one of the major ones.  However, given the present results and those from the recent hypercholesterolemia study (ALN-PCS02), I am much less concerned about this now, also in patient groups where liver health may be affected.

Moreover, the dose-related TTR knockdown in humans was essentially what has been seen in non-human primates.  Although a number of analysts were apparently puzzled by the importance of this observation, its value to a scientist is obvious: you can essentially eliminate the risk that an SNALP program fails in phase I due to lack of knockdown efficacy (and if you are one of those scientists that also dabble in the stock markets, you can start investing in the stock based on the monkey data that you’ll see presented at an informal presentation).   

The safety profile with ALN-TTR02 in this study was good.  There was one infusion reaction seen in other SNALP studies before and that was managed by slowing the infusion rate.  This was said to be possibly related to the pre-treatment with corticosteroids, and not the liposomal formulation itself.  In any case, while corticosteroids and other transient immune suppressions will not be an issue for the initial patient populations in diseases like TTR amyloidosis, Tekmira and Alnylam should start to try and wean themselves of it.  The improved, more predictive immune stimulation assays may form a sound scientific basis for this.

The reason for why the study was limited to 17 healthy volunteers was mostly due to competitive concerns.  In particular, the ISIS/GSK TTR antisense compound is said to skip phase II studies altogether and go straight into a pivotal phase III.  In data presented in May of 2011, this antisense compound was reported to effect a 44% mean TTR reduction at the 200mg/week dose level when given for four weeks, and 81% knockdown at 400mg which was the next higher dose tested.  400mg/week, however, is a dose that I believe is too high for chronic phosphorothioate oligonucleotide administration.  Although ALN-TTR02 should thus be 100- to 200-fold more potent on a weight basis than the antisense drug candidate, being the first-to-market is seen by some as an important factor for the commercial success in this orphan market, especially since these compounds work by the same mechanism of action.

The value of SNALP delivery

Some readers of this blog may be tiring by now of reading me sing the praises of SNALP delivery technology and Tekmira.  Yes, I’m invested in the technology via my Tekmira shares.  In all fairness, however, it is the clinical data from the SNALP compounds, really starting last November with the ALN-TTR01 data (SNALP Works!) that are reviving the RNAi Therapeutics field: ALN-VSP, ALN-PCS, and ALN-TTR.  We have heard a lot about exciting new, revolutionary RNAi delivery technologies, often claiming to solve the putative problems with  SNALP technology.  However, in the end, it is SNALP that is set to unlock the first wave of RNAi Therapeutics value creation.

Even Arrowhead Research and Benitec which develop competing delivery and RNAi trigger technologies, respectively, admitted so much in congratulating Alnylam to the results.  Sadly, they missed the opportunity to also congratulate the real innovator behind SNALP technology, Tekmira.  It seems that since Alnylam is getting all the media attention, it was probably not a good PR strategy to do so.

RNAi delivery technologies are tough to develop and rare to find, but when they work, they open up a range of therapeutic opportunities.   

Preview: For my next post, I am planning to write something about last week’s MC3 paper and how it reveals that covering all the tracks is hopeless when a scheme is that elaborate (RE Tekmira-Alnylam litigation), and why Alnylam the stock may be a great short here. 

ISIS Claims Scalp of Santaris CEO

After noticing that the former President of the US operations of Danish LNA antisense company Santaris, Art Levin, had just joined muscle microRNA Therapeutics company miRagen, I was alerted that this has been in the wake of a major shake-up at Santaris. Most notably, it appears that the CEO of Santaris, Soren Tulstrup, either handed in his resignation, or, more likely, was let go.


Privately-held Santaris, of course, has been a fierce rival of ISIS for supremacy in the antisense space.  Having former ISIS insider Art Levin join Santaris 2 1/2 years ago was in fact a bit of a coup for that company.  Its LNA technology is probably the most potent/high-affinity in the space for many applications.  ISIS, however, has had the benefit of increased financial might and having built a large patent estate that it is (ab-)using to sue, preferably smaller competitors with the obvious intention of making it difficult, if not impossible for those to raise capital and pursue business development opportunities.  It is rumored that ‘partner’/satellite company Alnylam has probably paid the price in the tens of millions for the privilege of going public.   


Not long ago, ISIS sued Santaris for unduly hiding under the Research Exemption in its various relationships including Pfizer, Enzon, GSK, and Shire.  In parallel, ISIS and microRNA spin-off Regulus have been haunting Santaris by questioning Santaris’ ability to develop its exciting lead candidate, miravirsen, a miR-122 antagonist for the treatment of HCV.  As a result, Santaris was dumped by GSK in favor of Regulus’ miR-122 program despite having the superior data package, which is also born out by the clinical data so far (here for the latest update at EASL).


Regulus announced this week that it was awarded another HCV miR-122 patent, in Japan, concluding with the remark that it was continuing to pay 'attention to hepatitis C'- somewhat ironical given that its clinical entry seems to be delayed forever.  Seeing that announcement, I had thought that this was one of the unnecessary PRs that I often take as a sign of weakness. In hindsight, it was probably just a case of adding insult to injury.


