Friday, December 21, 2012

Viruses, Beware! This time, RNAi Therapeutics Mean Business


Viral infections have long been thought of as an attractive therapeutic area for RNAi Therapeutics.  Unfortunately, with the exception of Tekmira’s Ebola biodefense effort funded by the US Department of Defense, this area has had trouble taking off: Nucleonic’s ddRNAi-based HBV program should never have gone into the clinic (and as expected was soon terminated thereafter), and there is considerable concern that the main mechanism of action of Alnylam’s ALN-RSV01 for respiratory viral infection is due to innate immune stimulation of the unmodified RNAi trigger, not RNAi-mediated gene knockdown.

As RNAi Therapeutics as a whole has turned the corner in 2012, so has antiviral RNAi Therapeutics. 

This assessment is based on two quality programs that have either made it into the clinic recently, Calimmune’s ddRNAi candidate for HIV (LVsh5/C46), or is close to it (Arrowhead’s DPC-delivered anti-HBV candidate ARC520 for which an IND is planned in Q2 2013).  In addition, there is expectation that Tekmira’s Ebola program will be able to take advantage of the significant improvements in SNALP delivery technology, thereby considerably increasing the odds for an FDA approval under the Animal Rule (note the recentapproval of a second drug under this rule).


Suppressing Immune Suppression

Antiviral RNAi Therapeutics have to overcome the important theoretical limitation that even a potent, e.g. 99% knockdown of a viral transcript or particles may not be sufficient as in theory a single infected cell may fuel viral rebound.  It turns out that rather than blindly aiming at knockdown potency, RNAi Therapeutics are likely to be more successful when targeting an important mechanism employed by virtually all viruses: avoiding detection or removal by the immune system.

In the case of HBV, a disease affecting North of 200 million patients worldwide, the virus produces large amounts of the Hepatitis B Surface antigen (HBsAg).  This is thought to suppress, by acting as a decoy, the development of a productive anti-HBsAg immune reponse.   It is thus widely believed in the industry that reducing HBsAg is required to finally generate a drug that can achieve a functional cure, essentially paralleling the recent developments in HCV.  Interferon-based treatment regimens may actually partially work via this mechanism, but cure rates are rather low and come with considerable side effects in the form of severe flu-like symptoms.  Moreover, protein-targeting anti-HBV agents such as small molecule-based polymerase inhibitors do not seem to reduce HBsAg.  This leaves RNAi Therapeutics as the most promising mechanism of action.

A recent article in PLOS Pathogen suggests that the Ebola virus similarly churns out decoy viral proteins so as to subvert the immune system into making antibody duds that do not effectively remove the real viral particles.  It is therefore intriguing that an Ebola drug candidate by Tekmira should not only aim at providing the immune system with more time, but also that it would facilitate it mount a more effective antibody response.


No Escape

Another attraction of the RNAi Therapeutics approach for viral diseases is the fact that such agents may be more successful in prohibiting the virus to mutate around the drug and thereby escape its actions (viral escape).  Consequently, all antiviral RNAi trigger selection strategies focus on sites that are conserved in the various genotypes and quasispecies.  Even if the virus is successful at mutating around conserved sites, it is then relatively simple to include a second (such as in Tekmira’s Ebola program) or third RNAi trigger targeting a conserved site such that the virus would have to mutate around two sites at the same time- a highly unlikely event.

In addition to these general antiviral mechanisms, RNAi Therapeutics may also work through more virus-specific mechanisms.  Calimmune’s ddRNAi-based HIV candidate LVsh5/C46 for example down-regulates the cellular receptor for viral entry, CCR5, such that HIV particles cannot enter cells and integrate into their genomes in the first place.  As an ddRNAi gene therapy approach, LVsh5/C46 further takes advantage of the fact that you can express a therapeutic protein along with the RNAi trigger, thus uniquely combining mechanisms of actions in a single drug.    


Smooth Sailing Ahead

Of course, it is impossible to tell whether an RNAi Therapeutic will actually overcome a virus in each case and receive regulatory approval.  Nevertheless, I believe that the above candidates for Ebola, HBV, and HIV stand a real chance. 

The Ebola program by Tekmira is arguably the most advanced, and it is difficult for me to see how based on the non-human primate data and the lower dosages required for SNALP delivery, which should widen the therapeutic window, approval can be denied under the Animal Rule.

For the HBV and HIV candidates that are being developed along more conventional regulatory pathways, I  am similarly optimistic that they will generate some excitement in the near-term.  This is because viral load is a powerful biomarker, often also an approvable endpoint, and even early clinical studies should be able to generate such outcome data (if Arrowhead could help it, they should go straight into patients with ARC520). 

After orphan diseases involving the liver and oncology, antiviral applications are therefore poised to become the third major support of the RNAi Therapeutics platform.

Thursday, December 13, 2012

Arrowhead and Alnylam Vying for Subcutaneous RNAi Delivery Success


The use of the intravenous route of administration for the currently leading systemic RNAi delivery technology, Tekmira’s SNALP technology, has been noted to be a drawback of the technology, especially for non-severe diseases and in therapeutic areas historically dominated by oral medicines (e.g. the cholesterol-lowering market).  As a result, the arrival of two delivery approaches that promise to allow for subcutaneous administration has been welcomed: Arrowhead’s Dynamic Polyconjugates (DPCs) and Alnylam GalNAc-siRNA conjugates which have shown data suggesting their clinical use for gene knockdown in the liver (at least initially; DPC with potential to go beyond the liver).  

A day ahead of Alnylam’s Roundtable on conjugate delivery, I thought it would be a good time to get into the mood and compare the two competing technologies.


Basic Chemistries

GalNAc-siRNAs consist of siRNAs to which a cluster of three N-acetylgalactosamine residues have been appended.  It is these GalNAcs that are recognized by the ASGPR receptor protein that is abundantly presented on hepatocytes.  The choice of three over just one or two GalNAcs is due to the synergistic binding of multiple GalNAcs to the receptor.

DPCs also comprise of siRNA conjugates, but involve an additional endosomolytic agent to facilitate siRNA release from the endosomes.  The two components can be mixed together so that the drug can be given as a single formulation.  This, however, also requires that both siRNA and endosomolytic agent end up in the same place.  For hepatocytes, this is achieved by conjugating the siRNA to a cholesterol moiety and the endosomolytic agent to GalNAc.  

The reason why two different targeting agents are employed are two-fold: reduced competition for the uptake receptor, and not requiring triantennal GalNAcs such as in Alnylam's case which seems to involve a quite costly chemistry. The reason why GalNAcs on the endosomolytic agent in DPCs are not so expensive is because as a polymer (a peptide in the latest versions) multiple mono-GalNAcs can be conjugated distributively and still achieve the same synergistic binding effect.


Potency and Safety

The Holy Grail in RNAi subcutaneous delivery appears to be to get formulations potent enough so that the desired level of knockdown can be achieved with volumes of 1ml or less: you can squeeze only that much liquid under your skin through a thin needle.

The first of Alnylam’s GalNAcs, ALN-TTRsc, achieves a 80% target gene knockdown (ED80) following repeat administration in preclinical animal studies.  This is below the (based on OTS 2012) 3mg/kg barrier that apparently would allow for 1ml or less volumes in humans.  What surprised me to see at the OTS meeting in late October was that the GalNAc potencies, both in rodents and non-human primates, varied quite a bit between the programs.  The TTR formulation actually had the poorest potency among the programs.  This surprised me even more so given that ALN-TTR01 and ALN-TTR02 (both SNALP programs) contained highly potent RNAi triggers.  In the case of PCSK9, ED50 of less than 0.1mg/kg were obtained.

