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.

Tuesday, August 12, 2014

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.

Thursday, August 7, 2014

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.

Monday, August 4, 2014

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.

Thursday, July 31, 2014

What TKM-Ebola Could Achieve in the Current Outbreak

(please read financial conflict at the end)

We are in the midst of the largest recorded outbreak of Ebola hemorrhagic fever virus ever and there is little evidence that its spread is being contained.   Here, I will make the case how TKM-Ebola, the most advanced Ebola therapeutic in clinical development, could help in avoiding ever more damage from the deadly virus.

How TKM-Ebola could help

1.       Give suspected cases an incentive to go to the treatment centers.
2.       Provide medical personnel with a stand-by and therefore help in their recruitment.

The continued spread of Ebola can be partly attributed to a breakdown in confidence in the authorities and fear by medical personnel. 

I have been viciously attacking the World Health Organization (WHO) that by downplaying the significance of the outbreak it has been a key factor in re-igniting viral spread.  It is the WHO who have tried to minimize the true numbers of infected, possibly in cahoots with local authorities, and laughed off the suggestion that the virus could get on a plane in arguing against any kind of travel restrictions.  Of course, it recently did and I am still waiting to see top WHO officials send their families on a vacation to Western Africa.  But probably the most outrageous insult was in suggesting that the spread of the virus is explained by ‘funny’ cultural practices in these countries such as kissing the dead during burials.

The WHO, probably in the comfort of their headquarters in Geneva, even attacked on-the-ground Medecins Sans Frontieres as alarmist for calling the outbreak ‘unprecedented’ in late March.

This notion that the virus is very bad in spreading from person-to-person and can only do so with the help of obscure practices is obviously wrong given that more than 100 medical personnel have become infected. While I am still waiting for an explanation by the WHO of how this could happen, I refuse to believe that they kissed the dead in the treatment centers or licked any other of their body fluids for that matter.  Has the WHO (and others) maybe considered the unthinkable, namely that the reason why this is the biggest ever spread of the virus is because the virus has mutated and new routes of infections are possible, such as by aerosol?  The US military will have its reason to believe that this could happen, otherwise why would they be so concerned about it being weaponized and spending hundreds of million dollars on the development and stockpile of an Ebola therapeutic such as TKM-Ebola?

And if people that protect themselves with space-suits get infected, how would you feel as a suspected case of Ebola? 

I know I would do anything NOT to go to these treatment centers, because what is obviously for the good of the overall population would only exponentially increase my risk of contracting the infection in case I was one of those wrongly suspected to have Ebola.

In my opinion, providing individual isolation wards with the best medical equipment possible, a dignified environment, and a drug as an option for the patient could make the difference in whether suspected cases will turn themselves in or not.  The argument that ‘this is Africa’ and you cannot expect good medical care there should not count in this day and age when equipment can easily be shipped between continents.  It’s probably far cheaper to do it now than further risking for the virus to go global (obviously, the WHO thinks this is impossible).

And for medical personnel, the benefit of making TKM-Ebola available is obvious and most tangible: since the onset of flu-like symptoms in this population is highly likely to be due to Ebola and because they have ready access to the necessary equipment such as infusion apparatus, they could be treated immediately with the agent.  Treating as soon as possible is thought to be critical for TKM-Ebola to be efficacious.


What is TKM-Ebola?

TKM-Ebola is an intravenously infused RNAi Therapeutic that has been demonstrated to save the lives of monkeys infected with an otherwise fatal dose of Ebola.  It is being developed under the ‘Animal Rule’ in efforts funded by the US government which is afraid that this virus could be weaponized and used as a bioterror agent.  The ‘Animal Rule’ is a development pathway instituted by the US FDA for diseases such as Ebola for which it would either be impractical or unethical to conduct efficacy studies in humans. 

Because natural outbreaks are unpredictable and experimentally infecting volunteers with the virus out of the question, these monkey studies are as good as it gets regarding drug efficacy (so much for the mantra that there are ‘no drugs for Ebola’).

An important second element of the Animal Rule is that human volunteer studies demonstrate acceptable safety at the doses corresponding to the efficacious dose in monkeys.  For this reason, pivotal phase I safety studies have begun this year in healthy volunteers (so much for the notion that licensed Ebola drugs are far off- ‘phase I’ is misleading).

Clinical Hold

Unfortunately, in the midst of the outbreak, the FDA instituted a Clinical Hold on the TKM-Ebola safety study because a case of dangerously high cytokine elevations was observed at the highest dose planned in this dose-escalating/dose-finding study (0.5mg/kg).  I agree that this is to be considered a serious adverse event in a volunteer that is not infected with the virus.

