Monday, September 15, 2014

Alnylam’s Venture into Preeclampsia Exudes Confidence about Safety of RNAi Therapeutics

Last week, Alnylam disclosed yet another one of their development candidates (press release here, slide presentation here), this time ALN-AGT for the treatment of preeclampsia.  While most previously disclosed candidates address severe orphan diseases of high unmet medical needs such as TTR amyloidosis and complement-related diseases, some candidates such as ALN-PCSsc for hypercholesterolemia already have the potential to go after larger patient populations with less severe diseases.

With the preeclampsia indication, Alnylam has gone one step further, not in the sense the preeclampsia was not a very serious condition, but because pregnancy-related drug development is an largely shunned arena as the safety stakes are particularly high here.  Alnylam’s decision to go after this indication therefore must mean that the company has high confidence that RNAi Therapeutics, at least their particular breed in the form of GalNAc-siRNAs, should be very safe and succeed where small molecules have failed before.

Preeclampsia and current management

Preeclampsia affects about half a million pregnancies annually in the developed world and is a leading cause of pregnancy-related death (16% of maternal mortalities), frequently the result of stroke or end-organ damage such as in the liver and kidneys.  Because the disease can be the death sentence for a previously young, healthy woman (and her child), preeclampsia is a much dreaded condition where better diagnostics (esp. those that predict which preeclampsia cases will progress catastrophically) and new drugs are urgently needed.

Preeclampsia is characterized by high blood pressure (>140/90 mm Hg), frequently accompanied by protein in the blood (proteinurea).  It is a disease of the vasculature (Silence Therapeutics- listen up!) and while the causes are not fully understood, overexpression of VEGF/angiogenesis inhibitor sFLT may be a key early event in the disease.  It is probably the combination of vascular abnormalities and high blood pressure that ultimately can kill a woman.

The only cure for preeclampsia is delivery of the placenta.  Unfortunately, this may be much too early for the baby or mean delivery as early as 25 weeks of gestation with all the attendant infant mortality and developmental deficiencies. 

Although large randomized trials are lacking (because they would be difficult to justify), it is widely accepted that lowering blood pressure with antihypertensives lowers the risk of stroke and end-organ damage and thereby can help buy valuable extra weeks for the fetus to further mature. 

How RNAi can help

The management of high blood pressure is typically a multi-drug approach.  This means that for a given patient multiple antihypertensives are attempted sequentially or in combination until the desired control is achieved.

The drug cabinet for pregnancy-related hypertension, however, empties rapidly to a few somewhat trusted ones such as hydralazine and labetalol due to the suspected or known side effects (to the fetus) of most of them.   This includes otherwise widely prescribed antihypertensives which tackle high blood pressure along the renin-angiotensin-aldosterone system (e.g. ACE inhibitors). 

The toxicity here is due to the small molecules entering fetal circulation and consequently interfering with blood pressure regulation in the fetus thereby causing often fatal cardiac and renal defects as well as a general failure to thrive.

By contrast, an RNAi therapeutic would allow you to target the angiotensin pathway in the mother only.  The target of ALN-AGT, angiotensinogen, for example is expressed in the liver and both angiotensinogen as well as a GalNAc-siRNA would be restricted to the circulation of the mother.   Given the biodistribution of oligonucleotide therapeutics, I could also imagine RNAi Therapeutics to go after targets in the kidney with the same benefit of being limited to the mother.

ALN-AGT good for both (rat) mom and baby

In data presented last week at High Blood Pressure Research 2014, GalNAc-enabled ALN-AGT was shown to inhibit angiotensinogen of the mother by 90% in rodent models of preeclampsia.  Importantly, this was accompanied by a 20mm Hg reduction of mean arterial pressure, in addition to a reduction in proteinuria.

Such a 20mm reduction is clinically meaningful, given that reducing blood pressure from 160 and 140mm Hg can be the difference of highly likely stroke to no stroke.

Interestingly, not only did the maternal manifestations of the disease improve, the placental blood supply and architecture was improved, too, resulting in considerable benefits to the fetus as seen by increased birth weights and normalized brain:liver weight ratios.  