It is possible that the differences are not just due to the natural sequence-specific differences in RNAi potency, but a result of advances in chemistry.  In particular, optimizing siRNA-conjugates for tissue/endosomal stability rather than serum stability as is often practiced in RNAi Therapeutics is critical.  Importantly, this consideration also applies to DPC technology.      

DPCs should be more potent than isolated GalNAc siRNAs.  This is because you are adding an endosomal release agent to the liver-targeted siRNA (e.g. GalNAc-siRNA) conjugate and unfacilitated release of nucleic acid out of endosomes is believed to be highly inefficient.

Arrowhead has reported various impressive potencies such as 99% knockdowns at sub-1mg/kg siRNA doses.  This to me is strong evidence of the superior potency of DPCs over GalNAc-siRNAs.  Moreover, it seems that DPCs may inherently require less frequent dosing compared to GalNAc-siRNAs for which Alnylam aims at weekly or twice monthly dosing.

What is unclear, however, is the amount and resulting safety and volume implications of the endosomal release agent.  In particular, the most impressive knockdown data seemed to involve saturating amounts of endosomal release agent (~6mg/kg).  The first-generation endosomal release agent, PBAVE, suffered from relatively high toxicity, partly as a result of premature unmasking in the blood.  It makes sense that the newer, ‘more natural’ peptide-based endosomolytic release agents are safer.  By contrast, assuming that the GalNAc sugar itself is harmless, I am not too concerned about the safety of Alnylam’s GalNAc conjugates.

In terms of potency, advantage Arrowhead, in terms of safety, advantage Alnylam.


Strategic Considerations

The challenge for Arrowhead will be to make the case of the benefit of increased complexity over GalNAc-siRNAs. Would the prospect of a 3- or 5-fold increase in potency e.g. be enough justification for the investment?  I say ‘prospect’ because Alnylam could obtain knockdown proof-of-concept data at least a year before Arrowhead, especially since Arrowhead is planning to conduct the first study with DPC (ARC-520 for HepB) in healthy volunteers and thus won’t be able to measure viral target knockdown.

In addition to potency, DPC has the important advantage that it may be a more widely applicable RNAi delivery platform.  This alone may tempt others to put some money down on the technology to see where it can go.

Although GalNAc-siRNAs and DPCs are currently clearly competing, there is also scope for them to synergize, especially in the area of oligonucleotide chemistry.  Curiously, Alnylam did seek access to DPCs earlier this year, supposedly for its evaluation in one of its 5x15TM programs.  Learning about DPC siRNA chemistry may be of at least equal, if not considerably more value to Alnylam.

Which of the two delivery technologies do you prefer for target gene knockdown in the liver?  Take the survey on the top right-hand corner.

Monday, December 10, 2012

SNALP Structure Reconsidered


SNALP delivery technology has not only been the subject of a heated fight over ownership and control, but also continued mechanistic, chemistry, and structural investigations.  

In terms of structure, I used to think of SNALP as simple, unilamellar liposomes with RNAi triggers captured in their aqueous interiors.  A patent application by Tekmira (WO2012/000104A1) and a recent paper by the Cullis/AlCana group (Leung et al., 2012) convincingly challenge this view.  Accordingly, SNALP particles are highly electron-dense entities in which pockets of RNAi triggers surrounded by positively charged lipid micelles lipids fill a lipid-enclosed vesicle.  

This new view should guide the future development of the technology, especially the ratio space of its components, targeted delivery approaches, and cytoplasmic release.


Numbers hinting at need for new model

One metric of Tekmira’s results that has always impressed me were its RNAi trigger formulation efficiencies, often well above 90%.  As Leung and colleagues note in their paper, this is at odds with the old simple aqueous encapsulation model which would predict that the likelihoods of an RNAi trigger to be encapsulated or not are roughly equal.  

It now seems that Tekmira’s manufacturing method of rapidly mixing lipids and nucleic acids is at least one critical factor allowing for this remarkable concentration effect.  Other groups largely failed to reproduce such data at least partly because the published method called for the use of costly amounts of reagents.  This is changing, however, due to the use of microfluidic formulation methods as was also practiced by Leung and colleagues. This method likewise allows lipids and nucleic acids to be mixed vigorously and consequently also generates electro-dense structures, but using much smaller volumes.    


Limit-size particles smaller than anticipated

A consequence of the new model is that SNALP LNPs may be as small as 15nm in diameter, whereas previously I subscribed to the view that 40-50nm was the limit.  While a 15nm spherical particles would still have problems in passively getting out of the vasculature in most tissues, it is in a dimension where it might become of interest for additional tissue targets than those that we have assumed to be suitable SNALP targets. 

While it is nice to think that SNALP LNPs has more tissues that it might be able to address, it seems that the more tangible, near-term value of the new insights is in how the particles can be designed to target the existing low-hanging fruit tissues such as liver, sites of inflammation, phagocytes and other cells in the blood/lymph, solid tumors (by systemic delivery), and local applications such as lung epithelia by inhalation.

You could for example imagine that this model makes the post-insertion method of adding a targeting ligand more attractive compared to a co-formulation one (both of which have been considered). Similarly, the model changes the space of lipid and RNAi trigger ratios that should be explored.  It also requires new models for the cytoplasmic release of the RNAi triggers from the endosomes to be considered.


Who was first?

While I do not wish to carry on with pointing out potential tensions between Tekmira, Alnylam, and AlCana, the fact that both Tekmira and the Cullis laboratory (‘AlCana’) came out with essentially the same discovery does not leave me much choice but to briefly comment on the coincidence (note that this clash was set in motion before the settlement).

In the patent application by Tekmira, the priority date is June 30, 2010 2011.  As this is also the international filing date, the invention/discovery must have occurred at least a few months, if not at least a year earlier.  The Leung et al. paper was received by the the Journal of Physical Chemistry approximately 2 1 year later (April 5, 2012).  This suggests that the Tekmira were the first to have these insights.  This, of course, could also be critical for the patent application.  If Tekmira were able to get patent protection for such ‘non-lamellar’ LNPs, this could be an important one and somewhat replace in importance the Semple/Wheeler patent estate which are about to expire over the next years.


The new findings illustrate that SNALP LNP delivery continues to be an area a rapid progress.  One would make a mistake to assume that the MC3 formulations currently in the clinic are as good as it gets and that other somewhat overlapping, and therefore competing approaches such as Arrowhead’s DPCs are about to catch up.  Here’s hoping that with the leading delivery technologies having so much obvious room to mature, 2013 will be at least as exciting if not more so than 2012 for RNAi Therapeutics.

Thursday, November 29, 2012

Arrowhead Research, Focus on RNAi Therapeutics


Arrowhead Research just announced the publication of a scientific paper detailing its fundamentally new DPC delivery approach (discussed here before).  Except for noting that this study is a proof-of-concept one only and, based on recent conference presentations, clinically more relevant formulations have been developed, I am feeling more compelled to talk about my thoughts on Arrowhead’s corporate strategy than to give you are run-down of the paper that you can also read yourself here.

A Dubious Past

When I used to think of Arrowhead, a company came to mind that was constantly chasing the latest hot area in science and technology to raise the next round of money, and never pursuing anything in great detail.  And it is great detail that is required if you really want to make a product, especially a drug.  It got worse during the economic downturn when the company claimed to be a broad RNAi platform play and did not even have a lab to show.  As Benitec is demonstrating, not having a lab while acting like a platform play means that you are wasting time while dwindling away cash resources.