I myself have had reservations about the wisdom of building the company’s financial future on a program for which it may be lacking access to some of the key patents in the space.  Although one may assume that in the wake of its scientifically leading efforts in miR-122/HCV, Santaris will have in turn built IP to which Regulus would need access if it were to develop its own miR-122 antagonist,  such a strategy has been a high-stakes gamble for sure.  Along with the ISIS lawsuit questioning Santaris’ fundamental business development strategy, I would therefore not be surprised if the CEO had faced some tough questions by Santaris' investors about his decisions and consequently was ousted. 


Clearly, an exit in the form of an IPO or takeover had long been overdue for that company.  The company’s technology, pipeline, and relationships, also in light of what’s out there already (i.e. ISIS and AVI Biopharma), should have easily supported this, if not for the lawsuit and threats thereof.

New Antibody Data Indicate Tough Battle for RNAi Therapeutic PCSK9 Approach

The past week has been a busy one for the hypercholesterolemia field. New clinical data for PCSK9-targeting monoclonal antibodies from Amgen and Regeneron/Sanofi-Aventis were presented at the 2012 American College of Cardiology meeting, while ISIS/Sanofi-Aventis separately presented long-term efficacy, and especially safety data on the ApoB antisense compound mipomersen (aka KYNAMRO) for which marketing applications have now been submitted in Europe and the US. As hypercholesterolemia also represents a significant medical and commercial opportunity for RNAi Therapeutics due to the advances in knocking down genes in the liver, I will briefly summarize the new data and discuss some of the implications for RNAi approaches, including the phase I candidate ALN-PCS02 by Alnylam.

If there ever were doubts as to the commercial attractiveness of PCSK9 as a target for treating high cholesterol, last week dispelled them all. When it seems that all the thought leaders in the field, including famed cardiologist Steven Nissen, otherwise known for his ultra-critical views of certain medicines, and Wall Street (expected annual sales as a class of up to $20B thrown out) hail the data as proof that PCSK9 will be the final nail in the hypercholesterolemia coffin and thus throw their support behind the class, the commercial success of PCSK9-targeting therapeutics seems a foregone conclusion.

Although pretty much every large pharmaceutical company sports a PCSK9-targeting antibody, the excitement this time centered around the phase II data of Regeneron’s/Sanofi-Aventis’ REGN727 and phase I data of Amgen’s AMG145.

The REGN727 phase II 8-12 week multi-dose studies were conducted in close to 300 patients with elevated ‘bad’ LDL-cholesterol (LDLc) on statins. Depending on the amount and schedule of antibody administered, mean LDLc reductions (note: it is not entirely clear to me whether this refers to the LDLc reduction over time, or the peak LDLc reductions) of 40-73% were achieved. Interestingly, one press report said that ‘727 suffered from relatively short-lived activity such that subcutaneous administrations every 2 weeks would be required.

The phase I studies with ‘727, just published in NEJM (Stein et al.) indicated that the apparent rebound effect after 2 weeks may be due to the concomitant use of statins and that without statins, 50-55% persistent LDLc reductions can be achieved with close to 4-week dosing intervals. This is relevant for example for the statin-intolerant population. With statins, that type of persistent knockdown (more on the higher end of that range) would probably require every 2 week dosing.

AMG145 seemed to attract even more excitement than '727. In the 6 to 8-week phase I studies in around 100 patients taking statins, mean LDLc reductions of 63-75% were observed. These numbers indicate more potent, and likely more persistent LDLc lowering compared to REGN727, although without having seen the LDLc response curves over time, it is difficult to conclude that for sure.

Route of administration and dosing frequency are often cited as important competitive criteria for this class of drugs. In the case of ALN-PCS02 which is enabled by Tekmira’s SNALP technology, the current data indicate that a range of 40-55% persistent LDLc reductions with intravenous dosing every 4 weeks are conceivable. This, however, would require further improvements in potency over the 0.25mg/kg dose level which was the highest dose for which data was presented by Alnylam at their early January update. For this indication, it will also be important to wean the SNALP formulation off the transient immune suppression currently used. This would not only address criticisms that such immune suppressions carry risks, but it also would likely get rid of the observed PCSK9 rebound effect that was apparently linked to it and would thus contribute to prolonging the efficacy.

Of course, an important wildcard in the competition between MAbs and RNAi pertains to the safety of each drug candidate, especially when they are used long-term. The monoclonal antibodies seem to perform quite well in this regard in the short-term, albeit multi-dose studies. RNAi, however, has the theoretical advantage in that it does not involve the formation of antibody-PCSK9 complexes which could eventually have an impact on both long-term safety and efficacy/dosing frequency.

In retrospect, RNAi has picked a tough battle here. As a target that acts extracellularly, readily accessible from the blood, it seems an ideal target for monoclonal antibodies and thus falls outside the undruggable target space that currently still drives the interest of large pharmaceutical companies in RNAi Therapeutic development. On the other hand, the PCSK9 field is populated with antibody approaches, and should there be antibody-specific class adverse effects, RNAi could be the last one standing (note: the antisense candidates by ISIS and Santaris have already dropped out of the race, at least for now), something that one of the few Big Pharma/Biotech that has not yet invested in a PCSK9 candidate may value from a strategic point of view alone (RE partnering).

