One Pitfall to Look Out for in RNAi Therapeutics

RNAi Therapeutics have turned the corner to being a generally accepted drug modality.  Following years of ridicule and benign neglect, the industry just celebrated the FDA approval of a 2nd drug, Givlaari for the treatment of acute hepatic porphyria. This was followed in short order by the $9.7B acquisition of The Medicines Company by Novartis for its cholesterol-lowering RNAi drug candidate, Inclisiran, a harbinger for how RNAi medicines will change the game in cardiovascular risk management.

Amid all the understandable investor excitement, it is probably a good time to reflect on the potential pitfalls as we move into a world where RNAi Therapeutics dominate liver-related diseases and advance into new big areas such as the CNS and ocular disease.  Remember, investor, it takes one safety event for the most highly valued programs like HBV RNAi candidate AB-729 by Arbutus Biopharma, Arrowhead Pharmaceutical’s ARO-AAT, or the Lpa program by Silence Therapeutics to decimate your investment by half or more.

Off-targeting safety, off-targeting safety, off-targeting safety

When the discovery of RNAi was rewarded with the Nobel Prize in 2006, the Andy Fire and Craig Mello hammered home a message that would resonate for the years to come: to make RNAi Therapeutics, it’s all about the delivery!

Delivery, delivery, delivery

But as the liver has been conquered and the industry is chewing on a number of liver-related therapeutic opportunities and opening up opportunities in new areas, we still have to fully understand the odds of seeing program failures due to the difficult to predict consequences of off-targeting.

Off-targeting is the moderate knockdown of sometimes dozens of genes sharing sequence homology to the target gene.  This was shown over a decade ago and soon thereafter shown to be addressed by using simple modification strategies.

This, however, was at a time when the delivery mantra was practiced and nobody wanted to risk reducing knockdown efficacy by simultaneously considering off-targeting in their RNAi trigger designs.  I was therefore upset, as it turned out rightfully so, that Alnylam let Nastech essentially go bankrupt a decade ago when it should have acquired rights to Nastech's then industry-leading off-target-mitigation technology.

Alpha-1 Antitrypsin as testbed

Alnylam would have to pay dearly for its pride in never treating a smaller competitor well, even if it served its own interest, with off-targeting manifesting itself in markers of liver toxicity in first-generation clinical programs like chronic HBV and alpha-1 antitrypsin-related liver disease.

These two indications, unlike the now approved AHP indication for Givlaari which addresses symptoms outside of the liver, represent the most exacting off-targeting challenge as they affect liver health directly and any further exacerbation of liver stress should be avoided.  

As a result, Alnylam dug out the old off-targeting literature and in their incarnation added a GNA modification at position 7 of the guide strand while, for experimental purposes, leaving the rest of the first-generation molecule unchanged. Translational science at its best (!) and the industry can be thankful for it.

Indeed, for both the ESC+ chemistry ALN-AAT02 and ALN-HBV02 no liver enzyme elevations (above 3x ULN) were observed in 49 subjects tested, whereas 3 of 33 had such elevations with the ESC-only versions.

In Arrowhead’s case, there also were no ALT elevations >3x ULN.  However, 3 treatment-emergent grade 1 ALT elevations not exceeding 2x ULN were reported in the 28 normal healthy volunteers receiving ARO-AAT.  I am curious to see the individual time curves and the doses at which they occurred.  It is certainly an area to watch for that program as it only now moves into patients. 

Add to this considerable uncertainty about patient selection, dose and duration required to have an impact on AAT liver disease, increasingly analysts topping their price targets by the day may have to re-think how they value this program (see disclosure below).

Further behind, we have to see how Silence Therapeutics and Arbutus have addressed off-targeting with first data expected in 2020.  While Silence has avoided indications directly affecting liver health (iron overload, cardiovascular disease), Arbutus with its HBV indication will have a steeper hill to climb, although the pay-off for investors in this $80 market cap company should be considerable should it climb it come Q1 2020.

Personally, my guesstimate for non-optimized GalNAc designs would be a 50% chance of off-targeting success, also depending on preclinical vetting (after all, the very clean $9.7B Inclisiran is such a design); a 85% off-targeting success for optimized designs. 

Disclosures: long Silence Therapeutics, Arbutus Biopharma; short Arrowhead Pharmaceuticals; no position in Alnylam.

Alnylam and ISIS Pharmaceuticals Divide Up a Small Lobe of the Liver Kingdom

For some time now, a GalNAc IP war has been brewing given that GalNAc conjugation has been used by both Alnylam and ISIS Pharmaceuticals to enable tremendous advances in targeting genes in the liver in an ever more potent and apparently safe and convenient manner.  This has suddenly opened the floodgate to numerous therapeutic targets (ApoCIII, Factor XI, HBV, TTR etc) and indications with gene knockdown likely to dominate drug innovation for cardiovascular, metabolic, plus a number of viral and rare genetic diseases in the years to come.

However, instead of escalating tensions, a partial armistice was declared this week when the companies announced that they will not get into each other’s hair for at least 4 gene targets in the liver.  According to the Agreement, Alnylam can use the two companies’ combined IP to support RNAi Therapeutics against the rare genetic disease targets antithrombin (for hemophilias) and ALAS-1 (for hepatic porphyrias) while ISIS can leverage the same for antisense therapeutics against factor XI (for anti-clotting) and Apo (a) (for cardiovascular disease). 

Interestingly, the press release talks about a reciprocal IP cross-licensing that extends to ‘RNA-targeting mechanism’ leaving open the possibility that Alnylam may use ISIS’ RNaseH antisense chemistry and ISIS in turn Alnylam’s siRNAs for these targets.  This would allow the companies to maximize the life-cycle opportunities for these targets by making available multiple routes of administrations (subQ, inhaled, oral) and pharmacodynamics (slow/rapid onset; different effects of gene/protein half-lives on required dosing frequencies).  Moreover, since some genes are easier targets for a given mechanism than others, chances are high that you can find a highly potent molecule with either RNAi or RNaseH ASO.   