The Roche Acquisition- A New Beginning?

Then suddenly, Arrowhead unexpectedly emerged with the Roche RNAi asset scalp in hand.  Overnight, the little company acquired assets from Roche on which the Big Pharma had spent ~$700M. including a vibrant research organization in Madison, Wisconsin.  Although this finally got the company that missing lab, it also seemed a huge financial gamble at a time when RNAi Therapeutics sentiment was at its bleakest (late 2011, a month before the ALN-TTR01 presentation).  How would such a small cap biotech company fund the enlarged operation?  Yes, you can buy castles in France for a token Euro, but can you actually afford to live in them?

That bet likely depended on the outcome of two issues: 1) RNAi Therapeutics sentiment had to turn around in time so as to provide upward pressure to the valuations of RNAi Therapeutics companies.  This would help Arrowhead Research raise the needed capital in a shareholder-friendly manner; 2) The DPC delivery platform needed to show signs that it was finally moving into the clinic.

The first bet worked out as we know.  RNAi Therapeutics sentiment improved dramatically over the last 12 months, albeit from a very low base.  Unfortunately, Arrowhead Research happens to be the worst performing publicly traded RNAi Therapeutics company over that period.  The second bet actually also worked out as the recently reported data on decent knockdown efficacy in non-human primates at acceptable safety strongly suggest.  Moreover, the latest advances in DPC subcutaneous delivery provide the technology with added value based on technical differentiation.  Without having maintained the research operation, we would not have seen such progress.      

When Arrowhead made the acquisition, I thought it was too bold and that it would destroy most shareholder value due to dilution.  Although the stock has kept decreasing and new shares were issued, it probably has not fully destroyed it (yet)- so I have to give at least some credit to Arrowhead’s management.


What the H…? Arrowhead Buys a Peptide Library

Turns out though that the boundary between what is a visionary or a delusional company can be a blurry one.  There is reason to believe for this to be the case here after Arrowhead decided to buy the peptide targeting library from Alvos.  As peptide drug conjugates and DPCs are both targeted technologies, the company is pursuing a strategy as a Grand Targeted Therapeutics company dominating all areas of targeted therapeutics.  It also claims that the peptides and DPCs can be used synergistically.  For various reasons, forcing the Alvos peptides on the DPCs does not make sense for the company- trust me.

I agree that targeted therapeutics is the future of the industry and it is a nice vision for an ambitious company with a strong cash flow and $3B on the balance sheet.  But even if this were the case, I doubt that a realistic Board of Directors would approve such a strategy.  The reality is that getting DPC delivery to work alone is more than enough on Arrowhead's plate.  Licensing out the peptide library would just be peanuts and consume much of the energy required for more worthwhile business development efforts.

Worse, the acquisition of the peptide library makes Arrowhead look like the old technology fashion-chasing company.  This impression is further strengthened by the emphasis of the peptide library over RNAi Therapeutics in the latest investor presentations. Biotech investors have learned to avoid such increasingly rare traps. 

Would you invest in Arrowhead Research?  Take part in the survey in the top right-hand corner.

Monday, November 26, 2012

Tuschl Patents Stage Remarkable Comeback in US


[please note the changes below after I discovered that I missed the last claim amendments, as correctly pointed out by a commenter]

Alnylam announced today that the US patent office had issued 3 Notices of Allowance in recent weeks pertaining to the Tuschl patent estate.  Before your eyes glace over, read on, it's actually one of the few important patent news.  

The granted composition-of-matter claims cover very broadly 19-25bp RNAi triggers, independent of structure.  This is a stark departure from the trend that had materialized in the Tuschl patent applications where Tuschl I seemed to get relegated to treating diseases of fly lysates and Tuschl II suffering from double-patenting issues over Tuschl I.

Claim 1 of allowed patent application 12/537602:

1. Isolated double-stranded RNA molecule, wherein each RNA strand has a length from 19-25 nucleotides, wherein said RNA molecule is capable of target-specific nucleic acid modifications.


1.  An isolated double-stranded RNA molecule, which is a non-enzymatically processed RNA molecule, wherein: (i) each RNA strand independently consists of 19-25 nucleotides in length, and (ii) at least one RNA strand forms a single-stranded 3'-overhang from 1 to 5 nucleotides, wherein said RNA molecule is capable of target-specific RNA interference.

The reason why such a comeback was possible can be traced back to the settlement over the Tuschl patents 'with Merck' in early 2011.  This allowed the Tuschl I and II prosecutions to be aligned such that the double-patenting issue for the 3' overhang claim could be overcome such that the broadness of the Tuschl I RNAi triggers could be rescued into the valuable human therapeutic uses. 

Among those without RNAi trigger licenses from Alnylam, Silence Therapeutics (and its partners, especially Quark) should be hit particularly hard by the latest development: 19bp RNAi triggers are no place to hide any more.   

There are thus two monsters of RNAi trigger patents that consequently co-exist in the commercially very important US market: Baulcombe for almost all types of therapeutically useful double-strand RNA lengths (20-24/30bp; see here) and Tuschl for the desirable 3' overhang feature of RNAi triggersIt is unclear to me how two patents can essentially claim the same.  Either the patent office is aware of this and is satisfied with ultra-fine semantics distinguishing the two, or this is an issue worth reconsidering.

Another indication that the last word might not have been spoken may be the multiple requests by Alnylam (i.e. their law firm) to expunge certain materials that were rendered during the Tuschl patent prosecutions (possibly relating to timing and nature of the claimed invention), but are deemed trade secrets by Alnylam (can’t help but smile, here). Has Utah taken notes in time?

Sunday, November 25, 2012

RNAi and Antisense Targeting the Same Gene: Not a Zero-Sum Game


Transthyretin-mediated amyloidosis has become the single-most important factor for the 3-5 year valuation of certainly Alnylam, and possibly ISIS Pharmaceuticals as well. After TTR, Z-alpha-1-antitrypsin (Z-AAT) is gearing up as the next Alnylam(RNAi)-ISIS(antisense) battleground. Expect to hear competitive language from the two camps why their approach will prove superior over the other.  It is, however, worth keeping in mind that having two candidates race towards approval can also have significant pie-enlarging benefits.


Raising Awareness and Pressuring Regulators

A good competition will attract attention, from patient groups, physicians, regulators, and the investor community.  Patient groups and physicians will be primed that after years of lacking treatment options, the time for change has come.  We have seen this with the trifecta ofweight-loss drug candidates, two of which have recently been virtually willed into approval.  Given the enormous problem of obesity, it became politically untenable to further withhold treatment options.  Eventually, the regulators were forced to give in to the pressure despite their original objections over safety.

Closer to home, similar awareness and pressure is being brought to bear on getting exon-skipping oligonucleotides approved for the treatment of Duchenne Muscular Dystrophy, stoked by the competition between Prosensa/GSK and Sarepta .  If you caution about hurried, aka accelerated approval, then you are readily labeled as a heartless misanthrope.  Similarly, the homozygous familial hypercholesterolemia population has already been educated, targeted, and friended by both patient-access specialist Genzyme and competitor Aegerion and probably strongly expect the new drug approvals.  

Understand that these comments are not meant to be judgments on the respective drug candidates, but just the way I view current dynamics.  In fact, I agree that developing drugs for many of the rare and severe (orphan) diseases requires more risk-taking, usually biomarker-based approaches, and that not too long ago patient needs were not sufficiently considered in a highly risk-averse regulatory climate.