Meanwhile, the mipomersen extension study data presented this week suggest that the high drop-out rates and liver fat accumulations* (both possibly linked to some extent) at modest ~30% LDLc reductions make it a niche drug for the very small indication of homozygous familial hypercholesterolemia in the US and Europe, and possibly severe hypercholesterolemia in Europe. Although ISIS continues to claim that the liver fat accumulations normalize after reaching maximum median increases of +13% over baseline at week 52 (note: according to the mipo-related Visser et al. 2009 study, 5.6% absolute intrahepatic triglyceride contents are considered the upper-limit of normal), the absolute values would call for caution, especially when there are outliers in whom almost half the liver consists of fat. In the related conference call, ISIS further claimed that the apparent normalizations were not the result of the patients that discontinued mipomersen (possibly due to high liver fat contents). However, since ISIS has a history of erring on the side of optimism when it comes to mipomersen safety and tolerability, I’d like to see the liver fat-related sub-group analyses that take into account the discontinuations, dose reductions and interruptions.

The liver fat data may also mean that ApoB is unlikely to be a suitable stand-alone target for an RNAi Therapeutic, especially if even more pronounced ApoB knockdowns can be achieved than with mipomersen. Consequently, in order to exploit the therapeutic utility of ApoB as the critical protein of atherogenic lipoproteins, an ApoB-targeting RNAi Therapeutic should involve at least one other target gene which will also reduce liver fat content. It remains to be seen whether ApoC-III can be that target.

* Note: Despite the caution expressed in this article, I should add that there is general controversy about the relevance of simply elevated liver fat (NAFLD) in the absence of inflammation.

Technology Trends: MicroRNA Inhibitors and Single-Strand RNAi

There have been developments in the areas of microRNA inhibition and single-strand RNA-mediated RNAi that might have strategic implications for delivery technologies and RNAi Therapeutics, respectively.

MicroRNA inhibition: naked antisense no more?

Currently, all development-stage anti-miR programs to my knowledge envisage the use of unformulated phosphorothioated antisense molecules with various high-affinity modifications such as LNA/LNA-type conformationally restricted nucleotides or 2’F and 2’MOE. To some degree, antisense and certain microRNA companies are making a living out of advertising that, unlike (most) RNAi Therapeutics, no intravenous administration was required.

At the same time, it is becoming clear that more complex structures such as Dharmacon’s miRIDIAN hairpin microRNA inhibitors or the tough decoys (also the synthetic versions that were newly developed in collaboration with Japanese RNAi behemoth Kyowa Hakko: Haraguchi et al. 2012) are considerably more potent on a per molecule basis. Because of their structural complexity, however, they would require delivery formulations for therapeutic use. It remains to be seen how often such formulations would need to be applied, but the early research by Haraguchi in tissue culture shows that the anti-miR effect with these structures can be relatively long-lived. Nevertheless, the in vivo pharmacology of these structured anti-miRs remains to be better explored, but I could imagine that especially for antiviral or oncology applications, the more rapid onset of action and the potentially improved targeting due to the delivery technology could yield positive surprises.

Single-strand RNAi Therapeutics: Stable 5’ phosphate and 2'F

A little more than a year after ISIS and Alnylam ended their ssRNAi Therapeutics collaboration (for which I believe Alnylam had greatly overpaid), ISIS and Merck have made progress in the area.

It had been well known based on particularly protein structural work that the 5’ phosphate modification in the guide strand is important for incorporation in the RNAi effector complex RISC. There has also been corresponding early evidence in ssRNAi research (Martinez et al 2002) that ssRNAs with a 5’ phosphate are more efficient inducers of RNAi, albeit at much lower efficicay compared to dsRNAi triggers. Notably, in the case of double-strand RNA-induced RNAi, prior 5’ phosphorylation is not necessary as this is efficiently accomplished inside the cells.

Based on work by ISIS presented at the Keystone conference in January it now seems that lability of the 5’ phosphate is partly responsible for the reduced efficacy of ssRNAi. Using a new 5’ phosphate mimic (and some other lipophilic modification strategies) that is much more stable than the natural counterpart, it was now possible, following subcutaneous administration, to achieve solid knockdown in rodents. However, the cumulative dose required to get there is still very high, in the range of 2^nd generation RNaseH antisense.

ssRNAi work just published by Merck (Haringsma et al. 2012) confirmed that the 5’ phosphate modification was important, but more importantly worked out the beneficial role of the sticky 2’-fluoro (2’F) modification in ssRNAi. The impressive efficacy-enhancing activity of the 2’-F modification (for which, I believe, Alnylam holds rights to an important patent via a license from ISIS, but a modification that has also raised genotoxicity concerns) was found to apply to both tissue culture and animal settings [Note: an earlier version mistakenly stated that the IP belonged to Alnylam]. Unlike the ISIS work, however, Merck studied ssRNAi in mice using SNALP-like liposomal delivery. It therefore seems as if Merck would need to extra work on adding chemistries such as phosphorothioates or conjugates so that its ssRNAi technology can be used in the ‘naked’ form. This is because the prospect of using ssRNAi in the naked versus formulated form would really be the only motivation for pursuing ssRNAi.

