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.
By Dirk Haussecker. All rights reserved.

Disclaimer: This blog is not intended for distribution to or use by any person or entity who is a citizen or resident of, or located in any locality, state, country or other jurisdiction where such distribution, publication, availability or use would be contrary to law or regulation or which would subject the author or any of his collaborators and contributors to any registration or licensing requirement within such jurisdiction. This blog expresses only my opinions, they may be flawed and are for entertainment purposes only. Opinions expressed are a direct result of information which may or may not be accurate, and I do not assume any responsibility for material errors or to provide updates should circumstances change. Opinions expressed in this blog may have been disseminated before to others. This blog should not be taken as investment, legal or tax advice. The investments referred to herein may not be suitable for you. Investments particularly in the field of RNAi Therapeutics and biotechnology carry a high risk of total loss. You, the reader must make your own investment decisions in consultation with your professional advisors in light of your specific circumstances. I reserve the right to buy, sell, or short any security including those that may or may not be discussed on my blog.