Tekmira’s SNALP Technology Once Again Highlighted in High-Profile Medical Literature

It is a good sign to see RNAi Therapeutics appear in high-profile medical journals with increased regularity (see also Tekmira’s TKM-EBOLA in The Lancet and Arrowhead’s CALAA-01 in Nature).  This reflects the fact that RNAi has moved well past being a laboratory tool and is coming closer to first regulatory approvals.  The TTR amyloidosis program sponsored by Alnylam and critically enabled by Tekmira’s SNALP delivery technology is arguably spearheading this effort and results from the ALN-TTR01 and ALN-TTR02 studies were published last night in the New England Journal of Medicine (Coelhoet al.).

As most of you know, the top-line results from ALN-TTR01 in November 2011 was unambiguous proof of RNAi-mediated knockdown of a liver-expressed gene (~40% median peak knockdowns in the highest dose; SNALP Works!), with top-line results for ALN-TTR02 (knockdowns in the 90% range) in the summer of 2012 translating it from proof-of-concept for RNAi in Man to a medically highly relevant approach for a disease of high unmet need, TTR amyloidosis.   

The RNAi mechanism of action of the knockdown was confirmed both by showing no TTR knockdown with an unrelated siRNA (but same delivery) and by demonstrating cleavage sites by 5’ RACE as predicted by the RNAi trigger sequence.  Notably, the assay was performed on RNA harvested from simple blood draws (‘exosomes’).

In terms of adverse events, there was also little new in the publication: dose-related infusion reactions were the most significant adverse events and were readily managed by slowing the rate of infusion. Importantly, at doses up to and including 0.3mg/kg, the current dose in the later-stage studies with ALN-TTR02, no such infusion reactions have been observed. 

The most notable part in the publication was actually what was missing from it: nowhere throughout the main text nor in the countless pages of supplementary material was it mentioned that the critical delivery technology had been developed by Tekmira.  Based on the ongoing  arbitration over a milestone payment related to liver cancer drug candidate ALN-VSP02 which was triggered by Alnylam refusing to behave like an adult, you can rest assured that this was no innocuous omission and that Alnylam is intent on applying pressure on Tekmira wherever it can.   

A Sign That Big Pharma Could Recognize the Low-Hanging RNAi Therapeutics Fruits

It was with much amusement and head-shaking disbelief that I read the Li et al. paper oncancer RNAi Therapeutics  development from Abbott.   The amusement stemmed from the fact that in the paper, the authors had come to the obvious conclusion: current technologies should allow you to develop real-world therapeutics based on the RNAi platform if only you judiciously combine the delivery platform with the right target and indication.  Duh…

As such, Abbott is representative of the various Big Pharma companies that guttered in-house RNAi Therapeutics development as they chose to only see the challenges instead of realizing the obvious opportunities.  Even those still in the game like Merck have long liked to go around and teach everyone how super-diligent, but super-slow their RNAi Therapeutics game strategy was. 

As RNAi Therapeutics have created market values at lightning speed (Alnylam now at a market cap of $3.5 billion), not based on just hype, but based on paths well-trodden by orphan drug companies, I expect more and more Big Pharmas to re-think their strategies.  Maybe even listen to this blogger who has always advocated a pipeline strategy that is based on where your delivery technologies can go to, instead of the traditional cart-before-the-ox-I-want-the-next-blockbuster-pill wishful thinking by people who may have risen to the corporate tops in companies like Coca Cola.
  

C’mon scientists from Merck, Novartis, and Takeda.  Inside yourself is an innovator that finally wants to see how your technology performs in humans.  Instead of just focusing on what could go wrong, don’t you also have an obligation to address diseases of high unmet medical need? And if not you, then who is going to transform your organization into a dynamic science-driven one that you can identify with as a scientist?  On a more practical level, I cannot see how many of you will be with your present employer in 2-3 years if you don’t have the goods to show.  Look at what happened to your peers from AstraZeneca whose last job it was to look for partnership in the more innovative pure-play RNA Therapeutics space before they were given the boot. 