The TTR Amyloidosis Race

Given that Pfizer’s drug candidate tafamidis does not appear about to revolutionize the treatment of TTR amyloidosis, I see similar pressure building for the approvals of gene knockdown approaches ALN-TTR02 and ISIS-TTRRx in 2015-6.  The intense competition between ISIS and Alnylam will provide good fodder for the press (just as it does for this blog), engage the minds and pad the pockets of key opinion leaders and consequently raise the expectations of the public and hopes of those suffering from the disease.

While I have 2015-6 approvals baked into my projections of the RNAi Therapeutics story, I wouldn’t mind a year’s delay if the competitive dynamics would allow for it.  In fact, I expect the FDA to manipulate the process in a way that both drug candidates will come before an AdCom panel together, making the obsession with speed over good science even more so nonsensical.

My problem with ISIS and GSK speeding straight into a phase III study after a single phase I study is that they do not seem to have identified a suitable dosing regimen: 400mg/week good efficacy (-81% knockdown), but probably too high a dose from what we know about the safety of phosphorothioate oligonucleotides; 200mg/week could fare better in terms of safety, but has shown only modest knockdown efficacy (-44% knockdown).  In my opinion, another dose-finding study would be in order under normal circumstances.

[Update 29Nov2012: The Clinicaltrials.gov entry for the upcoming phaseII/III ISIS-TTRRx trial shows that the companies have chosen the stab-in-the-dark 300mg/week dose.  Although the January 2012 phase I results press release made no mention of such a cohort, the June 2012 Annual Shareholder Meeting TTR poster shows data from a 300mg cohort (~70% knockdown).  Unlike all the other cohorts were 90+ day data, the 300mg cohort data was less than 50 days.  It thus looks like a 300mg cohort was included post-hoc. Unfortunately, ISIS has chosen not to list the phase I trial on clinicaltrials.gov.]

This concern over uncertain dosing schedules also applies to Alnylam's ALN-TTR02 (-87% peak knockdown, -67% knockdown after 28 days following single dose) where the current ‘multi-dose’ phase II study only seems to cover two doses.  You would hope that Alnylam presses hard to amend the study to include further doses to gain more experience with the repeat-dosing pharmacology of ALN-TTR02 before betting their house on a single pivotal phase III trial.

Once approvals are obtained, however, the beneficial aspects of competition should disappear quickly especially when small orphan disease populations are involved.  But until then, the race towards approval should free energies allowing TTR amyloidosis to become the first real commercial success story of either RNAi Therapeutics or RNaseH antisense.

Who do you think will win the TTR race? Take part in the survey (upper right hand corner).   

Tuesday, November 20, 2012

Demystifying ddRNAi Trigger Design


A somewhat vexing problem with DNA-directed RNAi (ddRNAi) has been the heterogeneous mixture of small RNAs generated from a typical expression vector.  Not only will this compromise knockdown efficacy through competition for RISC loading, it also poses a safety risk by increasing the number of off-target genes.  For a long time, heterogeneous ddRNAi trigger processing had been accepted as a way of life- as long as the efficacy was right you would not waste too many thoughts on those multiple bands on your Northern blot.  

Choosing the double-strand length of a ddRNAi trigger has also been more art than science.  Different sizes, usually between 19 and 29bp facilitate potent gene knockdown, but there have been few studies looking at the consequence of size on the uniformity of hairpin processing.   

Next-generation sequencing technology is changing these attitudes.  In a paper that appeared last week in CELL, Gu et al. from the Kay lab in Stanford showed that next-gen sequencing is a powerful tool to detect which small RNA species are generated from a given ddRNAi template plus their relative quantities.  Moreover, through an iterative process of structural change and sequencing, a key structural feature causing the Dicer enzyme make just one predictable cut could be identified.  Without going into much detail, a simple 21 base-pair hairpin yields the purest results.


Commercial ddRNAi trigger landscape getting rusty  

While the clinical validation of SNALP delivery technology has allowed synthetic RNAi trigger-based Tekmira and Alnylam to turn around their fortunes and should also increase general interest in the technology, there seem to be no commercial players left in the ddRNAi Therapeutics arena able to champion and refine the platform. 

Benitec is pre-occupied with trying to monetize IP that is rapidly losing in value due to its age and as a result failing to become a real biotechnology company with a lab.  The two viral delivery companies that once had some ddRNAi ambitions, AMT (AAV) and Oxford Biomedica (lentivirus), either went out of business and/or lack the requisite RNAi molecular biology expertise.  And the other groups developing ddRNAi Therapeutics, including Calimmune and Genables are de facto one-product, disease-focused companies.  This is unfortunate as there should be room for at least one or two ddRNAi Therapeutics platform companies.

Such a company would have expanded on the basic dsRNA concept and would thus have kept its IP estate fresh, eventually forcing others to take a license as simply running out the patent clock would not have been an option.  Such IP would include discoveries like the one made by Gu and colleagues.  On a more positive note, it is possible that the current revival of gene therapy after a decade of neglect and scorn will eventually carry ddRNAi Therapeutics along with it.

Tuesday, November 13, 2012

Tekmira Regains Control over Leading Delivery Technology

When Tekmira filed its lawsuit against Alnylam in March 2011, it found itself in an impossible position: Alnylam’s apparent mis-use of SNALP-related trade secrets not only diminished their strategic value to Tekmira, including depriving it of potential pharmaceutical partnerships, Tekmira may even not have been able to use the technology it invented.  As a result, it might have been left to die in a little cage with ‘1st generation’ technology as its technology was making rapid progress.    


Cash and Control

Based on the disclosed terms of last night’s settlement with Alnylam (Tekmira's version here, Alnylam's version here), Tekmira got essentially all that it needed: control over its leading systemic RNAi Therapeutics delivery technology, including the contested MC3 lipids of which the IP was assigned to Tekmira, and a $65-75M payment (~$16-18M of which will go to pay outstanding legal fees).  The payment, of course, will finally allow the otherwise financially conservative company to push forward their development pipeline with interesting therapeutic candidates such as TKM-PLK1.  Notably, as part of the settlement, Tekmira obtained full ownership over this candidate which I believe addresses one of the most exciting RNAi targets in oncology. According to the Q3 financial results, also released today, TKM-PLK1 has been making good progress, including showing signs of drug activity and will advance into phase II studies next year.

Beyond the cash, the new IP licensing arrangement between the companies is at least as valuable.  Importantly, Tekmira has regained exclusive sub-licensing rights to ‘LNP intellectual property’, an incredibly important part of the settlement as before that Alnylam enjoyed such control, mainly via its rights to the Semple-Wheeler IP covering important aspects of ionizable SNALP technology.  This should open the door for Tekmira to enter into the long-expected pharmaceutical partnerships. Novartis and Takeda are here the most obvious near-term candidates for platform-type relationships.

Assuming that the new relationship is not restricted to RNAi Therapeutics, an added bonus is that the settlement could similarly pave the way for relationships in the microRNA Therapeutics space, particularly for the delivery of microRNA mimics.  Only recently, privately-held Mirna Therapeutics made headlines by raising $34.5M to fund the development of a microRNA mimic into the clinic, indicating the significant interest in this area of drug development.