Safety and future development path

The data provided little discussion of the potential drawbacks and safety concerns around ALN-AGT.  One concern would be hypotension (lowering blood pressure too much) and related to this the reversibility and/or half-life of ALN-AGT.  Given that RNAi Therapeutics targeting genes expressed in the liver are typically active for weeks, close attention needs to be paid to hypotensive potential.
Encouragingly, the rat data indicate that the effect of ALN-AGT on blood pressure is more pronounced when high blood pressure is high (delta of 20mm Hg) compared to when it is normal (delta of 5mm Hg).  Another parameter that would be useful to consider in this context would be the dose/knockdown-blood pressure relationship and intrapatient/intra-rat variability.  E.g. would increasing the knockdown from 90% to 95% have a dramatic effect on blood pressure lowering or would it make little difference?

It will be safety that will guide the future clinical development path of ALN-AGT.  I can imagine Alnylam to first address women with a high likelihood of developing the devastating consequences of preeclampsia, perhaps with the help of a companion diagnostic.  Alternatively, it is not farfetched to think that ALN-AGT will first be used on top of other antihypertensives when they alone are not able to sufficiently control blood pressure.

Albeit little-loved by the pharmaceutical industry, once having been validated by proper clinical development in pregnant women, a drug like ALN-AGT would be poised to immediately become a mainstay in this indication.  From there, ALN-AGT could take on the rest of the $30-40B antihypertensive market.

Wild speculation

A drug like ALN-AGT is unlikely to be commercialized by Alnylam, both in the focused, likely hospital-based preeclampsia setting and in the wider antihypertensive market.  For preeclampsia, it would seem like a good addition to the portfolio of The Medicines Company, Alnylam’s partner for ALN-PCS in hypercholesterolemia.  

It is my suspicion that Alnylam rues the day it gave away ALN-PCS when times were hard. I don't believe Wall Street is anywhere close to grasping the potential of ALN-PCS to become a well-differentiated best-in-class in what is predicted to be a very large market.   And since Alnylam is known for its zeal to exploit all the RNAi value there is, with licensing partners regularly turning into fierce 'competitors', it could be just a matter of time before Alnylam will claw back control over ALN-PCS.  ALN-AGT looks like the perfect trade-in. 

Wednesday, September 10, 2014

RXi Provides Disappointing Clinical Update for Scarring Drug

At the Rodman & Renshaw investor conference today, the CEO of RXiPharmaceuticals dropped a little bombshell in the form of a disappointing clinical update on their lead clinical candidate, RXI-109 for the treatment or prevention of dermal scarring.  In that interim look for efficacy, RXI-109 and placebo were not really distinguishable in scar severity sending the stock down 30-40% in the middle of today’s trading session.

1301 study design

The 1301 study is the first of three phase IIa studies evaluating 109 in a number of different scar settings.  In this case, RXI-109 was administered following scar revision surgery on the lower abdomen.  Part of the same scar received either 3 injections of the self-delivering RNAi compound, another part placebo solution.  Half of the subjects (50% of the study/16 subjects have enrolled as of today), received 109 on days 1, 8, and 15 following surgery (cohort 1), the other half received injections on days 14, 21, and 28 following surgery, the latter apparently inspired by clinical design trends observed for competitor antisense drug from Pfizer/Excaliard.

I find that blindly adjusting your clinical design based on such competitive intelligence is a worrisome sign of lack of confidence.

1301 study results

The first interim look for efficacy took place at 1 month post-surgery.  Results were based on the blinded visual assessment of scar severity on a scale from 1 (good fine-line scar) to 10 (worst scar imaginable). 

Unfortunately, the close to 50% knockdown of target CTGF observed in a similar 3-dose phase I study, did not translate to an obvious improvement in scar severity: for the immediate treatment group, the VAS score was 2.0 for both the 109 and the placebo side of the scar; for the delayed treatment group, the VAS score was slightly better in the 109 side (2.0) than on the placebo side (2.5) even reaching statistical significance.

Before you get excited and buy into the biological rationalizations by the CEO for why it makes sense that delayed, but not immediate would exhibit such a benefit, note that a VAS difference of 0.5 on a scale from 1 to 10 would appear to be clinically meaningless despite the statistical significance.  Moreover, looking at the VAS scores across the board, it seems that the placebo side in the delayed treatment cohort is a statistical outlier as it should not have performed any different than the placebo cohort in the immediate treatment group.    