Progress in Predicting the Immune Response to an RNAi Therapeutics

When in 2010 Tekmira decided to prematurely terminate its first clinical RNAi Therapeutics candidate, TKM-ApoB, due to unexpected immune stimulation in a phase I, it was disappointing in a number of ways.  Not only did it destroy TKM-ApoB right there, it also cast doubts on how predictive our preclinical immune stimulation tests really were and whether as a result, other candidates may suffer similar fates.  Apparently, using rodents or even non-human primates for that purpose had not been all that useful either. It is also said that the event served as a red flag to some regulators which wanted the technology being developed more slowly.

Tekmira, however, soon claimed to have discovered the reason why their PBMC-based assay did not pick up on the immunostimulatory potential of TKM-ApoB and reported that they had have found a more predictive test tube assay instead.  Apparently, using heparin during the preparation of PBMCs (sample enriched for certain immune cells from blood) was the culprit and a whole blood-based assay (including things like red cells and albumin) would reflect much more reliably the situation in people.

A nice paper by Coch et al. from the Hartmann group in Bonn, Germany, now provides comprehensive insight into possible causes for false positives and false negatives when using in vitro immune stimulation assays.

Role of serum proteins  

It is known that phosphorothioate oligonucleotides, a particularly widely used chemistry in the antisense field, rapidly bind to proteins in serum.  Given its abundance and sticky nature itself, albumin is thought to be an important target of such oligos.  The group thus found that their binding in whole blood sequestered them from the immune cells such that the immune response was significantly lessened.  By contrast, in the PBMC-based assay, these phosphorothioates stimulated a robust immune response.

This insight is obviously particularly important if the interest was in developing oligonucleotides that are immunostimulatory on purpose, especially for cancer and viral applications (false positive in PBMC).  Whether the finding that in whole blood assay phosphorothioate oligos may not exhibit immune stimulation can be used to conclude that they will be safe is another question as the blood-based assays may not be informative on what is going on in the tissues.   

Role of anticoagulants

Closer to the TKM-ApoB story, another important insight was that the nature of the anticoagulant can have a big impact on the outcome of the assay.  When drawing blood, and especially when employing a whole blood assay, it is important to use an anticoagulant as otherwise standing blood would start to clot. 

EDTA and heparin are standard reagents for this purpose.  EDTA has been recognized that it can distort immune stimulation assays and thus is not used.  The surprise (well not that surprising in hindsight) is that heparin, due to its negative charge, could displace oligos such as RNAi triggers from their delivery vehicles, including liposomal nanoparticles (LNPs).  It is often the delivery agents that bring the oligonucleotides to the immune receptors.  Moreover, nanoparticle formulations such as LNPs can facilitate multivalent interactions as they hold together and present a number of oligonucleotides simultaneously.  This in turn can potentiate the immune signaling.

Accordingly, what probably caused the immunostimulation of TKM-ApoB to be missed is that heparin at some point displaced the ApoB siRNA from the liposomes thus destroying the potentiator effect of SNALPs.  Importantly, replacing heparin with hirudin (think leeches) did not suffer from the same limitation.  Therefore, human whole blood assays with hirudin as the anticoagulants appear to be the simplest and most reliable preclinical innate immune stimulation assay for Oligonucleotide Therapeutics development.

Growing the database

Of course, these insights are only a first, albeit critical step.  The next step is to understand what degree of immune stimulation in the test tube relates to a likely adverse event in the clinic.  For this, it is necessary to go back and forth between the test tube and clinical observations and correlate the clinical phenotype (cytokine production, fever/chill symptoms) with the test tube response. Complicating matters, different persons have individual innate immunostimulatory sensitivities, and depending on the state of the immune system (e.g. existing infection) there will also be intra-patient variability.

   

On a more positive note, not all delivery/RNAi trigger formats are equally prone to stimulating the immune system with LNPs probably being one of the more challenged formats with regard to innate immune stimulation.  Even so, the clinical track record with SNALPs after TKM-ApoB (lowered doses of steroids with ALN-TTR02, no steroids apparently used in the TKM-EBOLA trial) suggests that the new assays are having a positive impact already.