Value of the Trade Secrets

Less clear is the damage to the trade secrets that were at the center of the dispute and on which much of Tekmira’s strategic advantage rested.  Numerous groups and companies have attempted home-brew liposomal RNAi trigger delivery, but only Tekmira’s technology has made it into the clinic.  Especially the ability to formulate and manufacture the particles is what set Tekmira apart from the competition.  In their press release, however, Alnylam stated their intention to use its own LNP manufacturing capabilities.  Apparently, it has already established such GMP manufacturing capability over the last year (in a separate building apparently for added suspense).  Assuming that Tekmira did not assist them in establishing such manufacturing, this indicates that the number of people with critical knowledge to the trade secrets has grown.  This can be particularly a problem in the wake of lay-offs in a place like biotech world capital Cambridge, Mass. 

Still, I assume there to be significant hurdles for other companies to replicate Tekmira’s processes, and with its liposomal experts, Tekmira should continue to lead in advancing SNALP delivery, making it the liposomal delivery partner of choice.


With cash of around $50M (excluding the $10M which are contingent on likely 2013 development milestones for ALN-VSP02 and ALN-TTR02), partnerships likely to come in due to the clarified IP situation, the $141M DoD Ebola biodefense contract (a new more potent and safer formulation will be used) and the income stream from Talon Therapeutics, Tekmira has thus emerged as the vibrant RNAi Therapeutics company it should have become 3 years ago.  Nevertheless, today's terms underline the value of Tekmira and RNAi delivery in general.  Exciting times ahead.

Tuesday, November 6, 2012

Cautionary Statements in Alnylam's SEC Filing

Today, I would like to post excerpts from Alnylam's most recent quarterly filing with the SEC (10-Q, here), comments that are related to the trade secret litigation between Alnylam and Tekmira.  Since the use of cautionary language is strongly encouraged for public companies to protect themselves from shareholder lawsuits, such filings are a great way to glean some insights into how they think about legal proceedings.

Although Alnylam claims that a loss, including resulting from a settlement, is 'less than probable', they certainly acknowledge the significant potential liability.  For the first time, Alnylam discloses what it considers an appropriate damage estimate- a meager (less than) $4.4M, but which makes me wonder whether the manufacturing accusations have been more or less admitted to (highlights are mine):

        'The Company has not recorded an estimate of the possible loss associated with this legal proceeding. Although the plaintiffs have not specified in their complaint the amount of monetary damages they are seeking, they have served an expert report estimating their damages under a variety of theories, which was subsequently updated. These theories include the purported lost value of the plaintiffs’ trade secrets, calculated using two approaches, as well as the Company’s alleged unjust enrichment resulting from the purported misappropriation of the plaintiffs’ trade secrets, again using multiple approaches. The Company believes that under the applicable law the plaintiffs are not entitled to aggregate multiple theories, but the plaintiffs have argued to the Court that they are entitled to do so. The various theories of plaintiffs’ damages under the plaintiffs’ most recent expert report range from approximately $61 million to $234 million. If the theories of monetary recovery included in the plaintiffs’ most recent expert report were aggregated, it would result in damages within a range of approximately $310 million to $484 million. In addition, under one of the plaintiffs’ claims, any compensatory damage award sum could be subject to doubling or trebling, which could increase the potential damages up to approximately $1.5 billion, and the plaintiffs would also be entitled to recover their reasonable attorneys’ fees. As of June 30, 2012, Tekmira reported a contingent liability to their lead counsel in the amount of $12.5 million, which is likely significantly higher as of the date of this filing. The plaintiffs are also seeking prejudgment interest.
The Company denies any wrongdoing and believes that the plaintiffs’ estimate of their purported compensatory damages is grossly overstated and based on faulty reasoning. The Company has served expert reports supporting the Company’s position that it is not liable on the plaintiffs’ claims, as well as an expert report on damages, which opines that the plaintiffs’ damages estimates are unreliable and, if their expert conducted an appropriate analysis under one of his approaches, damages in the event of an adverse finding on the liability issues would be less than $4.4 million. This amount could be subject to doubling or trebling, and the plaintiffs would also be entitled to recover their reasonable attorneys’ fees and potentially prejudgment interest. However, notwithstanding the Company’s expert report, damages would be much greater if the case is tried to a verdict, and the jury finds the Company liable and also accepts the plaintiffs’ theories of monetary damages discussed above.
As the trial date approaches, the Company is pursuing all reasonable approaches available to it to reach resolution of this matter, including both settlement negotiations and full trial preparation. The Company does not currently believe that a settlement of this legal proceeding is probable, in accordance with ASC 450, Accounting for Contingencies, due to a number of factors; however, the Company intends to continue to evaluate all possibilities for resolution of this matter, including a potential settlement.
Significant judgment is required to determine both the likelihood of a loss and the estimated amount of any such loss. The Company currently believes that the likelihood of a loss is more than remote but less than probable, in accordance with ASC 450, Accounting for Contingencies, and is unable to estimate a reasonable range of loss for this legal proceeding due to many factors.'

It is also of interest that there was a statement on the recent Genzyme deal which strongly indicated that it got done without Tekmira's consent.  Note that Tekmira would stand to receive part of the financials of that deal just as ISIS got a cut, plus Tekmira would have to be involved as regards ALN-TTR02 manufacturing:
'The Company has agreed to indemnify Genzyme for legal costs and other losses or amounts required to be paid by Genzyme, if any, in connection with or related to certain of the Company’s ongoing litigation matters.' 

Finally, Alnylam discusses that it is important to be able to manufacture RNAi Therapeutics candidates themselves, including LNP delivery.  It would not be surprising if building such capabilities had already been a major effort at the company.  Such a move would also support that the two companies rather than merging will go their separate ways after all is said and done:
'Given the limited number of suppliers for our delivery technology and other materials, in the future, we expect to develop our own capabilities to manufacture drug substance, including siRNAs and siRNA conjugates, and/or finished drug product, including LNP formulations, as permitted under our manufacturing agreement with Tekmira, for human clinical use.'


The jury trial is slated to start next week.  Alnylam won't have to face analysts on a conference call for another 3 months, and tonight all the attention will be with the outcome of the US elections.  If there were a settlement, tonight would be the night to announce it.

Monday, November 5, 2012

Bullish on RNAi Therapeutics


Attending the Oligonucleotide Therapeutics Society meeting last week in Boston, I could sense a new bullishness around RNAi Therapeutics like I haven’t in a long time.   


Big Pharma: Tipping the Scales in Favor of RNAi Therapeutics

Over the last year, we have been witness to the clinical successes of Tekmira’s SNALP delivery that literally saved the sector.  After nightmarish years, these results had investors enjoy the doubling and tripling of the share prices of companies like Alnylam, Tekmira, and even non-SNALP players such as Silence Therapeutics.

For the next leg of expanding investments in RNAi Therapeutics, a renewed, publicly visible commitment by Big Pharma would be important.  With Genzyme taking a license to Alnylam’s transthyretin amyloidosis program, a first step in that direction has been just made.  On the platform side, it is worth noting that the two most significant Big Pharma players in RNAi Therapeutics, Merck and Novartis, were also represented at the conference.  One can only hope that the current RNAi Therapeutics clinical and scientific tailwind will give their internal champions the ammunition to push the technology into the clinic in the next 2-3 years and therefore escape the Sword of Damocles that surely must have been felt dangling above them.

Unfortunately, if these two companies, like so many others it seems, big and small, insist on using ‘their’ own delivery technologies, chances for that will be much reduced.  With all due respect, but the presentation on RNAi delivery by Merck was just a review of the most advanced systemic delivery technologies, SNALP (Tekmira), DPCs (Arrowhead Research), and GalNAcs (Alnylam) exemplified by Merck’s homebrew versions.  Somebody needs to show me the math behind it- I just don’t get it.  The only explanation for me is pride and the resistance against collaborating after having invested internally so much.