By thus avoiding duplication of efforts and competition in the marketplace, the economic value of these targets is likely maximized.  I do not expect, however, that the truce will extend to all targets in the liver given the advanced stage and importance of for example the TTR amyloidosis programs of the two companies.  Moreover, there might be partner (e.g. Genzyme, GSK) pressures to go after certain targets no matter what.  For these targets, it will be interesting to see whether the companies will resort to patent litigation or whether they agree to merely compete in the marketplace. 

While the economic rationale is obvious, there is a scientific risk to the non-compete.  This is because you might end up with a late-stage failure, e.g. due to an unanticipated side effect related to sequence-specific off-targeting that is only seen in larger patient populations.

This, of course, would be welcome news to 3^rd party competitors such as Tekmira, Arrowhead, and espcially GalNAc wannabe Dicerna who could then be the last man standing.  On the other hand, the concentration and coordinated use of the IP estates of the two juggernauts in the RNA Therapeutics space will make circumventing it more difficult to the competition.

GalNAc Advance by ISIS Means Oral Oligonucleotide Therapeutics around the Corner

Last week, the long-awaited first publication on ISIS’ GalNAc-targeted antisense oligonucleotides appeared in a ‘NAR Breakthrough Article’ (Prakash et al., 2014)  Living up to that label, the data with RNaseH antisense oligonucleotides (ASOs) conjugated to triantennary GalNAc sugars showed that this strategy increases potency by nearly an order of magnitude.  In combination with high-affinity chemistries (in this case cET) that facilitate the use of short (12-14nt vs ~20nt) oligonucleotides which has been shown to improve in vivo potency (Santaris research by Straarup et al., 2010), oral delivery for antisense modulation of hepatic gene expression has essentially been solved. 

Moreover, the lower dosages that can now be used in addition to the improved biodistribution profile that is now heavily slanted away from non-hepatocytic cell types in the liver and the kidney in favor of hepatocytes, means that the other major benefit of GalNAc-ASOs with immediate applicability is that the safety of therapeutic ASOs for liver-directed applications will be much improved.

The conclusion that oral antisense therapeutics are just around the corner is based on the observation that a relatively crude caprate-based phosphorothioate oligonucleotide formulation enabled a ~10% bioavailability following oral administration already (intravenous = 100%; Tillman et al., 2008).  This, in addition to some inter-subject variability that might have been linked to gastric emptying times, meant that too much oligonucleotide would have had to be administered orally with previous antisense oligonucleotides. 

Granted, this first piece of GalNAc-ASO literature did not explore oral delivery. The fact, however, that the combined use of GalNAc conjugation with high-affinity chemistry improved parenteral ASO potency by 60-fold is predicted to mean that oral GalNAc-cETs are already more potent (~6x) than subQ-administered 2^nd gen ASOs.  These have already produced impressive phase II data for targets such as Factor XI and ApoCIII.

The potency improvement in terms of oral delivery may actually be larger than 60-fold.  This is because oral delivery strategies such as caprate co-formulation which aim at increasing intercellular drug permeability are size dependent.  This means that there should be an added benefit of the smaller oligonucleotide size facilitated by cET chemistry with oral delivery.

Of course, the data leave open some questions.  My most pressing, applicable to both oral and non-oral uses of GalNAc-ASOs is how the rodent data translate into non-human primates and ultimately humans.  This is because for unconjugated phosphorothioate oligonucleotides, the potency on a mg/kg basis improves with the size of the organism.  The discussion above is based on an assumption that GalNAc-targeting will not change that.  I have been unable to make a determination yet as to whether this should hold true or not.

Other than that, the antisense weather forecast is blue skies ahead, though with a chance of thunderstorms in the form of IP skirmishes with their good friends over at Alnylam as they have pioneered GalNAc conjugation in oligonucleotide therapeutics, but may now be ironically penalized for it from a competitive point of view.  Alnylam is not the type of company that just lets competitive threats happen to them.

Tekmira Setting New Standards in mRNA Therapeutics…and They Have Only Started

Tekmira today presented impressive data on pre-clinical messenger RNA (mRNA) delivery to the liver and solid cancers (PR here, slide deck here).   The data presented at the AsiaTides in Tokyo greatly exceeded those reported in the literature, including a 4 log increase in potency of liver gene expression compared to the seminal paper by Kariko et al.paper (2008). 

Importantly, the formulation process and chemistry were optimized for mRNA delivery thus generating electron-dense ~80nm particles and easily surpassing >90% encapsulation efficiencies that are thought to be required to pass regulatory CMC muster.

Tumors giving liver run for its money

What had caught my eye when Tekmira presented firstmRNA delivery data at the Tuebingen mRNA conference in October 2013 and which was further substantiated in the present presentation, in terms of absolute gene expression, their mRNA delivery seems to perform as well if not better in liver cancer and non-liver cancer models compared to in normal hepatocytes.  This could be due to a less RNA degradative environment in tumors versus the liver of which an important function is detoxification.  This is supported by the fact that the duration of mRNA expression in tumors greatly exceeded that in the liver (a gene with a short protein half-life was used).

This made me think of the recent oncology spin-out (Onkaido)by mRNA Therapeutics company Moderna, the darling of mRNA Therapeutics which has raised around half a billion dollars in partnership money (AstraZeneca, Alexion, DoD) and fund raisings over the last 18 months alone.  The spin-out and internal focus on genetically defined orphan diseases may mean that Moderna would like somebody else to fund Onkaido, for example as part of a joint venture.  Remember, for most Big Pharma companies, RNA Therapeutics are first and foremost interesting for oncology as it allows them to go after the targets that their scientists have been dreaming about for so long yet were not able to drug.

Given that most of the de-risking in oncology drug development occurs late in clinical development, it is debatable whether Tekmira, already with a lead candidate in oncology (TKM-PLK1) should focus their in-house mRNA development attention towards oncology initially and whether this might not be better done as part of a spin-out/joint venture (Onkaido and/or Big Pharma).

