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.

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.  

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 1/10^th 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.

Dose of ARC520 HBV Drug Candidate to be Increased to 3mg/kg

Today, Arrowhead Research reported preliminary phase IIa safety and efficacy results of ARC520 in patients infected with HBV.  The goal of this study is to determine the level of viral knockdown, especially HBsAg, following a single dose of ARC520.

Efficacy in-line with animal studies

The results for the 1mg/kg and 2mg/kg cohorts indicate that knockdowns are in-line with what has been seen with 2 molecule DPC-enabled RNAi in non-human primates, including the HBV-infected chimpanzee.  The company could not be more specific about numbers since it is still a blinded study and the knockdown curves apparently haven’t stabilized yet after 8 weeks in the 2mg/kg cohort suggesting remarkably prolonged pharmacology.

In the chimp study, HBV load in serum was reduced by 1log at 2mg/kg, but determination of the HBsAg knockdown following a single administration was complicated by the fact that Arrowhead gave a second dose of ARC520 of 3mg/kg before the HBsAg knockdown at 2mg/kg had leveled off.  With 2 doses of 2mg/kg and 3mg/kg 2 weeks spaced apart, the final knockdown was ~80%.  This level of knockdown was likely an underestimate of the true efficacy of ARC520 since one of the two siRNAs was a mismatch and the chimp had very high viremia to start with.  For the non-human primate study with ARC-AAT, Arrowhead’s new development candidate for alpha-1 antitrypsin, the knockdown with a single dose of 1.5mg/kg was ~75%.

Taken together, I expect that the knockdown at 2mg/kg to be somewhere around 70-75%.

Arrowhead extending to higher doses

While this level of HBsAg knockdown in such a short period of time, would be superior to anything before in the HBV space, for comfort and competitive reasons, it is not all that exciting.  What is more exciting is that Arrowhead is doing what I’ve always said they should be doing, namely testing higher doses than 2mg/kg.

This is because for 2-molecule DPC-RNAi, it is the amount of the endosomolytic peptide that is rate-limiting for knockdown efficacy. As shown in animal model after animal model (e.g. Wooddell et al. 2013), the efficacy achieved with 2mg/kg was well below maximal knockdown efficacy.  However, when slightly extending the dose to 3mg/kg or 4mg/kg, the depth of knockdown increases tremendously. 

In the case of ARC-AAT for example, a 75% knockdown at 1.5mg/kg became a knockdown of 90% at 3mg/kg, which was the same knockdown as with 6mg/kg indicating that a plateau had been reached.  In the case of HBV, the plateau, based on 6mg/kg endosomolytic peptide was a virtual elimination of HBsAg in the serum.

Despite the various genes and doses, the picture is that the dose-response becomes very steep soon after 2mg/kg.  Whether it is 3mg/kg or 4mg/kg is another question.  Arrowhead is currently gearing up for these doses following phase I extension studies in healthy volunteers,   

The most important news today therefore is that 3mg/kg in healthy volunteers was as well tolerated as 1mg/kg and 2mg/kg before (4mg/kg in the works).  Indeed, it was even better tolerated since the skin reactions disappeared with the transient use of an oral anti-histamine. 

Results from the 3mg/kg cohort in this dose-finding study should be presented at or around AASLD in November. Stay tuned.

FDA Greenlights Use of TKM-EBOLA in Current Outbreak

Canadian drug developer Tekmira just disclosed that the FDA verbally notified them that it would partially lift the clinical hold it had placed on TKM-EBOLA a month ago.  The clinical hold had been instituted as the result of cytokine elevations that had been observed in a healthy volunteer study of the drug.

By partially lifting the clinical hold, the FDA ‘blesses’ the use of TKM-EBOLA in actually infected patients whose odds otherwise would be to die from the infection.  Since the FDA only has authority over health matters in the US, this is strictly only of relevance for the use in people infected with Ebola either brought back from Africa for treatment just as the two US aid workers, or in case the virus started to crop up in the US. 

I am, however, inclined to interpret the news that the FDA is encouraging Tekmira to consider the use of TKM-EBOLA in Western Africa at the epicenter of the epidemic.   You could imagine that if anything went wrong with the use of TKM-EBOLA, it might have had adverse consequences for the future clinical development of the drug candidate in the US.

Overall, I am pleased with this development as the clinical hold in light of the current health emergency was just unbearable. How to handle the practicalities of using TKM-EBOLA in the field, including who will bear the costs, we will have to see.  Remember, financially, it is Tekmira's HBV candidate that has priority over TKM-EBOLA and so I expect TKM-EBOLA to be part of a broader effort funded by the public health bodies.

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.