Sarepta Ditches Dystrophin Assay Getting Closest to Being Quantitative

I am aware that my interpretations of Sarepta’s exon-skipping data for Duchenne Muscular Dystrophy are not universally embraced.  But one point that I believe all of us can agree on is that it would be important to determine whether the antisense oligo eteplirsen can restore sufficient Becker-type dystrophin to have a therapeutic effect.  Although falling short of a controlled clinical outcome study, one way to gain accelerated approval for this devastating disease would be to first establish what this ‘magic’ level is through historical outcomes studies and then compare this value to the levels actually restored following drug treatment. 

Western blot not worth the film

But when you listened to last week’s presentation by Sarepta CEO Chris Garabedian (link to transcript), the company appears to have dropped the idea of demonstrating such altogether by declaring dystrophin Western blots as unreliable.  I agree that Western blots do not lend themselves to quantitation.  Moreover, I have sharply criticized a number of technical aspects with the eteplirsen Western blot: spliced gels, uneven loading controls, cropped band of unknown identity shown.  

The latter point is particularly bothersome as any molecular biologist would know that even two supposedly identically processed protein samples will show numerous bands that are present in just one of the samples if only you expose the Westerns for long enough.  Ergo, the particular Western blot does not live up to being even merely ‘supportive’ as the company had claimed until recently because it now seems that even the company is only guessing that the cropped band shown is the one based on predicted size (note: proteins do not even migrate on Western gels according to known size...charge etc).

All this begs the question of why Sarepta never developed a reliable quantitative assay (e.g. an ELISA) before entering clinical development and making the case for biomarker-based accelerated approval.

Counting dystropin-positive fibers by immunofluorescence does not address magic number

This alas leaves us with the immunofluorescence (IF) assay data.  Sarepta and their collaborator have counted and presented numbers, so this surely must mean that the IF data are ‘quantitative’?

Wrong.  

As I keep pressing the point, the IF assay (at best) can only determine if there was a increase in dystrophin.  Manipulate the exposure times and picture contrasts enough and you can magnify any difference in dystrophin levels out of proportion. Again, this is no revelation to the molecular biologists and hobby photographers here, and I won’t even start to discuss the issues arising out of comparing IF numbers from experiments conducted at different dates and from different muscles.

By way of example, say you obtain an average signal level of 1.5 due to naturally occurring alternative splicing noise when measuring background, with the vast majority of fibers being below 2.0 and with some rare revertant fibers exceeding that value manifold.  Then you treat with eteplirsen and you find that the signal level increased to between 2.2 and 2.8 in 70% of fibers.  You then set the background arbitrarily to be 2.0, subtract the few revertant fibers, et voila, you determine that 65-70% of fibers have started to express dystrophin following drug treatment although the absolute increase in dystrophin produced is well below what we would predict to be therapeutic: from 1.5 to about 2.5. 

    

Leaving it open what that ‘magic number’ actually refers to

Consequently, IF is ill suited to address the issue of the ‘magic number’ as Mr Garabedian called it.  It would have been incredibly helpful for him to have elaborated what he actually meant with the ‘magic number’.  Does he really believe that counting fibers allows you to get at this issue, as he implied in his answer*, or would he agree that it should be with respect to the absolute amount of dystrophin?  I hope the company will clarify that point almost as quickly as they declared the image duplication event an honest mistake.

What do you think?  Should the ‘magic number’ refer to counting dystrophin-positive fibers or quantitating the absolute amount of dystrophin?  Participate on the poll on the right hand top.

* Making a circular argument, the CEO said that ‘yes’ they know they have surpassed the magic number because of the claimed 6MWT improvements.  Note that in making the case for a biomarker like dystrophin, it should be the amount of biomarker that supports functional outcomes like 6MWT e.g. through some quantitative correlation- not the other way around. 

Marina Biotech to Stage Comeback

Arcturus Therapeutics Acquires usiRNA Technology from Marina

Today, Marina Biotech announced that it is assigning, i.e. is selling usiRNA technology to San Diego RNAi Therapeutics start-up Arcturus Therapeutics.   In addition, the company detailed recent progress in its patent estate encompassing various RNAi trigger- and delivery-related technologies.  Based on these accomplishments, and after extending the maturity date of a critical loan to 2014, it now wants to resurrect itself as a real biotech company by raising capital and relocating to, you guessed it, Cambridge, Mass.  