For the liver, mRNA expression was detected in essentially every hepatocyte.  This supports the use of SNALP-mRNAs not only for the use of the liver as a factory for protein production, but also for diseases with defects in single genes expressed in the liver, including those genes that act only in the cell that they were expressed in (cell autonomous).  Compared to oncology, such indications would also allow for earlier clinical de-risking and together with orphan status mean that they better match the profile of a (still) sub $500M market cap company.

More upside

Given that Tekmira has only been working on mRNA Therapeutics for a year plus (1-year stability data were presented) and since simple off-the-shelf mRNA chemistry was used, one can only dream which diseases could be addressed and where the company could go with this, including developing their own mRNA Therapeutics instead of viewing it as a simple monetization opportunity. 

Tekmira mRNA developments that I am looking out for include advances in mRNA delivery to the lung epithelium.  Tekmira has been, and according to the presentation is still actively working on inhaled LNPs, and it would be logical if they adapted that effort towards mRNA delivery.  Expressing CFTR in lung epithelia for the treatment of cystic fibrosis (CF) would be an obvious target here that could rally a lot of support from various stakeholders (important for companies like Tekmira).  Gene therapy involving the inhalation/instillation of DNA to the lung has failed, in large part due to the difficulty of getting the DNA from the cytoplasm across the nuclear membrane into the nucleus.  Just like in RNAi, mRNAs only need to get into the cytoplasm, a much simpler task.  Moreover, in CF you should not need to get the mRNA into every cell.

A final opportunity for Tekmira is the co-formulation of mRNAs and siRNAs into a single LNP.  Imagine an oncology drug targeting both cancer drivers such as PLK1 and overexpressing tumor suppressors.

Oyasuminasai.

Disclosure: long TKMR.

Tekmira Grabs Leadership Position in Messenger RNA Therapeutics

Messenger RNA Therapeutics is the latest addition to the RNA Therapeutics tool box.  A simple concept, it has only been recently revived outside the therapeutic vaccine realm when it was shown (e.g. Kariko et al. 2012 and Kormann et al. 2011) that certain nucleic acid modifications can minimize, if not abolish innate immune responses triggered by these long RNAs.

Of course, in addition to representing an increased innate immune challenge compared to RNAi Therapeutics, like RNAi Therapeutics, the key technical challenge for mRNA Therapeutics is the effective delivery into the cytoplasm of target cells.  When AstraZeneca paid Moderna Therapeutics $240M in upfront alone earlier this year for a limited technology license, I half rolled my eyes telling myself ‘not again’.  

The ‘not again’ refers to Big Pharma getting distracted by supposedly gate-keeping IP claims regarding the RNA inducer all the while ignoring the critical importance of delivery.  As we know, this mis-attribution of value has caused much trouble in the history of RNAi Therapeutics.

Enter Tekmira

I further believed that the SNALP LNP technology by Tekmira is most readily adapted for mRNA delivery among the RNAi delivery technologies.  So it was pleasing when Tekmira disclosed last week at the 1^st International mRNA Health Conference in Tuebingen, Germany, that they indeed have been pursuing mRNA delivery and presented first data indicating that they are most advanced in this effort.

As it is difficult to compare the amounts of proteins expressed with the different technologies due to the unique half-life of each mRNA and protein, the leadership hypothesis is partly based on an assumption, namely that there is a close relationship between SNALP LNP delivery of siRNA and mRNA.  More importantly, however, due to their ample experience of commercially translating SNALP LNP delivery for RNAi Therapeutics with over half a dozen SNALP-enabled candidates that have entered clinical development and with the demonstration of scale-up, they should have a first-mover advantage.

Liver and solid cancers

In particular, Tekmira presented data on mRNA delivery in mice for the liver and solid cancers.  The liver is of interest both as a target organ itself and also as a factory for the production of proteins secreted into the circulation.  The data showed that robust levels of the luciferase marker protein could be made in the liver.  However, the data also reminded me of one (certainly not insurmountable) challenge with mRNA Therapeutics: the relatively short period of time (<24 a="" as="" been="" boundary="" data="" designed="" expression.="" expression="" gene="" half="" has="" have="" hours="" however="" indicate="" life="" lower="" luciferase="" nbsp="" of="" ote="" p="" platform.="" protein="" reporter="" robust="" short="" so="" specifically="" that="" the="" to="">

In that light, the tumor expression data were particularly intriguing.  Not only were the tumor peak expression levels comparable to the liver, but they also were maintained over a longer period of time (~2 days) and generally declined more gradually.  Since the same mRNA was delivered, this observation would be consistent with delivery to the tumor over time, as one would expect if the EPR effect plays an important role here.  The data also support a depot effect following extravasation meaning that intratumoral SNALP-mRNAs can be stable so that they can be taken up in a delayed fashion and yet be functional.  Such observations incidentally should also prove quite useful for the development of RNAi delivery for solid tumors and I look forward to more such cross-fertilizations between siRNA and mRNA delivery.   

Tekmira the logical partner for AstraZeneca

The new disclosures would seem to warrant AstraZeneca partner with Tekmira on mRNA delivery.  After all, having spent $240M for IP puts them under pressure to do something with their rights.  As I do not think much of Moderna’s own LNP delivery efforts (order my latest OTS 2013 Report to see why), AstraZeneca would be well advised to look outside of that partnership for delivery solutions.

It also so happens that AstraZeneca’s IP rights relate to protein expression for metabolic disorders (--> liver as a critical metabolic organ) and cancers, precisely the two areas where Tekmira’s technology should be most useful initially.    

And who knows, maybe Big Pharma is not always that dense as it sometimes seems and all this had been in the making months ago and the CEO of Tekmira is about to make good on his partnership ‘promise’ (in that case, sincere apologies to AstraZeneca).