On the other hand, given that the scars have not had time to fully develop and the VAS scores were still so low, it is possible that real differences will emerge at later time-points such as month 3 when the next interim look will take place and it may thus be premature to declare the study or drug for that matter a failure.

RXi needs a plan B and investors patience

In light of the disappointing trial update, it may not be a coincidence that Geert Cauwenbergh today took the opportunity to talk more about their other, preclinical pipeline candidates in the dermatology and ophthalmology space in much more detail than had been the case.   

I am less excited about the prospect of RXi expanding their dermatology footprint given the relatively modest gene knockdowns observed, limited tissue penetration from the site of injeciton, and the cosmeceutical nature of their current line-up (acne, depigmentation etc).  By contrast, I am much more excited about  the prospect and value of self-delivering RNAi triggers in the ophthalmology space given the great unmet medical needs there and the highly encouraging tissue penetration/biodistribution data for self-delivering RNAi in that organ.

There will be more to talk about that in the future.  As stock market investors, however, one has got to wonder whether management is aware of that value and knows how to best exploit it (pro tip: VEGF is a no-no for RNAi in the eye).  Even more concerning is the fact that the majority shareholder (Tang Capital) still holds close to half of the company and is in the process of unloading it thereby putting constant pressure on the stock. And with only $10M in the bank, you know what the trip to the Rodman & Renshaw conference was all about.

So no more than a small starter position for me despite the steep sell-off today.

Wednesday, September 3, 2014

Regado Shock Raises Questions for Oligonucleotide Therapeutics Field

Ave biotech investor, morituri te salutant.

Last week, aptamer company Regado Biosciences imploded in a spectacular fashion after it had to permanently terminate a large pivotal phase III trial of its lead therapeutic program, REG1.   It followed notification that an unacceptable rate of serious anaphylactic adverse events had occurred.

Aptamers are oligonucleotides that recognize their protein targets based on their 3-dimensional shape, instead of sequence complementarity as is the case with most other oligo-based mechanisms of action.  

Because REG1 involves oligonucleotides and pegylation chemistry, the event warrants the close attention of the wider oligonucleotide therapeutics field.  This blog tries to summarize what is known about REG1 and the adverse events and aims to pinpoint potential safety landmines as well as suggest strategies to circumvent them for the benefit of ongoing and future oligonucleotide drug candidates.

REG1 a 2-component system for the tight regulation of hemostasis

The bane of anticoagulant drug development is that too much of it and/or inhibiting clotting at the wrong drug targets can lead to great morbidity and death due to uncontrolled bleeding.  REG1 aims to address this catch 22 by providing an oligonucleotide-based aptamer that binds and therefore inhibits a key regulator of the clotting cascade, Factor IXa, but with the twist that the inhibition can in turn be turned off at will by administering an antisense oligonucleotide that has sequence complementary to that of the aptamer.  This disrupts the 3-dimensional shape of the aptamer thus abrogating its ability to bind Factor IXa.

The aptamer (pegnivacogin) itself consists of a heavily modified 31mer oligonucleotide with lots of 2’-O-methyl and 2’-O-fluoro residues (~10kDa molecular weight).  However, the bulk of the drug is made of a 40kDa polyethyleneglycol (PEG) moiety appended to the 5’ end of the oligonucleotide.  

The antisense oligonucleotide functioning as the antidote meanwhile is an unassuming 15mer 2’-O-methylated RNA, hardly something that would catch the eye of an oligonucleotide toxicologist.

The reason why the aptamer had been modified with PEG was to extend its circulation half-life.  What otherwise would likely be a half-life of a few minutes for a simple 2'-O-methyl/fluoro oligonucleotide, obviously of little use for the intended regulation of blood clotting, could thereby be extended to over 24 hours when administered intravenously (à REG1) or an even more impressive ~1 week when injected subcutaneously (àREG2).

Serious allergic events in REGULATE PCI study

The REGULATE-PCI study that has just been terminated aimed to enroll approximately 13000 (!) subjects undergoing percutaneous (through the skin) coronary interventions (PCI) to unclog arteries around the heart.  Because things tend to clot around devices introduced into the human body and in the presence of physical stresses such as clot disruptions, such procedures have to be performed with the concurrent use of anti-clotting agents (anticoagulants).