A re-commitment towards RNAi Therapeutics should also be at the expense of ‘naked’ RNaseH antisense.  Although in his keynote speech, Alnylam’s CEO made a point of congratulating ISIS CEO Stan Crooke (sitting in the first row) on the recent mipomersen Advisory Panel, there was considerable talk during the conference about antisense-related toxicity, also as regards the high-affinity versions.   And even Stan Crooke could not help but admit that the RNAi Therapeutics results have ‘exceeded [his] expectations’.  However, since ISIS claims ownership over RNAi Therapeutics, as it does indeed over most of oligonucleotide therapeutics anyway, his pain should be limited if indeed he believes what he is saying [note: Dr. Crooke in discussing the safety of mipomersen went as far as saying that there has been no imbalance in its safety profile compared to placebo, and when there was an imbalance, it was in favor of mipomersen...for a starkly different view, see here].


A Breath of Fresh Air in Delivery

A conference highlight were the new results from Arrowhead Research on their new DPC delivery, a conjugate approach.  Potent gene knockdowns in the liver with an apparently reassuring safety profile in non-human primates using subcutaneous delivery is certainly deserving of some serious attention.  Having had systemic delivery of synthetic RNAi triggers almost for themselves for the last few years, Tekmira seems to be finally getting some company- although in terms of validation, SNALP is still years ahead and more de-risked.  Such increased diversity of approaches should also be good for attracting general interest to the sector as it would be viewed as more vibrant and with more disease opportunities.


5 Reasons to be Bullish on RNAi Therapeutics

1)      Clinical results show that RNAi Therapeutics in Man can be made to work (Tekmira’s SNALP delivery);
2)      RNAi Therapeutics ideally suited to address orphan disease, the hottest category in drug development;
3)      RNAi Therapeutics has taken the lead over RNaseH antisense for gene knockdown;
4)      Big Pharma coming back to RNAi Therapeutics;
      5)      Vibrancy of sector increasing (e.g. recent results on Arrowhead’s DPC technology). 


A Side Note on the Conference

To some degree reflecting the increasing maturity of Oligonucleotide Therapeutics, the 4-day program did not include important programs in the field.  Not only was Tekmira notably missing, but also efforts such as Dynavax’ important HepB vaccine candidate that could soon allow oligonucleotide ‘therapeutics’ to touch many lives.  Moreover, the industry-academia balance was tilted in favor of industry like I have not seen before.   

Friday, October 26, 2012

The Mipomersen Briefing Docs: Gymnotic Delivery Revealed


After Alnylam’s CEO John Maraganore famously failed to deliver on his Big Pharma platform partnership promise in 2009 (aka ‘SRTL’), and ISIS Pharmaceuticals announced a preferred partnership with GSK in March 2010, it seemed that Dr. Maraganore salesmanship was outdone by one of the best biotech salesmen ever: Dr. Stanley Crooke. 

In addition to likely having deprived Alnylam of a deal, what is this salesmanship claim based on?  It is the fact that not all that long ago, antisense technology had fallen out of favor in Big Pharma.  This was because it required large amounts of oligonucleotides and, partly as a result of that, was associated with non-specific immunostimulatory effects.  And this is where the more elegant RNAi Therapeutics was supposed to take over. Despite this poor track record, the technology managed a comeback, ironically partly on the back of RNAi Therapeutics which was seen stumbling at the ‘delivery’ stage.  In some cases out of the frustration of making RNAi delivery work, the 'naked' delivery approach of antisense would be embraced by the likes of GSK, BiogenIdec, Pfizer, Genzyme, and VCs making the application of Santaris' LNA technology the basis of start-up biotechs targeting emerging classes of non-coding RNAs such as microRNAs or long non-coding RNAs.

The reason, however, why I had always favored RNAi as the more ‘natural’ gene silencing modality over antisense is that antisense, to this day, relies on achieving extremely high steady-state tissue concentrations (>100-300mg/kg of liver and kidney tissue e.g., see ISIS TTR patent application US2011/0294868) either by using reactive/sticky chemistries such as the phosphorotioate backbone or more gentle backbones such as the morpholino chemistry that, however, require even larger amounts of oligonucleotides.  


Mipomersen Briefing Docs Surprises

Unfortunately, the publication last week of the FDA Briefing Document related to the new drug application (NDA) for Mipomersen (tradename: KYNAMRO) by ISIS and partner Genzyme, should serve as a reminder that giving an old technology new names such as ‘gymnotic’ or saying that the algorithms now allow for the discovery of much more potent antisense sequences without any new chemistry (despite the fact that narrow tiling had always been practiced) are telltale signs that the biggest change that has occurred was in the marketing department.

The selective disclosure strategy by ISIS Pharmaceuticals on mipomersen has certainly contributed to the impression that antisense technology had indeed made important progress.  Dr. Crooke claimed in a recent interview on Mad Money with Jim Cramer that ISIS had been very transparent with mipomersen.  I could agree that there has been a lot of scientific data released on mipomersen by ISIS and their clinical investigators, but in hindsight this was arguably not for the claimed sake of transparency, but for providing the impression that mipomersen was a safer drug than it probably is. 

Here are a few examples of the ‘surprises’ that were revealed in the Briefing Docs:  

1) Liver Fat: More and Persistent

While the significance of the non-alcohol-related accumulation of fats in the liver in predisposing towards liver fibrosis is still hotly debated among experts, ISIS tried to dispel the still understandable fears about ApoB-knockdown-related fat accumulation in the liver by claiming a) that it was only modest, and b) that feedback mechanisms allowed the liver to adjust and that liver fat would decrease over time.  Supporting the impression that ISIS was concerned about transparency was the publication (Visser et al. 2010) of liver fat data from a 13-week study in a small, 21-patient study population which only showed a trend of such accumulation (median control-adjusted increase from baseline to day 99 of < 1%).  

Compare that to the actual results (as revealed in the Briefing Docs): In study ISIS301012-CS7 and CS12, 61.8% (63/102) in the mipomersen group had a > 5% increase in hepatic fat content.  This already shows that the median increase is definitely higher than 5%, not < 1%. Moreover, in study CS6, 16% had average liver fat of > 20% measured at least once.  30-40% liver fat contents also were not uncommon.  Such values were practically not observed at baseline or in control subjects despite ISIS’ repeated claims that such values are of no concern since NAFLD is so common anyway.

Furthermore, the data such as capture in Figure 10 of the Briefing Document show that there is no basis for claiming that the liver adapts and liver fat contents come down over time.  They only come down after you discontinue treatment.

Suggesting that the fat accumulations could be of clinical significance, a fibrosis-related biomarker score (ELF) was elevated in the mipomersen population.

To be clear, at least the liver fat accumulations (not sure about  the ELF score) is very likely a target-related side effect and cannot be attributed to the phosphorothioate antisense platform per se, but it is an important issue for mipomersen and raises suspicions about the way ISIS Pharmaceuticals has dealt publicly with platform-related safety issues.


2) Partial Clinical Hold: Vasculitis

Were you surprised by the fact that, according to the Briefing Doc, the FDA issued a Partial Clinical Hold for the non-severe LDL patient population in January 2008?  I certainly was taken by surprise and would also think that this should have been disclosed in an SEC filing as a Partial Clinical Hold for your lead program should be considered a material event.  Not only can shareholders be pitied, but also Genzyme which less than a month before invested more than $300M in mipomersen! I cannot imagine that they would have closed such a deal had it known that there was data that would trigger such a Hold (I do not exclude that the data was there under their nose, but in that case it was probably either buried under a pile of paper, or Genzyme just did not do their job well).