Antisense Therapeutics Dressing Up with RNAi Delivery

The boundaries between RNAi Therapeutics and Antisense Therapeutics have become blurrier.  Originally, the basic practical difference between RNAi and antisense was as follows: on the one hand, RNAi is very powerful, but it requires special formulation chemistry which often is the tox-limiting factor and necessitates that the drug be administered intravenously.   On the other hand, antisense can be utilized in unformulated/naked form and be administered subcutaneously, but it is less potent and involves the accumulation of large, problematic levels of phosphorothioate molecules in a range of tissues, primarily the kidney, liver, spleen, and probably the vasulature.

   

Recently, however, some RNAi approaches have become more antisense-like in terms of patient convenience in that conjugate technologies and ‘self-delivering’ RNAi triggers that can be administered subcutaneously have advanced into the clinic.  Over the last two weeks then, two pieces of evidence emerged of the reverse trend of antisense technologies adopting delivery technologies originally developed for RNAi Therapeutics.  The is done for improved potency and could also bring critical safety advantages.

Evidence 1: ISIS and Alnylam Find SNALP Delivery to Increase RNaseH Potency by ~20-fold

In a paper by Prakash et al. in ACS Chemical Biology, scientists from ISIS Pharmaceuticals and Alnylam demonstrate significant potency enhancements for RNaseH antisense and single-strand RNAi to be gained from the use of Tekmira’s SNALP technology.  As a reminder, the two companies once collaborated on single-strand RNAi before Alnylam terminated that relationship with ISIS continuing work on the subject, so the present publication relates to historical work during their partnership, but is relevant to ISIS' continued efforts.

The study simply compared the knockdown potencies of RNase H gapmers (phosphorothioate 2’ MOE chemistry), single-strand RNAi triggers (heavily modified), and dsRNAi triggers (ssRNAi molecule as guide together with complementary unmodified passenger strand), either as unformulated oligonucleotides or when formulated with SNALP containing the DLin-KC2-DMA lipid.  This lipid, developed by Old Tekmira (see Semple et al. 2010, Nature Biotech), was a critical ingredient of the first ‘2^nd-generation’ SNALP showing significantly improved liver knockdown potency over the original DLin-DMA SNALPs. 

When targeting PTEN in mouse livers, none of the oligonucleotides alone exhibited knockdown activity at the 4.5mg/kg dose tested.  Only when increasing the dose up to 100-200mg/kg did the RNaseH gapmer and ssRNAi trigger exhibit marked knockdown.

   

In stark contrast, profound knockdowns were achieved with SNALP-formulated RNaseH gapmer and the ssRNAi trigger at the 4.5mg/kg dose.  Remarkably, for the RNaseH gapmer, the activity was obtained at liver oligonucleotide levels that did not differ much from that of the unformulated oligonucleotide (which did not exhibit PTEN knockdown).  This means that SNALPs not so much increases the delivery to the liver per se, but the functional delivery of RNaseH gapmer, possibly by liberating phosphorothioate oligos along the endosomal cellular uptake pathway.  This finding also makes it difficult to believe some reports that phosphorothioate oligos administered without special formulation accumulate in the nuclei as this is the site where they are supposed to be mostly active.  Where the non-functional oligos actually end up is an important question as this also relates to the safety of these molecules.

Overall, the study shows that formulation with RNAi delivery technologies can greatly enhance knockdown potency.  It should be added here, however, that this single-dose study somewhat underestimated the potency of RNaseH antisense as normally a loading dose schedule would be performed to reach threshold tissue levels required for knockdown activity of unformulated PS-RNaseH gapmers.

The lower dosages and more targeted delivery with RNAi delivery may also reduce the typical toxicities associated with phosphorothioate antisense.  Indeed, one may even be able to do away with the phosphorothioate modification altogether.  Of course, toxicity related to the delivery vehicle also needs to be considered, but the authors noted in their paper that SNALP was ‘well tolerated’ and that no changes in liver enzymes or in body weight loss were seen.

To further gauge the value of SNALP and RNAi delivery in general for gapmer antisense technology, a more detailed time-course analysis of the knockdown and the tissue concentrations would be useful.

Evidence 2: Regulus Therapeutics to Employ Alnylam GalNAc-Conjugation for Mir-122 HCV Program

In another example of the value of delivery technology originally developed for RNAi Therapeutics for single-strand oligonucleotide Therapeutics, Regulus Therapeutics disclosed at last week’s Needham conference that it would use Alnylam’s GalNAc conjugation for the delivery of anti-miR122 in its HCV program which is to enter clinical development in 2014.

With this, Regulus expects to achieve once-monthly-dosing with their single-stranded anti-miR at lower doses (0.3mg/kg) than the weekly LNA-based phase II compound by competitor Santaris (>1mg/kg).  Importantly, Regulus claims that HCV patients go to see their doctors to check their antiviral responses on a monthly basis anyway.  Consequently, these routine visits for patients on anti-HCV therapies could then be combined with the subcutaneous administration of the anti-miR.  This obviously would increase compliance in an era in which oral HCV meds reach high cure rates in clinical trials, but the actual cure rates in the field are significantly lower due to rate-limiting compliance issues (h/t to John Alan Tucker from Zacks Investment Research).   

Without showing the data, it remains to be seen whether and by how much GalNAc conjugation improves anti-miR potency.  The once-monthly dosing, however, indicates that once the single-stranded oligonucleotide is in the cytoplasm (where the target is), it is trapped and stays there for quite some time instead of being washed out again into the interstitial space (outside the cells) which would be the case if extracellular and cellular oligonucleotides were in dynamic equilibrium.     

It will be interesting whether the GalNAc approach will utilize the phosphorothioate backbone which ISIS Pharmaceuticals is using at all.  If not, the decision would indicate that ISIS satellite company Regulus is concerned about the safety implications of that chemistry, especially in patients where the liver is already diseased.

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.