The study was terminated when after about one quarter of the target enrolment (~3200), an imbalance in what the company described as ‘serious allergic adverse events’ in its press release was noted by the body tasked with ensuring the safety of clinical trial participants (DSMB).

Unfortunately, I was not able to retrieve a replay of the conference call held by the company to discuss the trial termination to gather more information on the nature of the adverse events.

It is, however, logical to assume that the serious allergic events are the same that had already been noted in the phase II RADAR trial.  Specifically, 3 of the ~500 subjects that received REG1 had allergic reactions ‘shortly’ after infusion of the pegnivacogin aptamer, 2 of which were considered ‘serious’ and in fact led to a stopping of the phase II trial after it was deemed that the study already had enough statistical power.  The affected patients were successfully managed with antihistamines and steroids (Povsic et al. 2013).

Immune reactions not foreign to Oligonucleotide Therapeutics

Immune stimulation, of course, is also the main safety risk of Oligonucleotide Therapeutics as a class.  We are all familiar with injection site reactions, alternative complement activations and attendant cytokine elevations, antibody generation to phosphorothioated oligonucleotides etc.  Some of them, e.g. TKM-Ebola most recently, have led to Clinical Holds or the discontinuation of drug candidates.  On the other hand, thousands of subjects have been given oligonucleotide therapeutics, some for years, with apparently acceptable safety profiles.

So is the oligonucleotide component of pegnivacogin the culprit for the ‘serious’ allergic reactions?

After reviewing the data, the safety issue is unlikely due to the aptamer itself.  A naked, in this case 2’-O-methylated, 2’-fluoro oligonucleotide alone is expected to be very safe, at least in terms of acute toxicity.  This conclusion can be drawn for example from the administration of naked, non-phosphorothioated oligonucleotides, including the systemic programs of Quark Pharmaceuticals. 

On the other hand, the reason for the apparent safety of unformulated oligonucleotides could be simply because such an oligonucleotide is rapidly excreted into urine and therefore has little chance to be toxic.  This is, however, unlikely since the allergic reactions were reported to occur shortly after drug administration, at a time when there would still be considerable concentrations of naked oligonucleotides left in circulation, therefore allowing for such a comparison.

It is interesting that only REG1 has been terminated so far, but not REG2.  Although this shoe could yet drop due to an abundance of caution, it suggests that the serious allergic events are dependent on the intravenous route of administration, a route of administration that is more generally plagued by immune-related ‘infusion reactions’ from small molecules to large biologics.

...enter Omontys...

This brings me to the topic of Omontys, aka peginesatide.  Omontys, of course, is a pegylated peptide that had to be withdrawn from the market 18 months ago following similar (rare) allergic events, including fatalities due to anaphylactic shock.  

Like pegnivacogin, the pegylation in Omontys had a molecular weight of 40kDa.

Like pegnivacogin, there were both intravenous and subcutaneous versions of peginesatide, but only the intravenous version was associated with the severe allergies that occurred shortly after drug administration.  

Like pegnivacogin, the allergies only occurred during the first administration of peginesatide (note: for pegnivacogin there is just 1 administration).

So taken together with the well-known hypersensitivity often seen in response to PEG, the evidence strongly points towards PEG as the culprit, not the oligonucleotide.

Pegylation widely used in RNAi Therapeutics

This unfortunately does not entirely exonerate oligonucleotide therapeutics.  The reason is that pegylation is a widely used tool in the biotech industry, and within Oligonucleotide Therapeutics, RNAi Therapeutics has made ample use of it and promises to support advances in delivery, especially beyond the liver. 

This raises the question of whether such problematic compounds can be spotted earlier.  If not, or if the decision is to continue development, can simple allergy tests identify the subjects that should not get the drug, or would anti-histamine and/or steroid pre-treatment be practical and acceptable?  Or is it even just a manufacturing/quality control issue or a matter of the molecular weight of PEG? I’m sure some of these questions sound familiar to the readers of this blog, and I expect that the field will learn over time if and when to use these and other strategies. 

As both fearlessness and fear can be fatal to drug development and investment returns, it is worth reminding ourselves here that numerous pegylated drugs have been approved and are being commercialized.  Also, there are various degrees of allergic events, some more serious than others.  