The usiRNA modification arguably allows you to circumvent the Tuschl siRNA structure and related patent estate by Alnylam and is claimed to even improve upon the Tuschls by allowing for greater specificity.   In addition to Arcturus and Marina, Tekmira, and likely Arrowhead Research (through Roche), have access to such RNAi triggers.

Financial details of the transaction were not disclosed.  Through the assignment of the patents, Arcturus stands to collect the milestone and royalty streams from Marina’s existing licensees.  This includes Tekmira which in its quarterly filing disclosed this week that it would have to pay $3,250,000 in milestones plus single-digit royalties for the technology.  Although these numbers pale in comparison to what Alnylam has asked for RNAi trigger licenses in the past, this suggests that the undisclosed financials and likely upfront payment by Arcturus should have been substantial for a company like Marina…at least enough to continue to pay its patent prosecution and other bills. 

How Arcturus paid for it is unclear to me given that its first significant capital raise involved merely $1.3M.   

For Arcturus the deal seems to make a lot of sense.  If the usiRNA workaround theory holds up, you would be very hard pressed to find such inexpensive RNAi trigger IP (because from a distressed seller) allowing you to broadly go after your targets of choice.  Obviously, this is to complement the liposomal delivery technology on which it was founded.  With the right financial support and scientific talent, Arcturus will be an interesting story to follow.

Why Appointing a Chief Medical Officer Early On could be a Good Thing

Platform-based biotechnology companies, especially those in the early stages, are often criticized that they are about technologies in search of a medical application.  RNAi Therapeutics has also been the subject of such criticism.  

Think for example of an RNAi Therapeutic going after run-of-the-mill hypercholesterolemia with an intravenously infused formulation because this is what the technology allows you to do.  Obviously, you might end up with a marketing approval, but no physician is going to prescribe it. Tekmira’s first clinical RNAi candidate, TKM-ApoB, may be thought of in this way, especially since it had been conceived long before homozygous FH and the like entered the public mind. 

As we are in the middle of the perfect storm for ultra-orphan drug development (genetic, regulatory and reimbursement-wise), riches can be created almost instantaneously in RNAi Therapeutics by just finding the appropriate severe ultra-orphan, genetically-defined indication to match your technology.  This should not be lost on the Tekmiras, Silences, and Arrowheads which all have promised that they would come out with their next development candidate in due course (I am waiting...).

As an investor, I worry whether they put adequate resources into this task.  I have yet to see job ads looking for full-time medical geneticists that do nothing but hunt for and evaluate potential development candidates.  In addition to genetics, such a person should understand the capabilities and limitations of the (delivery) technology of the company, be able to think of how best to navigate the regulatory maze, and ultimately have an inkling of whether physicians and patients would welcome such imaginary drug candidates.

My impression has been that RNAi Therapeutics companies, except for the cashed-up, chest-pounding 800-pound gorilla Alnylam, have done too little in that regard.  I therefore hope that the appointment of a Chief Medical Officer (CMO) by Tekmira announced yesterday was not done merely to help sketch out and execute the clinical development of its lead clinical candidate, TKM-PLK1, but also to provide critical input into the early pipeline.

Of course, given the enormity of genetics and the proliferation of consultancies catering to the orphan drug revolution, you also would want to tap into outside knowledge for added inspiration and critical feedback.  Considering, however, the importance of drug candidate selection, RNAi Therapeutics companies, especially those with around 30 employees and more, would be remiss not to have internal staff with an understanding of medical practice entirely focused on this task.

Tekmira and Alnylam in Binding Arbitration Over $5M ALN-VSP Milestone Payment

The Alnylam-Tekmira dispute may not be over yet.  After de facto losing in the trade secret litigation over SNALP delivery technology, $3B heavy weight Alnylam seems intent on making life for Tekmira difficult and earn the milestones it is entitled to according to the settlement the hard way.