An Exemplary RNAi Therapeutics Target

A paper by Alnylam and collaborators at Harvard and the MIT on a strategy to treat anemia recently appeared in Blood and involves an exemplary RNAi Therapeutics target gene selection which takes into account both the scientific and technical strengths and challenges of the technology (Querbes et al. 2012).  In addition, it is yet another demonstration of the powers of Tekmira’s SNALP technology and the various therapeutic opportunities that it can address with gene knockdown in the liver alone. 

Before going into the narrative of the paper and risk losing your attention, here are the main reasons why the target gene(s), PHD1-3, uniquely lend themselves to an RNAi Therapeutics approach (as opposed to competitive small molecules and recombinant proteins/MAbs) and why SNALP delivery is a particularly good match for them:

1. The target genes (PHD enzymes) are cell-autonomous negative regulators of the expression of a secreted factor (here: erythropoietin/Epo).  This means that even if efficient RNAi knockdown were only achieved in a subset of cells, the secreted factor that will be generated in this subset of cells can still act globally through the systemic circulation.  This is in contrast to other gene targets, such as certain intracellular targets involved in cancer cell proliferation, where efficient gene knockdown would be required in almost every cell to achieve a measurable outcome. 

2. Specific multi-targeting. In order to exploit a genetically intriguing mechanism for the treatment of disease (PHD regulation for the treatment of anemia), it was necessary to simultaneously knock down three gene family members.  Small molecule inhibitors with such multi-targeting activity would most likely cross-react with additional family members, increasing the risk of (A) obtaining confounding biological outcomes as other family members are more likely to play roles in related biological pathways than e.g. a spurious RNAi off-target event that is pathway-independent, and, of course, of (B) off-target toxicity.  The next time you hear about that exciting multi-targeting kinase inhibitor approach in oncology, remember that many of these were not multi-targeting by design and that there will be a number of additional kinases being hit that you will hardly hear about.  In summary, it is not just the capacity of RNAi Therapeutics for multi-targeting (e.g. as in the clinical candidates ALN-VSP02, TKM-EBOLA), but it is also the more ‘wholesome’ multi-targeting mechanism that is an advantage of the technology here. 

3. Tissue-specific delivery (here: SNALP and liver). Non-specific, but also specific targeting when it occurs in the wrong tissues, may cause toxicities.  In this example, because the gene family to which the target genes (PHD enzymes) belong play roles in various non-targeted biological processes throughout the body, there is great concern that a small molecule PHD inhibitor would cause a number of toxicities.  With regard to on-target toxicity in the wrong tissues, the HIF transcription factor that is regulated by the PHD1-3 enzymes plays an important role in cancer biology and inhibiting it all over the body may elevate the risk of developing cancer.  On the other hand, with the SNALP-formulated PHD siRNAs most of the knockdown can be directed at the desired liver so that the numerous on- and off-target toxicities in the rest of the body become irrelevant. 

4. Being an exclusively intracellular target means that it is not druggable by monoclonal antibodies = less competition. 

5. Recombinant protein therapy (e.g. rhEpo) often involves periods of supraphysiologic exposures which can be associated with adverse events (this is thought to be one problem with rhEpo therapy).  By contrast, (genetic) PHD knockdown allows for more physiologic regulation of the targeted biological pathway making it a potentially safer and also more efficacious therapy.  This advantage of affecting a more physiologic pathway compared to the competing recombinant protein approach is largely the result of having a larger target space to choose from.

Summary of paper

The motivation for the study by Querbes and colleagues is the fascinating insight that even in adulthood the liver continues to be a potential source of physiologically relevant levels of erythropoietin (Epo), a key red blood cell-stimulating factor that is almost exclusively produced by the kidneys in adults under normal conditions.  Nevertheless, it is the liver that is the site of Epo production until around birth when erythropoiesis switches from the liver to the bone marrow. At that point, hepatic Epo transcription is suppressed by the degradation of the important Epo transcription factor HIF following the activity of PHD enzymes 1, 2, and 3.  When these enzymes are removed, e.g. by genetic excision, the liver can produce similar levels of Epo as the kidneys even in adult mammals.

Clearly, constitutive genetic excision is not an option when it is important to achieve just the right level of a physiologic process (here: red blood cell volume).  The anemia drug development field in particular has been tarnished by companies and physicians having excessively pushed the use of high-dose recombinant Epo (rhEpo) and other erythropoiesis-stimulating agents (ESA).  In some patient groups, this has been shown to greatly increase the risk of thrombotic events and other side effects that may be unique to ESAs.

Oral small molecule inhibitors of HIF-related PHDs (HIF-PHIs) have been tested in clinicaltrials by Fibrogen for the treatment of anemia in end-stage renal disease along a more conventional treatment paradigm.  Although the clinical experience so far suggests a good safety profile, until larger patient numbers have been exposed for prolonged periods of time, there will always be concern about toxicities from the inhibition of on-target and off-target PHDs, especially in non-liver, non-kidney tissues.

By contrast, the SNALP-siRNA approach taken by Querbes and colleagues targets PHD1-3 almost exclusively in the liver by virtue of  short PEG-anchored SNALP* biodistribution.  Moreover, the nature of siRNA selection and off-targeting makes it much less likely that confounding or adverse off-targeting will be encountered.  

70-80% target gene knockdowns were achieved for PHD 1 and 2 and a somewhat less pronounced ~50% knockdown for PHD3 (probably due to a previously observed feedback mechanism) when measured across the liver in rodents.  Despite the incomplete overall knockdown, this resulted not only in several log-fold increases of liver Epo mRNA, but also pronounced, highly physiologically relevant levels of serum Epo.  These responses can be likely attributed to the RNAi Therapeutic candidate targeting negative regulators of gene expression.

Commercial potential

The anemia market has long been dominated by rhEpo and related protein-based ESAs.  Only recently, a peptide-based ESA by Affymax (Omontys/Peginesitide) was approved by the FDA as a new treatment option and should help bring down costs.