Impressions from the Abstracts of the 7th Annual Meeting of the Oligonucleotide Therapeutics Society (Part 1)

The Annual Meetings of the Oligonucleotide Therapeutics Society are among the best on the conference circus related to, well, oligonucleotide therapeutics drug development. One benefit of bringing together RNAi Therapeutics, traditional RNaseH and steric block antisense, aptamers, and a few other oligo-based approaches is that researchers can benefit from sharing lessons in safety, how pharmacology relates to chemistry and formulation, manufacturing etc. Remember, it is the experience with older oligonucleotide technologies that allowed RNAi Therapeutics to take 10, instead of 20 or 30 years, to get to where it is today: over a 1000 patients and healthy volunteers dosed with more than a dozen of RNAi candidates exhibiting a decent, and improving safety profile; the ongoing Atu027 and ALN-TTR01 trials having reached dose levels where, based on sound science, robust target gene knockdown, technologically the primary objective, can be expected. Moreover, data from hypercholesterolemia, solid cancer, ocular and respiratory disease studies have provided evidence of dose-related therapeutic efficacy.

Not able to attend the 7^th Annual Meeting to be held this week in Denmark myself, I eagerly went through the abstract book to learn of new developments and trends. Here are my thoughts on a few select abstracts that I thought might be of interest to the readers of this blog (presented in the order they appear in the book). Note that if you are a Tekmira investor, keep reading until the end. Part 2 of the discussion can be found here.

Oral presentation: Expanding the structural diversity repertoire of siRNAs (Dong-Ki Lee, Sunkyunkwan University, Korea)

This presentation highlights the realization that a number of non-Tuschl RNAi trigger structures are not just IP workarounds, but can be used to achieve novel biological outcomes such as targeting multiple genes with one RNAi trigger molecule (multipodal structures), inducing select innate immune stimulation while at the same time silencing genes (long siRNAs), and reducing off-targeting (asymmetric siRNAs and ‘wobbly’ siRNAs).

Oral presentation: Activation of RNA interference in animals with single-stranded oligonucleotides (Erice Swayze, ISIS Pharmaceuticals)

For some indications, the intravenous application of the nanoparticle RNAi formulations which are leading in terms of in vivo RNAi gene silencing potency may be a commercial drawback (for the purpose of long market exclusivities, I believe it is a widely underappreciated benefit). ISIS Pharmaceuticals, until recently in collaboration with Alnylam have been working on naked single-strand RNAi (ssRNAi) solutions that can be administered subcutaneously.

It has been long known that ssRNAs can induce RNAi gene silencing, just 100-1000 less efficiently, which is not surprising since RNAi has evolved as a dsRNA-induced mechanism. The abstract claims that using fully modified, partially phosphorothioated ssRNAs, they were able to come within 5-fold of the potency of corresponding double-stranded structures. The initial animal experiments, however, seem to have failed due to ssRNA instability, but after further modifications they have now achieved activity at ‘pharmacologically relevant doses with subcutaneous administration in saline formulations’.

Certainly an interesting abstract and it remains to be seen just how pharmacologically relevant these doses are and the related safety profile. Similar, or better to their current RNaseH antisense? ssRNAi...ISIS’ antisense 3.0? Another interesting question is at what point did Alnylam drop the ball on ssRNAi after considerable investments- before or after the initial animal experiment failures?

Oral presentation: Delivery of Nucleic Acids (Muthiah Manoharan, Alnylam)

Alnylam’s oral presentation will be, you already guessed, about RNAi delivery. By listing 25 papers on two pages without any meaningful comment or discrimination, the abstract obviously wants to make the point that Alnylam is the leader also in RNAi delivery. Somewhat reminiscent of Alnylam’s press releases that used to list seemingly all their RNAi trigger-related patents, no matter how relevant to their gate-keeping potential which was the reason for listing them in the first place. As such, the abstract carries the dubious distinction of being the longest one of the conference, but the one with arguably the least content.

It is not the amount of money spent, the numbers of patents (‘thousands’), or papers published that makes you a leader in RNAi Therapeutics.

Oral presentation: Non-covalent peptide-based delivery systems (Divita, CRBM-CNRS-UMR5237, Montpellier, France)

This abstract concerning a non-covalent cell penetrating peptide-siRNA systemic delivery technology to me has firstly sentimental value. This is not a specific criticism of the work to be presented, a body of work that is buttressed by some credible data, but the abstract still reminds me of the early days when RNAi Therapeutics was hot…hot, hot, and all kinds of, sometimes wild, delivery claims were made: oral, blood-brain, all organs to name a few keywords.

While I wished that there was more excitement around RNAi Therapeutics right now as the negativity, particularly in the commercial arena, threatens to choke deserving technologies, the one benefit of RNAi being less hyped and exploited for fund-raising purposes by the biotech promotion machinery is that the overall scientific credibility index has increased. This can also be seen from the abstracts at this year’s OTS meeting.

