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.

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 5x15^TM 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.

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.

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.

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 triggers.  It 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?

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).   

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.