Although all these ESAs do a good job in elevating hemoglobin content, the opportunity for new agents is in doing just that, but with a better safety profile as all currently approved ESAs suffer from the above-described concerns arising from supraphysiologic exposures.  Thus, the hope with the new anemia drug candidates is that the targeting of alternative biologic pathways won’t suffer from the same limitations.  They may also increase the quality of the hemoglobin elevation, especially with regards to iron metabolism.

Despite the promising data, it is as yet unclear whether Alnylam will develop PHD inhibition to treat anemia.  Although refractory anemia is among the 5x15^TM programs, Alnylam hasindicated that it is more interested in modulating the hepcidin pathway for that indication (hepcidin affects erythropoiesis via iron metabolism).  I’m not sure though whether the choice is not partly 2due to the fact that Fibrogen seems to have a respectable IP position in PHD inhibition.  Incidentally, the corresponding author of the paper, Prof. William Kaelin from Harvard, has a financial interest in Fibrogen.  Also of note, some of the transgenic mice used in the study originated with Regeneron.

*Alnylam tight-lipped about specific SNALP formulation used in study

It is not surprising that, like all of Alnylam’s other commercially interesting applications, also the anemia program is based on Tekmira’s SNALP technology.  Because of the litigation between the companies, it is of interest that while the methods section described much of the SNALP formulation, it conspicuously failed to mention the identity of the ionizable lipid, although based on the observed potency it should be a ‘second’ or ‘third’ gen lipid:

‘siRNA Formulation in Lipid Nanoparticles

The LNPs were prepared with an ionizable lipid, disteroylphosphatidyl choline,

cholesterol, and PEG-DMG using a spontaneous vesicle formation procedure as

previously described at a component molar ratio of ~50/10/38.5/1.5 Ref 17,18.’ 

Ref 17: Akinc A, Querbes W, De S, et al. Targeted delivery of RNAi therapeutics with

endogenous and exogenous ligand-based mechanisms. Mol Ther. 2010;18(7):1357-1364. Study on the ApoE-mediated mechanism of SNALP delivery to hepatocytes (Tekmira claims this was their trade secret which Alnylam chose to publish and claim to be its own insight)

Ref 18: 18. Semple SC, Akinc A, Chen J, et al. Rational design of cationic lipids for siRNA delivery. Nat Biotechnol. 2010;28(2):172-176. Rational SNALP design paper by Tekmira (the contested ionizable MC3 lipid was derived from the rational design approach among others)

Alnylam said that it was planning on partnering the anemia program for phase I studies.  By disclosing their intention to partner at such an early stage, it almost seems like they have been in advanced talks with potential partners.  Otherwise, promising partners at such an early stage would only set the company up to disappoint.

I would therefore assume that Tekmira will be looking at this paper for evidence for whether, also in this case, Alnylam inappropriately shared SNALP reagents and insights with ‘3^rd parties’.  As a reminder, according to the Manufacturing Agreement between Tekmira and Alnylam, Tekmira is the sole SNALP supplier, also for pre-clinical research purposes, and Alnylam is prohibited from providing ‘third parties’ with such formulations (and insights).  

‘Third parties’ ought to include academic collaborators such as Prof. Kaelin and the other researchers from Harvard and MIT on this paper.  It is interesting to speculate that this was a reason for the secrecy around the ‘ionizable lipid’?     

MicroRNA-26 yet another Small RNA in the Fight against Liver Cancer

When you combine the fact that current systemic small RNA delivery technologies should work best for the liver and solid cancers, that RNAi opens up many of the well validated, but hitherto un-druggable cancer targets, and that endogenous small RNA regulatory pathways (microRNAs in particular) turn out to play central roles in cancer biology, then it should not come as a surprise that various Small RNA Therapeutics approaches are poised to greatly advance the care of liver cancer patients. Surgical resection is the most common treatment, while the pleiotropic small molecule inhibitor Sorafenib that has been shown to increase median survival from 7.9 to 10.7 months is the most advanced drug for this treatment. Clearly, in light of this and the over 600,000 deaths from primary hepatocellular carcinoma (HCC) alone each year, many of them in East Asia (HBV-related), and many more deaths from the metastatic spread of other cancers to the liver, the unmet medical need is significant.

This potential has not been lost on the RNAi Therapeutics industry. There are now a number of RNAi Therapeutics liver cancer programs already in or approaching the clinic, most prominently Alnylam’s VSP-02, but also others such as one by mdRNA. At the same time, there is increased validation for microRNA-based therapeutics for liver cancer both of the antagonist type (e.g. Rosetta-Regulus collaboration) and the agonist type. In the case of the latter, two recent high-profile papers in Cell (Kota et al., 2009: Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model) and the New England Journal of Medicine (Ji et al., 2009: MicroRNA Expression, Survival, and Response to Interferon in Liver Cancer) suggest that microRNA-26 mimicry is a serious contender.

Kota and colleagues got started after making the observation that miR-26a is strongly down-regulated in a mouse model of liver cancer driven by the myc oncogene. Importantly, miR-26a levels were subsequently found to be also reduced by about 50% in liver cancer samples compared to the matched healthy liver tissue. While global down-regulation of microRNAs is a well known general property of cancer, what made miR-26a a particularly attractive candidate for microRNA replacement is that a) it is a broadly expressed microRNA and over-expression in non-target cells therefore should be well tolerated; and b) its over-expression in a liver cancer cell line decreased cell proliferation which was attributed to miR-26a directly targeting the cell cycle regulators Cyclin D2 and E2.

They then chose AAV technology to deliver a microRNA mimic to the livers of the same mouse model. AAV delivery to normal liver can be extraordinarily efficient, with essentially 100% transduction using so-called self-complementary vectors in mice. The track record for HCC has been more mixed, but using a self-complementary vector of the AAV8 serotype the authors achieved more than 90% transduction. To their great satisfaction, tumor growth was greatly retarded following systemic administration of the mimic compared to an expression vector in which the microRNA cassette had been deleted (60% tumor burden to 20% tumor burden). Importantly, whereas there was wide-spread apoptosis in the liver cancer, the surrounding normal tissues and more distant tissues with a high proliferative index (e.g. testis) appeared to be untouched. This was the first demonstration of efficacy for a systemically administered microRNA mimic that was not targeted against the cancer-initiating oncogene itself.