Oral presentation: Investigating the potential of therapeutic oligonucleotides for pulmonary diseases (Clark, GSK)

GSK and AstraZeneca are probably the two Big Pharma companies most interested in RNAi/oligonucleotide Therapeutics for pulmonary diseases. This is an area with high unmet medical needs and new therapeutic approaches are needed here more than anywhere else. There are fundamentally two different approaches to knocking down genes in the respiratory tract: local delivery by aerosol inhalation, or through systemic delivery. Based on the abstract, GSK seems to be primarily interested in inhalation methods.

Among the companies having explored inhalation are Alnylam, ISIS’ respiratory disease spin-off Altair, and most recently Tekmira. It has become obvious that Alnylam’s naked siRNA approach (e.g. in ALN-RSV01) is sub-optimal and conjugation plus chemical modifications need to be applied to give such ‘semi-naked’ routes a chance. Altair meanwhile has closed down following phase II results with their naked MOE gapmer antisense candidate for asthma. Based on Tekmira’s track record of publicizing only meaningful scientific progress, Tekmira's aerosolized LNP approach is to be considered a serious contender for the leadership position in gene knockdown in the respiratory epithelium. Tekmira this year has presented data that its aerosolized LNPs retain the ability to knockdown genes in tissue culture. It is quite possible that GSK was/is the undisclosed Big Pharma collaborator for this program.

Silence Therapeutics, probably more by necessity than choice, takes a systemic approach towards gene knockdown in the lung using their intravenously administered lipoplexes (DACC). Actually, since Silence’s and Tekmira’s technologies may be best suited for endothelial and epithelial cell knockdown, respectively, the two approaches are complementary. It would make sense if AstraZeneca had some familiarity with Silence’s DACC technology.

Abstract #9: [3H]-radiolabeling of siRNA (Christensen, Novartis)

Abstract #86: Characterization of side reactions during the annealing of siRNA (Noll, Roche)

I list the two abstracts from Novartis and Roche here together because I believe they illustrate the cultural differences between Big Pharma and pure-play RNAi companies. While pure-play companies emphasize biology and developing new RNAi trigger and delivery solutions, the established pharmaceutical companies are apparently more concerned about manufacturing and pharmacology methods. It is obvious that manufacturing and pharmacology is an essential part of the game, and such work is also happening at pure-play companies and their outsourcing partners, but such work obviously does not address the rate-limiting challenges and Big Pharma, perhaps with the exception of Merck, willfully relies on accessing that from the pure-play companies.

Abstract #16: Inhibition of complement C6 synthesis in the liver using antisense oligonucleotides affects neuro-regeneration (Fluiter, Academic Medical Center, Amsterdam, Netherlands)

This abstract highlights that by knocking down a gene in the liver, one can have therapeutic benefits for a wide range of non-liver diseases, such as neurodegenerative diseases. This is not really surprising given that all organs almost exclusively depend on their development and function on what they are provided for by the blood. Proteins made in the liver constitute the majority of free proteins in the blood and consequently impact all organs. Complement proteins which play a critical in immunity are one example of such proteins. As most diseases contain a complement-related immune/inflammatory component, RNAi Therapeutics could be a tool for modulating a wide range of autoimmune and other hypersensitivity disorders.

This principle of inhibiting a target in one organ to address disease in others (see e.g. transthyretin amyloidosis) is in contrast to other, post-translational therapeutic drug modalities that target the liver for which the therapeutic benefit is almost always restricted to the liver. As such, the medical and commercial potential of RNAi delivery technologies that work well for gene knockdown in the liver is larger than widely appreciated.

Abstract #27: Thirteen week non-clinical testing of miravirsen in cynomolgous monkeys (Hildebrandt-Eriksen, Santaris)

This abstract concerns the toxicological evaluation of Santaris’ exciting phase II LNA anti-miR122, a LNA-modified phosphorothioate steric block antisense, for the treatment of HCV infection. Despite the successes of the recently approved protease inhibitors for genotype 1 HCV, there is still considerable unmet medical need, including for those with less drug-responsive genotypes or those high-risk patients that have failed on established therapies.

Presenting on home soil, the reported toxicities were in line with expected class effects of phophorothioate oligonucleotides, including slight, but relatively persistent clotting abnormalities which was not judged an adverse side effect because of the apparently small extent of the increase; reversible kidney toxicities at doses above 10mg/kg (the effective dose of miravirsen is likely between 2 and 5mg/kg); and finally some enlargements in macrophages which does not appear to be of too much concern. Note that because miravirsen is not intended for chronic use, this safety profile may be adequate. In addition to liver toxicity, it appears however that the kidney toxicity will be something to watch out for in the development of miravirsen.

The first phase II study of miravirsen has just completed enrolment according to clinicaltrials.gov and I look forward to learning about the results in due course.

Abstract #30: Lipid nanoparticle formulations of minimal-length shRNAs show potent inhibition of HCV-driven, liver-specific gene expression in mice (Johnston, Somagenics- in collaboration with Tekmira)

This abstract concerns the evaluation of 40-50 nucleotide hairpin RNAs with Tekmira’s LNP delivery technology for liver gene knockdown (in this case using HCV as a model system). It is not a surprise that the abstract shows that Tekmira’s LNP technology works with various RNAi triggers. The real new insight for Tekmira investors, however, is that Tekmira did not go into this litigation in a way that its access to payloads would be threatened as a loss of access to Alnylam’s RNAi triggers may very well be one of the outcomes that could facilitate a settlement. Instead, Tekmira must have been evaluating various RNAi trigger structures and presumably other nucleic acid payloads as well, and when it chose to exclusively license Halo-Bios multivalent RNAi triggers one has to assume that this was after an extensive evaluation of their safety and potency.