Apparently (à Briefing Doc), the ‘final interim report’ from the 52-week monkey tox study that was submitted in June 2007 showed vasculitis (inflammation of the vasculature which can impair blood supply and therefore organ function, possibly leading to organ failure) at clinically relevant doses of 3mg/kg and up, mainly, but not exclusively, in the gastrointestinal tract. Although the Hold was lifted after the actual clinical safety data did not indicate vasculitis to be an issue, subsequent skin biopsies around the injection sites showed an accumulation of inflammatory cells around the vasculature.

Although similar to the liver fat issue, the clinical significance and degree of systemic vasculitis due to phosphorothioate oligonucleotides remains unknown in Man, the potential for sustained inflammatory processes in the entire vasculature is a serious safety concern that needs to be studied in more detail before the technology can be applied to less than the most severe, 'orphan' patient populations- if at all.


3) Liver Enzyme Increases

That mipomersen increased liver enzymes in the serum, a measure of liver tox, has been widely known.  But ISIS used to claim that in a given individual these were one-off findings that could just as well have come after a night of heavy drinking.  By contrast, it seemed to me new news that there were cases where elevated (>3x ULN) liver enzymes were found on at least two subsequent investigations.  In fact, the FDA feels that the liver enzyme increases warrant them be tightly monitored as part of a REMS program should mipomersen be approved in the homoFH population.


4) Injection-Site Reactions and Discontinuations

One of the major marketing tools, and in fact actual draws, of ‘gymnotic delivery’ has been that it can be subcutaneously administered, while the leading RNAi Therapeutics delivery technologies, foremost Tekmira’s SNALP, still rely on intravenous infusion (for most applications at least).  Turns out that injection site reactions ranging from pain, redness, swelling, to skin discolorations and haematomas were a major factor for study drug discontinuations, especially in the open-label phases of the studies which should be indicative of what will happen in a real-world setting.  Overall, 61% of HoFH patients (23 of 38) discontinued study drug during the open-label phase, and 77 of 141 discontinued in the pooled Phase 3 population (including non-hoFH subjects).  Other adverse events attributable to the inflammatory potential of the technology, such as flu-like symptoms, contributed to the high discontinuation rate.

A curious, hitherto undisclosed finding, were injection site recall reactions with mipomersen (but not control).  This refers to an inflammatory reaction at an old injection site when the drug was administered at a new site.  The molecular basis and importance for such recall reactions seem unknown.  In fact, my literature searches suggest that they have almost exclusively been described for the TNF-alpha blocking antibody Enbrel.  However, to me, they once again indicate the systemic inflammatory potential of phosphorothioate antisense chemistry, likely involving adaptive immunity.  Accordingly, 30/50 patients (60%) in the CS5 and 6 studies exhibited mipomersen antibodies compared to none in the control population- another ‘surprise’ that should be applicable to the phosphorothioate platform in general.

5) Carcinogenicity

While there were ~2-3x more cases of tumors in the mipomersen-treated population compared to control, I agree with the reviewers that they are not the basis for concluding that mipomersen increases the risk of developing cancer.  This is because of the nature of the reported cases and the relatively short treatment duration after which many of them occurred.

However, what would worry me more is that in the preclinical carcinogenicity studies in rodents (mice and rats), mipomersen clearly increased cancer risk- despite ISIS’ claim that they have been ‘clean’.  Among the findings were hepatocellular adenoma and subcutaneous tumors.  Having a report by your CRO stating the findings are likely species-specific issues and not applicable to humans, does not mean that the findings are not material in a regulatory sense, especially as they were made during a period at the FDA when the agency was much more risk-averse.  I expect the clinical and preclinical findings to cause carcinogenicity to be included in a REMS program.    


6) Kidney Damage

The liver, spleen, and kidneys are the major sites of phosphorothioate accumulations.  Consequently, it is not surprising that kidney damage has been an important safety concern of such antisense technology.  Although the clinical safety findings did not demonstrate that mipomersen impaired kidney function, the study drug did increase the amount of protein found in the urine (proteinuria).  This finding supports that kidney function remains a safety concern and such monitoring should be included in a REMS.

In summary, the safety findings were not all that surprising given what we know about phosphorothioate safety in animals and study discontinuations by competitor Santaris, very likely due to safety.  However, if you indeed have taken the reassurances by ISIS CEO Dr. Crooke literally, you may be justified in feeling misled.  Even on that count, however, I am not all that surprised, and his salesmanship may indeed be one of ISIS' great assets.  

Disclosure: The author is 'long' the stock ($ISIS).

Clarification: The term 'gymnotic delivery' in the linked paper refers to a specific protocol for optimizing gene knockdown in tissue culture in the absence of assisted delivery.  However, the term has been more broadly used by Santaris to refer to unassisted delivery in general (including clinical applications), obviously in an effort to express that in contrast to phosphorothioate oligonucleotides, RNAi needed assisted delivery.  I found it suspect, however, that they used a new term to refer to an over decade-old industry practice.  In a further clarification, I have not seen ISIS Pharmaceuticals adopt the term either. But clearly, not using (in most cases) assisted delivery has been a greatly exploited marketing tool of the antisense industry built on the back of RNAi Therapeutics.

Monday, October 22, 2012

Genzyme Taps RNAi Therapeutics for TTR Amyloidosis


Orphan disease gold miner Genzyme has just partnered Alnylam’s promising TTR amyloidosis drug candidate, giving it the right to commercialize resulting medicines in Japan and ‘other Asia-Pacific countries’.  In addition to a healthy $22.5M upfront, Alnylam stands to receive the customary development milestones and actually high royalties for a post-phase I program, plus Genzyme bears responsibility for the regulatory process in the licensed territories.

The basis for this deal, as also expressly stated in the press release, were the potent, ~90% knockdown efficacies obtained in the (single) dose-ranging ALN-TTR02 phase I study (for review, see 'Simply SNALP').  Critically, it was Tekmira’s SNALP delivery technology that made these results possible and catapulted Alnylam’s market cap by ~300M (50% increase in share price) at the time the phase I results were announced this summer.  I should add, however, that in addition to ALN-TTR02, the preclinical subcutaneously administered candidate ALN-TTRsc has been included in the deal. 

Clearly, if they are really serious about the competitiveness of the subcutaneously administered GalNAc-siRNA conjugate, Genzyme has got to be hoping that there won’t be a repeat of the mipomersen (antisense) injection site reaction fiasco which, in addition to the liver fat and immune stimulation issues, is endangering the approval for mipomersen even in the very high unmet medical need population of homozygous familial hypercholesterolemia.  In this case, the adverse event that could drastically lower the deal value soon after its close comes in the form of the Tekmira litigation risk (note: Genzyme is ISIS' partner on mipomersen and should know, also about the strength of the competing subQ ISIS-TTRRx program which is partnered with GSK).  

In case you are wondering what I am alluding to: less than a month after Genzyme invested more than $300M in ISIS and their hypercholesterolemia candidate mipomersen, the FDA issued a Partial Clinical Hold due to significant safety signals with the systemic phosphorothioate oligonucleotide, according to last week’s Briefing Documents for the mipomersen Advisory Committee meeting.   The risk in the TTR case, of course, is the pending litigation with Tekmira, involving the critical RNAi delivery technology enabling the ALN-TTR02 results, its manufacturing and other (e.g. 2’-O-methyl) IP associated with the TTR amyloidosis program.  You would expect that Genzyme would have at least waited for another month or so before closing the deal (trial date in November).  