Finally, as a former Affymax (and Lehman Brothers in case you wanted to know) investor who lost a few feathers, I and Affymax just were incredibly unlucky.  It is my contention that Omontys would be widely used today if it had not been for the conservative market entry strategy by Omontys-distributor Fresenius Medical Care which involved the close monitoring of the first commercial Omontys patients which picked up the very rare events.  

Also, why simple strategies such as the use of anti-histamines or just going with the subcutaneous version were not attempted to salvage Omontys remains a mystery to me.  Blame it on the damaged goods theory.

For the sake of Regado investors and medicine, let’s hope that the parallels end here and Regado won’t follow in the footsteps of Affymax to bankruptcy and class action lawyers.  

Tuesday, August 26, 2014

Black Box Therapeutics Finds Small Molecule Cure for Spinal Muscular Atrophy

After curing Cystic Fibrosis and Duchenne Muscular Dystrophy with nonsense drug ataluren, PTC Therapeutics is moving on to relieving the sufferings of many more children afflicted with severe genetic diseases.  This time it is spinal muscular atrophy (SMA).  In a publication that recently appeared in SCIENCE, PTC Therapeutics has stumbled upon another orally bioavailable small molecule that is claimed to modulate an RNA processing event for therapeutic purposes.

The reason why I’m interested in the PTC story is that their small molecule approach to RNA modulation is counter-intuitive as it instinctively calls for a nucleic acid-based solution.  On the other hand, an oral alternative to what are usually more invasive routes of administration could have certain advantages such as patient convenience and access.

Naryshkin paper

The paper by Naryshkin and colleagues is on the discovery of RG7800, a compound partnered with the new LNA antisense owner Roche.  It is an illustration of the general strategy taken by PTC Therapeutics of finding RNA sequence-specific small molecule modulators of genes of interest. 

In this example, the company began the panning process by hooking up RNA elements from the SMN2 gene that are involved in the splicing of an exon of interest with a luciferase reporter gene so as to enable large-scale small molecule library screening.  If left untouched, this reporter construct will produce very little light emission due to luciferase expression.  On the other hand, if the small molecule is successful in biasing splicing towards the inclusion of exon 7, the intended outcome of this particular SMA treatment approach, then light is produced.

As one might expect there are numerous ‘hits’ that come out of such primary screens.  In this case, 2000 small molecules from the library increased luciferase expression.  Since such expression changes can be due to a myriad of causes, selected candidates were put through a number of tests such as whether the increase was dependent on the SMN2 sequence elements (reporter constructs without the SMN2 sequences would take care of this), whether the selected small molecules change the expression or splicing of other RNAs (à off-targets; e.g. by RNA seq) etc.

RG7800 apparently survived all these tests and was found to increase the desired SMN2 splice form by about 70% in a number of cell models (including patient-derived cells) and in a mouse model.  It also has entered clinical development.

Comparison with ISIS drug

Since RG7800 is in direct competition with antisense drug candidate ISIS-SMNRx by ISIS and Biogen as both aim to increase SMN2 exon inclusion, a brief comparison is warranted.

While RG7800 has the obvious advantage of being orally bioavailable versus the need for intrathecal administration of ISIS-SMNRx, in terms of molecular outcome, exon inclusion in spinal motor neurons, it appears to be lacking: a ~70% increase compared to ~100-150% increases in the good SMN2 isoform achieved by ISIS-SMNRx in two clinical trials in SMA infants and children, and even more than that in rodent studies before (Passini et al. 2011).    However, RG7800 achieves SMN2 splice modulation not just in the motorneurons of the CNS, but in many other places, in and outside the CNS. 

There is ongoing debate as to whether such body-wide modulation is required, a question also prompted by a study by ISIS and collaborators on the systemic/subQ application of the antisense drug (Hua et al. 2011).  Parenthetically, this also means that if ISIS/Biogen wanted global SMN2 regulation, they have an option with subcutaneous administration in addition to intrathecal administration.  Interestingly, with the subcutaneous administration of ISIS-SMNRx, the mice lived much longer than when the oligo was given intrathecally (days/weeks versus months).  Indeed, consistent with those studies, the PTC small molecule also prolonged the lives of SMA mice considerably.  