Whether there will be a similar arrangement with SomaGenics remains to be seen. Synthetic shRNAs are credible RNAi triggers and may in fact have some advantages over two-stranded approaches, e.g. highly efficient unimolecular annealing. However, their development has been held back by increased cost of goods associated with such long oligonucleotides and concerns about clogging up the RNAi enzyme Dicer (probably not an insurmountable challenge). A licensing decision may also depend on how broad SomaGenics' intellectual property is with regard to shRNAs. It is highly unlikely that SomaGenics has any gate-keeping claim in this area, and partnering with them would have to be driven by their shRNA-related know-how.

To be continued...(for part 2 click here)

Marina Biotech Continues to Drive Sector Consolidation, Acquires Novosom’s Liposomal Delivery IP

Marina Biotech, formerly known as mdRNA, continues to snap up assets in the oligonucleotide therapeutics and diagnostics space, this time acquiring the liposomal delivery IP from Novosom, a privately held drug delivery company based in Germany. This comes only a week after shareholders approved mdRNA’s merger with tkRNAi company Cequent Pharmaceuticals to form Marina Biotech. It will be curious to see whether Marina’s strategy of challenging Alnylam's industry-leading position by taking advantage of the depressed market for oligonucleotide therapeutics will succeed in luring Big Pharma and pay off for shareholders. The investment this time: $5M in newly issued stock.

Similar to Silence Therapeutics, Marina acts on the premise that in order to capture those lucrative partnerships, being able to provide a choice of delivery technologies, plus some claims in RNAi triggers, too, is key. This, of course, is in contrast to Tekmira’s so far quite successful strategy of doing one thing very well, meaning clinically maturing and expanding the applicability of its industry-leading SNALP delivery technology and avoid some of the deal frenzy and dilution of effort.

By the looks of it, Novosom has to be considered one of the more bona fide delivery companies. Similar to Tekmira’s SNALP, Novosom’s SMARTICLES can change their surface electrical charge and therefore reconcile (serum and storage) stability with endosomal release functionality. Unlike SNALPs, however, these liposomes also contain anionic lipids, in addition to cationic and neutral lipids, and do not employ PEG stabilizers. This stability can be attributed to the negative charge of SMARTICLES at phyisiologic pH which ought to avoid various interactions with host factors and resulting toxicities often associated with positively charged lipids. However, as the pH acidifies upon endosomal uptake of these particles, they become positively charged and competent for membrane disruption and cytoplasmic release. For similar reasons, ionizable SNALPs as practiced by Tekmira not only employ PEGylation, but are also essentially uncharged at physiologic pH.

In terms of IP, from the looks of it, Novosom has assembled a respectable IP estate with various fairly broad patents granted also in the important US market. This should provide Marina with considerable options to leverage its other liposomal assets, trp-cage targeting technology and amino acid-derived lipids, that I have felt lacked robust patent protection when used in liposomal formulations similar in composition to Tekmira’s SNALPs.

Theory and IP, of course, are only part of the equation. In terms of actual data, the literature bears out the tolerability of these liposomes. In terms of in vivo knockdown efficacy, I haven’t really seen much for liposomal delivery of siRNA in the peer-reviewed literature. There was, however, a paper on the liposomal delivery of a CD40 antisense oligo (under license from ISIS) in a rodent inflammatory disease model and that supported specific CD40 knockdown and disease amelioration while the unformulated antisense oligo appeared to be inactive. In general, based on the literature and also Novosom’s website, their technology seems to be in the late rodent stage and yet to be validated in larger mammals including non-human primates and Man.

There should, however, be an open IND for the delivery of a DNAi compound (no mis-spelling) by PRONAI which makes use of Novosom’s technology. However, it appears that PRONAI’s funding situation may have delayed actual dosing.

Novosom also entered into at least 2 collaborations with RNAi companies. One with (now defunct) DNA-directed RNAi company Nucleonics for treating hepatitis viral infections, and one with Boehringer-Ingelheim for the validation of drug targets in the liver and lung. Again, because Novosom is a private company I can only speculate whether the lipids used by Nucleonics in their soon aborted phase I study stemmed from the Novosom collaboration. Similarly, the status of the Boehringer-Ingelheim collaboration is unclear to me. Who knows, but maybe Boehringer-Ingelheim is actively looking for (liposomal) delivery and triggers for the development of RNAi Therapeutics, which raises another point: although the press release did not specify this, but selling what looks like the entire IP estate would appear to be tantamount to selling the entire company, existing partnerships and programs included.

But back to the fundamental question: Consolidating into a one-stop-shop, almost an ‘anti-Alnylam’, a la Marina and Silence versus technological deep-dive a la Tekmira- which strategy will create more shareholder value? The next 3 months should provide for some of the answers.

Please let your voice be heard and vote on the right.