I doubt that the deal value would have been much higher without the overhang given the size of the Asian TTR market.  But then, of course, nothing can surprise me here any more and a resolution to the Alnylam-Tekmira troubles may already have been hammered out.  A clue may be how Tekmira responds to the news.  If there is tonight a we-are-pleased-that-Genzyme-has-partnered-ALN-TTR02-for-which-we-provide-the-delivery-technology-PR, then it's probably nothing to get excited about.  But if there is silence, then maybe the real news may come with a slight delay.  

Talking of Tekmira, this company, as it is customary in biotech sub-licensing, is likely to receive a partial payment out of the $22.5M upfront (plus a cut of the future income stream related to ALN-TTR02).  Another partnership implication is that Alnylam has talked to Takeda, and Takeda declined on ALN-TTR.  As you remember, in 2008 Takeda forked over $150M to Alnylam, also for being the exclusive Asian partner for 5 years with a right of first refusal for Alnylam drug candidates in the Asian territories.   You’ve got to wonder how comfortable that conversation was, especially after handing over some of that for the ‘technology transfer’.  There might be even less room to hide from Genzyme, as I believe Genzyme and Alnylam may be sharing one building now.


Wednesday, October 17, 2012

Arrowhead Research Breaks Up Dynamic PolyConjugates into Two


Arrowhead Research announced yesterday that it had received Notice of Allowance from the USPTO for a Dynamic PolyConjugate-related patent application (for systemic RNAi delivery).  Instead of it being your run-of-the-mill patent PR involving known technology that finally received patent protection, it was really about revealing Arrowhead’s fundamentally new approach towards DPC delivery (for my take on the original form of DPC, see here).

The patent, part of a series of patent filings that have come out recently, shows that under Roche’s ownership, the technology has morphed through a number of iterations from the original complex polyconjugate chemistry combining endosomolytic polymer, masking groups, RNAi trigger, PEG, and cell targeting ligand all in one molecule (schematic shows such molecule and presumed mechanism of delivery), into one where a RNAi trigger and the masked polymer, both targeted individually, are administered as separate agents. 

One problem with the original design has been that combining all the functional groups, including negatively charged RNAi triggers and positively charged polymers, into one molecule was not particularly easy.  The tendency to aggregate and poor yields made it a quite expensive and difficult-to-scale proposition.

Turns out that such complicated chemistry wasn’t needed after all.  As long as the RNAi trigger and the masked endosomolytic agent end up in the same place, it does not make much of a difference whether they are getting there as one molecule or separately.  In the example provided, namely for gene knockdown in hepatocytes, the RNAi trigger could be conjugated to either cholesterol or a cluster of galactose sugars, whereas the masked endosomolytic polymer was targeted to the hepatocytes by galactose.  Viewed differently, the polymer allowed the cholesterol-siRNA that apparently gets trapped in the endosomes when alone to be released into the cytoplasm.  Hence, the multi-fold increase in potency (Arrowhead Research says it’s 500-fold) over Alnylam’s original 50mg/kg cholesterol-siRNA report (Soutschek et al., 2004).

Another potential advantage of this separated approach is that it makes each component smaller, perhaps 'one day' enabling subQ dosing.  However, as mipomersen's FDA AdCom meeting briefing docs show, when it comes to subQ dosing, be careful what you wish for.

Manufacturing appears to have been one of the issues delaying the clinical translation of DPCs for quite some time (Arrowhead Research says they are ready to file an IND in Q2 2012 for Arc520 in HepB).  Toxicity, mainly due to premature unmasking in the blood instead of in the target cell endosome, seems to have been the other main reason.  Such premature unmasking also adversely affected circulation times, thereby rendering attempts to get beyond the liver, one of the original promises of DPCs, futile.  We will probably get word from the company soon what solutions it found for this challenge, but it seems that, based on the emerging patent literature (including non-human primate data; e.g. WO 2012/083185), masking the membrane penetrating peptide mellitin with endosomal protease-sensitive groups, is a promising approach.



Saturday, October 13, 2012

Sarepta Discloses Dystrophin Expression Data at World Muscle Society Congress


Last week, I criticized Sarepta Therapeutics for failing to disclose critical dystrophin expression data regarding their exon-skipping drug candidate eteplirsen for the treatment of DMD.  Instead, they merely disclosed the percentage of muscle fibers staining positive for dystrophin, i.e. fibers that expressed some dystrophin, which, of course, would be a much more impressively sounding number.  Adding to what I perceived as dubious data presentation was 6-minute-walk test (6MWT) data of which the integrity seemed compromised based on cherry-picking the patients and the fact that the apparent improvement occurred in the open-label extension phase of the study.

Admittedly, the percentage positive fiber number was a previously defined primary endpoint whereas the amount of overall dystrophin relative to normal/healthy was not.  Therefore, like other biotechs frequently do, it might always have been Sarepta’s intention that only the primary endpoint data be presented initially ('top-line data'), with the balance to be disclosed at a future scientific conference or peer-reviewed presentation.

Accordingly, Sarepta today presented additional data at the World Muscle Society Confress in Perth, Australia.  I was pleased to see that at least some dystrophin expression Western blot data were revealed (slide 12)- with a direct comparison to ‘normal’ at that.


First of all, you can see on the left the ‘normal control’ (healthy) comparison.  The labeling seems to be somewhat off as I believe the outermost left lane represents the sample.  You can also see that the normalization control ‘actin’ was less abundant in the control sample compared to the patient samples (by naked eye about 3-5 fold).  This means that the 'normal' dystrophin signal is an under-estimate relative to the patient samples. What is more, because the dystrophin signal in the ‘normal control’ is over-saturated (big black blotch), the ‘real’ relative dystrophin amount should be even more compared to the patients'.  Because there is no standard curve and because of the apparent loading differences (à actin), it becomes an art to estimate the relative dystrophin amount restored by eteplirsen treatment, but with 7 years of doing the same type of experiments at the bench under my belt, I would guesstimate that the true value is about 2-5% of normal.  Certainly not the 20% believed to be necessary to start impacting the disease phenotype. [Correction 10/13/2012: This was an inadvertent mis-characterization of the quoted source, please see the discussion in the Comments section].

Supporting the notion that the Western Blot overestimates restored dystrophin levels, is the RT-PCR experiment on the right.  Again, the larger band which represents the mutated transcript is much more abundant than the re-spliced signal below, indicating that only a minor fraction of the mutant transcripts were re-spliced.  Moreover, smaller PCR products amplify more efficiently compared to larger ones, meaning that the apparent splice correction is an over-estimate.  Similarly, because the mutated and ‘normal’ PCR signals are almost the same (you would expect an mRNA with premature stop codons to be destabilized relative to wild-type), it is likely that the normally spliced bands have reached saturation, further over-estimating the splice correction in the RT-PCR experiment.

Overall, whatever Sarepta’s motives were behind failing to disclose the absolute dystrophin data last week, they clearly provided some of the necessary transparency at the WMSC presentation.  Having said that, I believe the data support the underlying concern that the splice correction may not be enough to be therapeutic.  I would not exclude it, however, as the 20% value, of course, is also an estimate.  Blame a less than rigorous trial design for not knowing for sure for quite some time to come. 
By Dirk Haussecker. All rights reserved.

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