I guess it will have to be the clinical trials which will be most informative as to whether this is an artefact of the mouse model or not.  If so, systemic drug exposure would add no benefit and only increase the risk of adverse events from off-targeting.

At the end of the day, what I find remarkable is that it is apparently possible to find small molecules that can modulate gene expression in a fairly sequence-selective manner.  While I don’t doubt that you can change splicing with small molecules, it is the apparent specificity of a simple molecule such as RG7800 that perplexes me.  

PTC Therapeutics did not disclose how many compounds they had to sift through to find RG7800.  If they really just picked one or a few after the initial screen that yielded ~2000 hits and then got lucky, I’d be quite skeptical.  Adding to my skepticism is that drug concentrations in the CNS were reported that greatly (>10x) exceeded those in the plasma (supplementary figure S7).  I’m no small molecule guy, but for an oligo guy who has been following drug development in general, that seems very unique. The blood-brain-barrier apparently does not exist for PTC.  

As a result, and also given the controversy around the discovery of ataluren (artefact or not) and the fact that PTC Therapeutics itself can only remotely speculate on the mechanism of action of RG7800, I acknowledge the publication as interesting, but am not ready to jump on board just yet.

Friday, August 22, 2014

Dicerna and Arrowhead Gain Broad Dicer-Substrate Patent Rights

A day after Alnylam claimed ownership over Dicerna’s Dicer-substrate technology, Dicerna shot back with the announcement of a newly allowed US patent that gives it and most likely its co-licensee Arrowhead Research (via mdRNA/Marina Biotech and then Roche) broad control over the Dicer-substrate RNAi trigger space.  Specifically, claims from the Rossi-Kim invention were awarded that broadly cover ~25-30bp dsRNAs with essentially no other structural limitations.

Below 25bp, Dicer does not effectively recognize dsRNAs for processing into small siRNAs; above 30bp, the risk of inducing immune responses is thought to increase considerably as do manufacturing costs.

If you are wondering how Dicerna and Alnylam could be claiming possession over the same trigger structures, it is worth noting that while Dicerna’s patent cover 25-30bp in general, Alnylam is limited to 25bp dsRNAs that further contain a 3’ overhang.  Moreover, it is expected that since the invention by Tuschl explicitly aimed at using dsRNAs downstream of Dicer for RNAi gene silencing purposes, Alnylam’s 25bp claim is unlikely to prevail if the company should file an infringement suit against Dicerna (or Arrowhead Research should it adopt Dicer substrates).  

A simple way to get around all the uncertainty is to simply use RNAi triggers of 26bp and more.  Of course, for Dicerna’s lead candidate DCR-MYC, a 25/27 design, this train has already left the station and I am not sure whether Dicerna was forward-thinking enough to consider 26bp and longer for the orphan PH1 indication, its next clinical candidate.

Assuming that Arrowhead Research has access to the same patent, the adoption of 26bp and longer Dicer-substrate RNAi triggers could be a relatively simple strategy to remove the IP uncertainty lingering around usiRNAs: is a UNA a 'nucleotide analogue' or not?  Moreover, there is a scientific argument for Arrowhead considering Dicer substrates given that they should synergize with a conjugate approach like DPC.

Should we care about all this IP stuff?

Yesterday, I read a comment on InvestorVillage that investors would do best to ignore all the IP noise, including on my blog :(.  I agree that as long as the capital markets are a valid source of capital for small biotech companies, the issues around fundamental RNAi trigger IP, which will largely expire in 2019-2021, will be mute by the time products have come to the market and/or a court decision has been rendered. 

However, the outcome from the lawsuit by ISIS against Roche/Santaris where ISIS accused Santaris (now part of Roche) of monetizing ISIS IP by entering into partnerships with Big Pharma, could remove a lot of business development from Safe Harbor protection.  From there, challenging capital raises on similar grounds is only one step away. Personally, I don't think it will come to this since essentially all business development deals and capital raises in this industry ultimately aim at helping get drugs onto the FDA-regulated market.

And at the end of the day, as is also illustrated by mega-blockbuster Sovaldi, as long as the scientific data is compelling, none of this will prevent a drug from reaching the market and biotech companies making deals.  