Antisense or Guide Strand? That’s the Billion Dollar Question

As somebody who has spent the better part of my last decade in academic science, which in its ideal form is guided by the self-less pursuit of the ‘truth’, I must admit that I am somewhat irked when I read a paper on Argonaute 2 enzymology and find science being used also (though not exclusively) as a tool of corporate propaganda. You may think that this is nothing new at all. However, I’d like to use the latest publication by ISIS Pharmaceuticals on RNAi as an excuse to highlight a few of the issues that come up when ISIS Pharmaceuticals, by sheer repetition, claims that RNAi Therapeutics is an antisense technology.

In the paper by Lima and colleagues in the Journal of Biological Chemistry (‘The Binding and Cleavage Specificities of Human Argonaute 2’), the investigators study the effect of sequence length and modifications of what they call the ‘antisense’ strand on Argonaute 2 binding. What the authors call here ‘antisense’ is widely referred to by the rest of the field as the ‘guide strand’, that is the small RNA of the double-stranded RNA trigger that gets loaded onto Argonaute 2, the enzyme in RNAi that carries out the target mRNA cleavage. Admittedly, the ‘antisense’ is sometimes used in the RNAi literature to operationally describe the relative polarities of the RNAs involved (guide strand, passenger strand, target mRNA). However, when ‘antisense’ comes up 243 times within a manuscript, but ‘guide’ not at all, even as a verb, I believe it is a valid hypothesis that this cannot be a coincidence, but is calculated corporate speak.

I have explained before why RNAi by its very nature is a double-strand RNA technology, most tellingly illustrated by the fact that the discovery that what had been thought was mediated by antisense was actually mediated by the double-strand RNA contaminants of antisense preparations was awarded the Nobel Prize, so I will not repeat here myself. This is also not meant to criticize the scientific merits of the paper which furthers our understanding of the chemistries that could be used for the less efficient form of RNAi, namely single-strand RNAi and must be seen in the context of the recently announced ssRNAi initiative by ISIS and Alnylam, except maybe for the observation that the finding that single-strand RNA binds purified Argonaute 2 apparently better than double-strand RNA does not reflect Argonaute 2 loading in a living cell.

The point is that whether RNAi is labeled as an antisense technology or not can have significant consequences for the interpretation of the scope of patents, some of which date from before the discovery of RNAi in worms. It is all good for Alnylam for example to implicitly subscribe to ISIS notion as long as they have exclusive access to that IP as it helps constrain the freedom-to-operate of competitors such as Merck and Silence Therapeutics. However, when these early patents are ultimately not deemed as critical, can this leave Alnylam with patents that were pursued less rigorously so as not to conflict with the ISIS’ IP? One of the Tuschl patents comes to mind, I believe dubbed ‘Tuschl IV’, which for the first time explicitly described the use of single-strand RNAs for the induction of RNAi, but, to my initial surprise, had received very little attention among all the ssRNAi talk.

Aside from this political minefield, labeling RNAi Therapeutics as ‘antisense’ just does not do justice to the many ground-breaking discoveries this field has seen, and the people and institutions that made them and that you would think deserved their rightful place in RNAi history and a larger piece of the royalty pie. Specific modifications when used within an siRNA yes, but not the fundamental mechanism of action.

I leave it up to the reader whether this makes ISIS Pharmaceuticals an even better investment, and I must say they clearly deserve credit for their pioneering role in oligonucleotide chemistry and clinical application, and are now harvesting the financial fruits not only from their own drug development pipeline, but also from licenses for all types of therapeutic RNA approaches, including microRNAs (progress so far have exceeded my expectations, and according to a just published comments by Regulus’ CEO on HCV-miR122 this looks set to continue for the foreseeable future) and aptamers. Regardless, 243 times antisense does not make RNAi such.

RNAi Therapeutics versus Antisense- Where Delivery Makes a Difference

Regulus, the microRNA joint venture between Alnylam and ISIS Pharmaceuticals, is the most visible manifestation of the scientific overlap that exists between antisense and RNAi. The overlap, however, is not just limited to the science, but also extends into the capital markets. To better help the investor differentiate between the two technologies, I’d like to use this blog to provide an overview of some of the fundamental differences underlying the development of RNAi and antisense into therapeutics and their long-term prospects, with an emphasis on delivery. Needless to say, beware this discussion will be heavily biased in favor RNAi, but then again these are the reasons why I’ve been attracted to RNAi in the first place.

Certainly, progress in both areas in the last 3-5 years has mutually benefitted the investment climate for both technologies as it has heightened interest and increased confidence in RNA therapeutics in general. However, the two technologies also compete for investment dollars with many of the same investors, which are typically upbeat about the future of gene-based medicines but unsure where to place their bets, allocating their investments based on where they see most promise. One issue that often comes up in making this decision is the observation that while for systemic applications antisense, as practiced in the most advanced programs today, is typically administered without a particular delivery formulation, the development of specialized delivery technology is frequently cited as the key challenge for RNAi to realize its ultimate therapeutic potential.