Further support that miR-26a is a good candidate for mimicry in liver cancer comes from a bioinformatic study just published in the New England Journal of Medicine which confirmed in a population of HBV-related HCC in China the down-regulation of miR-26 in liver cancer. Moreover, as predicted by the Kota et al. study, liver cancer patients with the least amounts of miR-26 had a worse prognosis. On the other hand, it is the same miR-26 lo patients that benefited most from interferon-alpha therapy. Such a finding may have its clinical use since for a drug with borderline efficacy like interferon in liver cancer. Being able to exclude those patients that will not benefit from a given drug may give them an opportunity to try other drugs or at least be spared of the side-effects. When used during drug development as a companion diagnostic, of course, such a test would moreover have major benefits for increasing the success rate of developing a drug candidate. Companies in the microRNA Rx/Dx space like Rosetta Genomics, Asuragen, and Regulus may want to take a look at being part of a companion Dx-Rx development program where patients are selected for a miR-26 mimicry liver cancer trial based on their miR-26a status. More generally, I shall look forward to seeing a wider adoption of microRNAs as companion Dx and, looking out from my soapbox, even a tighter integration of microRNA Dx/Rx capabilities.

In summary, miR-26a mimicry is yet another promising small RNA approach to treating a disease with a high unmet medical need, not just sometime in a distant future, but very tangible. Again, the lesson here is that once a given organ can be addressed by small RNA delivery, a flood of therapeutic targets immediately become available to keep Small RNA Therapeutics busy for some time on this one organ alone.

Journal Club: Therapeutic RNAi Delivered via Vitamin A-Coupled Cationic Liposomes Reverses Liver Cirrhosis in Rats

Illustrating how the basic concept of liposomal RNAi delivery can be modified to target specific cell types at low dosages, a study by Sato and colleagues published last week in the online version of Nature Biotech shows how the addition of vitamin A to cationic liposomes carrying siRNAs against Hsp47 can not only efficiently knock down the gene in hepatic stellate (HS) cells and block disease progression, but that this can actually reverse cirrhosis in a number of rat models for this disease [Sato et al. (2008). Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone. Nature Biotech doi:10.1038/nbt1396].

It is known that by adjusting their stability and therefore pharmacokinetics, liposomes can be adjusted to shift their relative accumulation in tissues such as liver, tumour, or lung. For example, recent improvements in formulation methods now allow for the delivery of around 90% of the injected dose to the liver. While for liver applications, this already minimizes the exposure of non-target organs to the RNAi therapeutic, the ability to knock down genes in specific cell types at low dosages should further improve its therapeutic index.

Adding small conjugates such as sugars (e.g. Mirus Bio’s DynamicPolyConjugates to target RNAi to either hepatocytes or Kupffer cells) to a basic delivery formulation appears to be particularly promising. The authors of the present paper reasoned that since the HS cell which is central to liver cirrhosis efficiently takes up vitamin A, adding this vitamin to liposomally formulated siRNAs may facilitate their uptake in HS cells, in addition to endowing the nanoparticle with favorable pharmacokinetics by binding to retinol binding protein in circulation.

Indeed, while some non-specific uptake was observed into phagocytic cells, the modified A-liposomes efficiently entered HS cells in a vitamin A-dependent manner while largely avoiding other cell types of the liver, including hepatocytes. Actually, delivery was truly targeted since overall drug uptake was greatly enhanced in cirrhotic rats versus non-cirrhotic rats, likely a reflection of the HS cell activation/proliferation state.

Importantly, knockdown of Hsp47, required for collagen production and therefore a potential target for treating cirrhosis, was achieved at the clinically relevant low dosages of 0.1mg/kg to 0.75mg/kg, resulting in the survival of rats concomitant with resolution of the cirrhosis both by histology and normalization of liver enzymes.

The paper also shows that as we are learning more about the molecular biology and pharmacological properties of synthetic RNAs, the scientific standards for publishing papers on therapeutic RNAi have risen. Consequently, the sequence-specific nature of the therapeutic effect was demonstrated by the use of two additional Hsp47-targeted siRNAs. Cytokines and interferon-alpha were also looked at, including IL-12 which has recently gained notoriety
as a TLR3-related pro-inflammatory cytokine. Reassuringly, no IFN-alpha, TNF-alpha, or IL-12 induction was detected at the reasonably early 7 hour time-point with this unmodified Dicer-substrate (Dicer substrates are longer and therefore should be more prone to immune recognition). However, some non-specific elevation in the apoptosis of rat liver HS cells was observed following treatment with VA-liposomes carrying a control siRNA. Albeit small, it remains to be determined whether this was related to sequence-specific cytokine induction, or liposomally or vitamine A-induced cytotoxicity.

It is likely that the systematic screening for improved VA-liposome chemistries and optimized siRNAs should considerably improve the therapeutic index of this interesting variation on the cationic liposome for siRNA delivery. Similarly, vitamin A derivatives that are still taken up by HS cells, but do not cause potential vitamin A-related toxicities may be warranted for actual clinical use.

A Framework for Progressing RNAi Therapeutics into the Clinic

This week was quite busy for the RNAi Therapeutics space with important corporate developments for CytRx/RXi (succesful spin-out/IPO), Nastech/mdRNA (announcements of patents filings and scientific advisory board), Silence Therapeutics (extension of AstraZeneca collaboration to Atuplex delivery), and Arrowhead Research/Calando (filing of first cancer IND). I hope to provide a wrap-up soon, but as I am contemplating the further developments in delivering RNAi to the liver, I would like to use the clinical development of liposomal RNAi as a case example of how one might think about development timelines for RNAi Therapeutics in general. While this discussion focuses on SNALP/liposomal-siRNAs just because it is an area that I am following more closely than others, it can easily be translated to for example to PEIs and delivery to the lung, or for that matter AAV/lentivirus and delivery to the brain.