Wednesday, August 20, 2014

Alnylam Once Again Clutches at IP Straws to Support Valuation Gap (with correction)

(21Aug14) Yesterday, I mistakenly stated that Alnylam wrongfully concluded that Dicerna was infringing on a newly issued Tuschl patent.  Following comments in the comment section below, it came to my attention that indeed there was a claim that I missed, claim 81 (and some contingent claims), that covers RNAi triggers of 25 base-pairs as follows:

81. An isolated double-stranded RNA molecule, comprising:

(i) a sense strand and an antisense strand that form a double-stranded region of up to 25 
base pairs, said sense strand having an identity in the double-stranded region of at least 85 
percent to a target RNA molecule; and

(ii) at least one strand having a single-stranded 3’-overhang, wherein said 3’-overhang 
has been stabilized against degradation; and

(iii) at least one nucleotide analogue, 

wherein said RNA molecule is capable of target-specific RNA interference. 

Note that Dicerna's RNAi triggers make use of the 2'-O-methyl modification which sometimes is found in the 3' overhang and can also have stabilizing activity.  Taken together, this claim indeed questions Dicerna's RNAi triggers, and although I would expect vigorous debate around whether 25 base-pairs are covered by the patent's description requirements should it come to a patent litigation, the assumption is that Alnylam's new patent rightfully questions many, if not most of the RNAi triggers used by Dicerna currently.  

Since I'm at it, the new patent also comes awfully close to the asymmetric RNAi trigger designs by RXi Pharmaceuticals and others (asiRNAs).  RXi e.g. uses dsRNA lengths of below 15bp with the guide strand having a long 3' overhang.  I am a bit surprised that Alnylam got just enough extension both below and above their traditional 19-23bp stronghold to start overlapping with some asiRNA and Dicer-substrate designs.

Regardless, I stand by my point that Alnylam has re-invigorated their patent-related press releases in order to explain the valuation gap to its peers in the public markets.  The original blog entry follows here:

This morning, Alnylam greeted the competition with another IP-related press release.  It wrongly claims that a patent it just obtained covers competing technologies.  This suggests that it either lacks an understanding of RNA technology basics or that it is afraid that the market will come to understand that the valuation difference to its peers has no basis in either a commercially more attractive clinical pipeline, a superior patent estate, or simply better technology.

Dicerna’s Dicer-substrate technology not in 14-24bp range

Today’s press release concerns US patent application 13/725262 which is part of the Tuschl patent estate covering certain RNAi triggers with 3’ overhangs.  Although the patent has not finally been published, based on the latest submitted claim set, the RNAi trigger covered by the main claim should comprise the following features:

a)      a dsRNA length of 14-24 base-pair; 
b)      at least 1 3’ overhang;
c)       at least one ‘nucleotide analogue’;
d)      and the dsRNA is non-enzymatically processed.

Clearly, in citing the Rose et al. and another paper by Dicerna (actually their scientific founders from the Rossi lab at the City of Hope) as proof of Dicerna’s infringement, Alnylam hopes that its investor and business development audience does not actually read scientific papers. 

‘Specifically, the newly allowed patent application broadly covers small interfering RNA ("siRNA") molecules of various designs, including so-called "dicer substrate" RNAi triggers (Amarzguioui et al., Nat Protoc.2006;1(2):508-17; Rose et al., Nucleic Acids Res. 2005 Jul 26;33(13):4140-56)…’

Otherwise, it would quickly become apparent that Dicerna’s version of RNAi triggers have a dsRNA length of 25 base-pairs and, well, are enzymatically processed: Dicer substrates!

[Note: in the original entry I mistakenly said Dicerna's triggers were 27 base-pairs; to be precise, they are 25/27 designs with 25 base-pairs and a 2 nucleotide 3' overhang on the guide.]

So as the actual clinical pipelines of Arrowhead Research and Tekmira are about to look more attractive in terms of commercial value (HBV alone), look forward to more Alnylam patent-related press releases to help the market understand why Alnylam has a market cap of $5 Billion and its competition only about 1/10th of that.

PS: the claim that usiRNAs infringe on this and other patents by Alnylam largely depends on the definition of ‘nucleoside analogue’ and ‘modified nucleotides’.

PPS: this patent does not change Alnylam's position as very similar ones related to 3' overhangs have already issued.  However, by slicing and dicing a patent application, it is possible to get issued a set of highly similar patents which, of course, is great fodder for the PR department.