Antisense for gene knockdown works largely by two mechanisms: interfering with translational initiation (e.g. AVI Biopharma's morpholinos) or through an RNase H-type mechanism (e.g. Santaris and ISIS Pharmaceuticals). For this, the key factor is to achieve efficient hybridization of a single-stranded oligonucleotide antisense with its target mRNA which either prevents productive ribosome association to the mRNA (inhibition of translation initiation) or may be recognized as a substrate for the RNase H enzyme which may degrade the RNA portion of the mRNA-DNA duplex, but normally functions in the degradation of the RNA primer during DNA replication.

Various oligonucleotide chemistries have been developed to optimize these processes for in vivo applications. Essentially all of these are single-stranded oligos of which the sugar phosphate backbone is heavily modified to a) increase their in vivo stability; b) improve their pharmacokinetics and avoid rapid renal excretion by promoting their association with components of the blood; c) similarly allows them to be retained in tissues; d) facilitate crossing of cell membranes; and finally e) increase their target mRNA binding. By contrast siRNAs, because of their charge and more rigid double-stranded nature and with apparently some exceptions that include mucosal epithelia, do not cross cell membranes efficiently on their own and therefore need to be specially formulated for most applications.

The use of unformulated antisense is consistent with their mechanism of action. Since antisense does not harness a naturally existing endogenous gene silencing pathway, it relies on achieving concentrations of oligonucleotides in the target tissue over a prolonged period of time that are high enough such that, as a result of the rules of thermodynamics, a sufficient fraction of target mRNA will be recognized. Similarly, unlike RNAi, the specificity of antisense is largely governed by biophysics and benefits only relatively little from biological proof-reading.

In practice, to achieve the necessary tissue concentrations, patients are typically dosed frequently at the initiation of therapy so that the tissue concentrations reach steady-state therapeutic levels. Targeted delivery of antisense into cells of interest may allow one to achieve a knockdown earlier, but any benefit would only be short-lived as antisense is not retained in specific gene silencing complexes but will soon redistribute according to their partition coefficient throughout the entire tissue so that ultimately similar amounts have to be administered and a formulation would only be a nuisance with little benefit.

By contrast, RNAi harnesses an endogenous and catalytic gene silencing mechanism, which means that once it has been delivered, either by conjugation or in nanoparticles into the cytosol, they are efficiently recognized and stably incorporated into the RiSC silencing complex to achieve prolonged gene silencing. In fact, measurable RNAi-mediated gene silencing can be observed at siRNA concentrations so low that it becomes difficult to detect them (e.g. fluorescently-tagged siRNAs by microscopy). This means that as the majority of siRNAs that do not reach the cytoplasm may disappear quite rapidly, the total exposure of the body to the nucleic acid can be much lower compared to antisense which should be beneficial both in terms of safety and pharmacodynamics (activity profile of drug over time).

This is not to say that chemical modification is not practiced in RNAi. However, unlike in antisense, the purpose of modification in RNAi is mainly to avoid triggering innate immune responses, making the siRNA sufficiently stable so that they survive their journey into their target cells, and also to stabilize them as part of RiSC (Merck has been talking about that concept on several occasions); and as we learn more about the biochemistry of endogenous RNA silencing pathways, modification is also increasingly used to increase the inherent biological specificity of RNAi. Unfortunately, it is surprising to me that compared to RNAi only very little, if at all, is reported about the specificity of antisense and I would be grateful if somebody here could point out pertinent studies that I should be aware of.

Targeted delivery may also avoid unnecessary drug exposure of non-target tissues. For unformulated antisense, no matter what the indication and target tissue, the biodistribution is essentially the same, and toxicities of the liver and kidney due to extended exposure to large amounts of the heavily modified antisense compounds is well known.

Certainly, improving the therapeutic index is an important issue for RNAi Therapeutics, too, but as the many transgenic mouse models which express ample and highly efficient RNAi throughout their life without causing overt toxicity attest, ultimately the improvement in the therapeutic index of RNAi is not limited by its very mechanism of action.

While it is a certainty that antisense companies will come out with 4th and 5th generation antisense technology, advances after decades of antisense research aiming to improve target mRNA recognition will only be marginal and based on trying out yet more nucleic acid modifications, although it appears to be a challenge to improve upon the efficacy of probably the most potent antisense modification that have now been known for a while, namely LNAs and their derivatives.

While RNAi efficacy in animals has already surpassed that of antisense for applications of the liver and lung as well as other tissues, I am confident that future advancements in RNAi will be more than marginal. For example, even as recent liposomal formulations achieve 90% gene knockdown in the liver at 1mg/kg, this still means that only about 1 in 10,000 siRNAs that have reached the liver makes it into the cytoplasm (assuming it takes about 1000 cytosolic siRNAs to achieve that level of knockdown according to a recent presentation by Phil Sharp). Alone a better understanding of the endosomal uptake of these nanoparticles, which is only in its infancy and starting to be explored, should allow for more than incremental improvements in the therapeutic index of RNAi Therapeutics.

And if you are still undecided on where the future is heading, numerous transfection studies in vitro where it can be assumed that equal amounts of antisense and siRNAs are present in cells, have shown that RNAi is quite a bit more potent on a mole-by-mole basis comnpared to antisense.

I am aware that some in the antisense community, including investors, may take offense with this blog, but since I am often asked about this issue, I think a more straightforward approach is better than to keep beating about the bush. And, of course, there is always the comment section.