The reason for the cytokine stimulation is likely due to TLR-mediated, lipid-amplified innate immune stimulation, a known risk of liposomal RNAi delivery, especially at doses of 0.5mg/kg and higher.  It is also the reason why all other active development candidates by Tekmira and their licensee Alnylam are conducted in the presence of transient immune suppression with steroids and the like which in many cases is acceptable given the severe diseases these treatments go after.

As indicated, the adverse event at 0.5mg/kg should by no means spell the end of TKM-Ebola.  Firstly, the company argues that the pharmacologic corresponding dose to those curing the monkeys is lower than 0.5mg/kg.  Secondly, a side effect that is not tolerable in healthy volunteers (usually ~20-year old male students) could be well tolerated in subjects with a 70-90% likelihood of dying from a disease in a matter of days.  It is ethically more troubling to involve healthy volunteers in such drug development just as aggressive experimental cancer drugs are hardly ever tested in healthy volunteers.  Lastly, there may be ways to avoid the side effect altogether, such as by using transient immune suppression.  However, I do not know whether transient immune suppression is possible for Ebola, but I expect Tekmira will have the answer for this from their large-scale animal experience.

Next steps

For TKM-Ebola to have the best impact on the current epidemic, the first step would be to start manufacturing it at scales sufficient to treat at least ~1000-5000 patients.  This takes time and given the uncertainty around the future course of the epidemic, the investment needs to be made now instead of waiting until it is too late for a treatment center-focused approached involving an intravenously infused agent.

The next step depends on the feasibility of steroid pre-treatment during an Ebola infection.

In case that it is known that steroid pre-treatment was of little concern (e.g. based on infected monkey studies), start treating rather aggressively (e.g. start at doses of 0.25mg/kg).  In case it was not, go about more slowly by starting at sub-therapeutic doses as low as 0.025mg/kg and treat the initial experience like a dose-escalation study in actually infected patients.  Of course, everybody would need to provide informed consent.  In general, any semblance that the use of TKM-Ebola was imposed by the Western world and the local population used as guinea pigs is to be avoided which is probably a key reason why Tekmira to my mind has been almost in hiding during this whole episode almost to the point that they (and the FDA) are glad about the Clinical Hold.  

To support the accelerated development of Ebola therapeutics and vaccines, including TKM-EBOLA you can add your signature to the following change.org initiative: http://www.change.org/en-GB/petitions/food-and-drug-adminstration-fast-track-drug-and-vaccine-research-for-ebola-hemorrhagic-fever

Disclosure: Tekmira constitutes a meaningful part of my investment portfolio and I have to credit the WHO for greatly increasing its value.


Tuesday, July 29, 2014

Quark’s Failure in Kidney Study Heralds End of Naked RNAi Triggers

Quark Pharmaceuticals today announced that RNAi Therapeutic candidate QPI-1002 has failed in a fairly large phase II kidney transplant study to meet its primary efficacy end-point.   For those interested, it was a pre-defined threshold of reducing the risk of having to go on dialysis after receiving a kidney transplant (30% pre-defined, 15% achieved with no p-value provided).  Following failures with similar 'naked' RNAi candidates by Quark and others (e.g. Opko/Acuity wet AMD candidate and Alnylam’s ALN-RSV01), this probably puts a nail in the coffin of a na├»ve, but convenient approach to RNAi delivery taken by quite a few in the early days of RNAi Therapeutics. 

QPI-1002 is a blunt-end 19 base-pair AtuRNAi targeting p53, thereby aiming to protect struggling kidney cells trying to take hold in their new host from dying.   It carries simple alternate 2’-o-methyls on both strands and is given by intravenous infusion reconstituted in saline.  While it is well established that unformulated/naked oligonucleotides without special modifications or delivery formulations to make them ‘drug-like’ will concentrate in proximal tubule cells of the kidney, the declared target cell for QPI-1002, it is still a mystery how the RNAi trigger is supposed to cross into the cytoplasm of those cells. 

Of course, a drug may fail for reasons other than target engagement, which couldn't be determined in this study, but I believe Quark in private would also side with the view that this most likely had to do a failure to deliver. This is because Quark itself has been adding chemistry to their more recent molecules to turn them into more credible self-delivering RNAi triggers.  And even if it wasn’t due to a failure to deliver, there is now little reason to pursue it any more.


RIP naked RNAi, RNAi Therapeutics has moved on.
By Dirk Haussecker. All rights reserved.

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