At least this is how I interpret the disclosure in the 10-Q quarterly report filed today with the SEC by Alnylam where the company discloses that it has entered into binding arbitration with Tekmira over a $5M milestone payment related to liver cancer candidate ALN-VSP02:

‘In addition, we recently initiated binding arbitration proceedings to resolve a disagreement with Tekmira regarding the achievement by Tekmira of a $5.0 million milestone payment under our cross-license agreement relating to the manufacture of ALN-VSP clinical trial material for use in China.’

As a reminder, Tekmira was to earn the milestone for enabling Alnylam’s ALN-VSP partner Ascletis to enter clinical development in China. 

I do not wish to speculate on the specifics of the dispute, but it is worth remembering that Alnylam is heavily dependent on rapid clinical development of SNALP-enabled ALN-TTR02 to support its valuation, especially since the GalNAc platform is showing additional blemishes (ALN-AT3 delay; admission in the conference call of certain side effects seen with ALN-TTRsc).

Dicerna in Record $60M Fund Raising for a Privately Held RNAi Therapeutics Company

Although we are still only in the middle innings of the RNAi Therapeutics resurgence according to my Mayan RNAi investment calendar (late 2011-late 2014), the investment appetite in the space shows no signs of letting up.  With over $400M raised since 2012, privately held Dicerna today announces a record $60M venture capital investment.  This follows an $18M VC investment in RNAiTherapeutics start-up Solstice Biologics and confirms that VCs, a group for which RNAi Therapeutics had become untouchable following the Roche pull-back, have now fairly good visibility regarding RNAi Therapeutics drug development and Big Pharma interest in the space.

Persistence paying off

Although the valuations of the fund raising are unclear, given the recent, in many ways similar private offering by Arrowhead Research, those investors holding through the dark days and who are adding capital now are standing to be rewarded handsomely for their persistence.  In fact, all five existing investors (Abingworth, Domain Associates, Oxford Bioscience Partners of Sirna Therapeutics fame, Skyline Ventures, and GSK VC arm SR One) have participated in this Series C.  It is their investments plus a few milestone payments from Japanese partner Kyowa Hakko that have kept the company alive.   

Oversubscribed offering

It is important to note that this capital raise was oversubscribed.  Remarkably, it was led by RA Capital which featured prominently in the Arrowhead Research ~36M secondary.  It thus seems that aside from Alnylam, the size of the other RNAi Therapeutics players and their offerings are not large enough (yet) to satisfy the investment appetite of some funds.  Witness also the rapid increase in the share price of Arrowhead Research (almost a double over the last month) which strongly suggest that at least one good-sized fund is gobbling up shares on the open market.

There are a number of factors driving this interest.  Firstly, it is the clinical gene knockdown results by Alnylam, utilizing Tekmira’s SNALP delivery technology, which de-risked the once ridiculed RNAi Therapeutics approach for many observers.  The ~$3B market cap by Alnylam also represents a juicy target for those that believe 20-30x differences in valuations grossly undervalues the likes of Arrowhead Research, Tekmira, and Silence Therapeutics.

For those companies that have technology ready for prime time and are thus only capital constraint in terms of expansion, the lowered cost of capital makes them even better investments.

Of course, this is happening within the context of a wider boom in biotechnology investments, orphan drug development, and an environment where small caps are outperforming large caps on the public markets.  However, it is my strong opinion, supported by the fact that RNAi Therapeutics share prices have lagged the general market recovery, that none of that would have helped without the clinical results by Alnylam.

Dicerna, just a cancer company?

Dicerna stated that the proceeds would be used to bring at least two RNAi Therapeutics candidates into the clinic starting in 2014.  An interesting question will be whether Dicerna will exclusively focus on oncology or whether it will also embrace severe orphan diseases which are so attractive for a genetic platform like RNAi Therapeutics.  Based on their work in delivery, liposomal nanoparticles for cancer delivery, it would surprise me, however, if the first two clinical candidates were not in oncology.

In any case, congrats to the fund raising, and who knows, maybe we will see a Dicerna IPO in the not-too-distant future.