Stabilizing RNAi Triggers against Cytoplasmic Degradation Pays Dividends

In describing the preliminary phase IIa results of ARC520 for HBV, Arrowhead Research noted that the duration of gene silencing (2 months and more) was surprisingly extended in Man compared to the preclinical experiences in rodents and non-human primates.  Alnylam hasnoticed the same with its GalNAc-siRNA conjugates, especially the highly modified ESC version. 

The extended gene silencing activities, of course, bode very well for RNAi Therapeutics in general when in the early days (~2002-2003) I was a bit apprehensive when gene silencing in my transfections of cancer cell lines persisted for only 2-3 days (as we now know largely due to their rapid cell division).  To maximize the duration of gene silencing, thereby opening up RNAi Therapeutics to new applications and increasing its competitive profile, it is important to understand the factors underlying it.

Alnylam explained the differences to the preclinical experience because rodent and monkey hepatocytes seem to have a more hostile, degradative cytosol compared to human hepatocytes (hypothesis 1).  In one experiment, only 6% full-length ESC-GalNAc-siRNA remained after a given time in rodent and monkey cytosolic extracts while in human liver cytosol extracts more than 60% persisted.

This, however, was only a correlation and I have considered it equally likely that the difference in gene silencing duration might be a function of more stable RISC complexes in humans (hypothesis 2) or increased stability in the endo-lysosomal compartment (hypothesis 3).  Especially for GalNAc-siRNAs, I would think that the reason that it works in the first place is due to them being able to accumulate in endo-lysosomes from which they only get released in the wake of natural vesicle membrane turnover.  So chemical stability here would be a critical factor since the endo-lysosomal compartment is known to be highly degradative.

DPC and SNALP: two endosomolytic technologies with different durations of gene silencing

While I still consider that endo-lysosomal stability of the naked RNAi trigger is critical for approaches like GalNAc-siRNA conjugates, the new DPC-enabled ARC520 results strongly indicate that another critical factor lies downstream of endo-lysosomes.  This is because in the DPC approach which involves strong endosomolytic activities that should activate soon after endocytic uptake, the risk of the RNAi trigger being degraded in the endo-lysosomes should be low.  Similarly, there should be little contribution to gene silencing from RNAi triggers that get released into the cytoplasm in a delayed fashion.

SNALP is another delivery technology where the RNAi triggers that become active in gene silencing get released into the cytoplasm soon after endocytic uptake.  However, while clinical data supporting 3-4 week dosing frequencies have been obtained with SNALPs (e.g. ALN-TTR02), the silencing does not appear to be as extended as with DPCs.  So given that one marked difference of the payloads used with SNALPs and DPCs is the modest degree of chemical modification historically used with SNALPs, this, too, points towards cytosolic stability of the RNAi trigger being important for the duration of gene silencing.  Parenthetically, it also suggests that Tekmira may want to similarly explore heavily modified RNAi triggers while being mindful not to step on the McSwiggen patent toes of Alnylam.

RISC-optimized ultra-stable single-strand RNAi triggers

In the case of traditional double-stranded RNAi triggers as e.g. used with DPCs, the stabilized RNAi triggers get used up over time as they are recruited into RNAi effector complex RISC.  Part of this process involves their unwinding into single-strand RNAs with the guide strand being retained.   It is known that once used, a 'normal' guide strand (or microRNA) is not recycled into another RISC complex and will likely suffer metabolic destruction once the protein components of RISC have become degraded as part of natural protein turnover.  And even if the guide strand had been stabilized, because a standard single-strand molecule that had relied on being part of a double-strand structure for RISC recognition, old age will eventually catch up here, too. 

What a waste after all this effort of getting the RNAi trigger into the cytoplasm.  So why not take a cue from the single-strand RNAi practitioners who optimize single-strand RNAi triggers also based on being able to be recognized by RISC?  If a corresponding dsRNA contained corresponding recognition elements, then the guide strand could contribute to another round of gene silencing, thus extending and enhancing knockdown.  On the other hand, the lessons learned from stabilized dsRNAi triggers should also benefit the single-strand RNAi approach as increased cytosolic stability should also increase their duration of activity: RISC-optimized ultra-stable single-strand RNAi triggers.
By Dirk Haussecker. All rights reserved.

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