RNAi Therapeutics are thought to speed up drug development, because once a target is identified, finding an appropriate siRNA should be a straight-forward process. However, it is clear that right now developing ways to get the RNAi triggers into the cells is the rate-limiting factor, meaning that the benefits of rapid drug development won’t necessarily all be realized for a good fraction of the first crop of RNAi Therapeutics. In order to assure that the next batch may more readily move through development, it will therefore be important to establish a number of predictable delivery technologies for the different target tissues. Once such a delivery system has been identified for a given tissue, virtually any gene of interest could be targeted in a manner that reduces development risk mainly to target risk (should be quite low given the virtually unlimited target space of RNAi) and safety profile of the particular RNAi trigger (particularly due to the less predictable off-targeting effect).

The question for Alnylam now is whether to choose liver cancer or hypercholesterolemia as the indication for their first SNALP/lipidoid-RNAi IND. Assuming that they feel quite confident with the gene targets and that they intend to use the same delivery formulation since the target tissue is fairly similar (normal versus tumour liver tissue), why choose one over the other? As we know, safety and finding a SNALP/lipidoid formulation with a good therapeutic index is as much of a concern as is efficacy. One could argue that starting with liver cancer may be a less risky business decision since the length of dosing and nature of the disease should be more forgiving in terms of the tolerated safety profile. By contrast, to derive a cardiovascular benefit through cholesterol lowering requires long-term chronic treatment which is why tolerability is so important for this indication. On the other hand, the PCSK9 program would provide a host of information as to the efficacy of this novel delivery system. Circulating PCSK9 levels could be readily measured and this information be used to adjust dosing strategies not only for the late-stage development of this particular program, but also all the other programs, including liver cancer, making use of the same delivery system.

I can imagine that the decision won’t be an easy one to make, but with improvements in the therapeutic index of SNALP-RNAi, the PCSK9 program becomes relatively more attractive. If successful, it should be possible to realize the anticipated advantages of RNAi Therapeutics in terms of increased development speed. Although true for drug development in general, from a technology and business development point-of-view it is therefore particularly important for the field of RNAi Therapeutics that its early clinical candidates, using unproven delivery systems, target genes where proof-of-concept can be established at an early stage.

Liposomal RNAi Delivery to the Liver Making Progress

[Disclosure: I chose to own both Tekmira and Alnylam stock, of which particularly Tekmira should benefit considerably from progress on liposomal RNAi delivery due to their IP position on cationic liposomes and relationship with Alnylam.]

Following my blog on the RSV-01 experimental infection results, I received a number of emails noticing my apparently less-than-usual bullish assessment of this Alnylam result, a concern heightened by a falling Alnylam share price.

The problem with calling the results a definite proof-of-concept for RNAi in humans is that this program targets a virus, and it is well documented that particularly unmodified siRNAs can have profound antiviral effects independent of their gene silencing activity, i.e. by inducing cytokine responses. However, I acknowledge that Alnylam and their clinical collaborators have done almost all humanly possible to (statistically) exclude the influence of various parameters such as inflammatory cytokines on the antiviral efficacy, although it is not clear e.g. when and where these markers were measured.

I guess I am just too much of a trained scientific skeptic here when I can agree with proof-of-concept for a clinical effect of an RNAi Therapeutic in man, but remain cautious on proof-of-concept for gene silencing in man. Maybe proof-of-concept should not be seen as a one-time event, but something that will emerge over time.

Especially with some of the other early clinical RNAi trials, one has to be aware that these were entered into the clinic before the entire scope of non-silencing activities of unmodified siRNAs were known (and some of the trials lacking supporting sound scientific data, btw). For those that would like to learn about this topic, I would warmly recommend the freely available, and very critical review article by Protiva’s Adam Judge and Ian MacLachlan from Protiva on the immune-stimulatory potential of siRNAs, including their non-specific antiviral and antiangionic (e.g. relevant for wet AMD applications) potentials. It is important to note that critics, also called “shorts” in stock market lingo, may exploit these issues to portray them as fatal short-comings of the RNAi Therapeutics platform, but like delivery, there is every indication that these will be overcome in a timely manner and the field should not shy away from openly addressing them.

As I indicated in my 2008: Year of the Liver , I believe that the demonstration of RNAi gene silencing in the human liver may very well steal the thunder from the RSV program this year. It was gratifying therefore to hear at today’s Alnylam’s R&D Day that by working together with Tekmira and applying a systematic, industrial-type evaluation of different liposomal nanoparticle formulations (LNPs, aka SNALPs), the potency of these particles has been improved such that only 0.1mg/kg dosages can effect over 50% knock downs. This is very important since these are now concentrations that should be well below the concentrations previously associated with toxicities by these cationic liposomes and likely the cause for delays in filing for the much anticipated liver INDs. Not only was it then confirmed that an siRNA administration led to silencing over an entire month, but equally important was the demonstration that repeat-administration of these particles over 3 months was able to cause sustained gene silencing, meaning that neutralizing antibodies, typically facilitated by concomitant inflammatory cytokine induction, are not generated to blunt repeat administration.

As a result, Alnylam today gave the best indication thus far that they are now ready to enter the clinic with such a systemic RNAi formulation, most likely for treating hypercholesterolemia. In addition to demonstrating safety, this phase I study may indeed provide the most impressive evidence for RNAi gene silencing activity in humans simply by measuring the level of circulating PCSK9, the gene target of this program.

Btw, comparable 0.1mg/kg efficacy and repeat-administration data with the essentially same underlying SNALP technology was also reported on Protiva’s website which I give an ‘A’ for being scientifically engaging, and possibly an ‘A+’ when Protiva and Tekmira can finally come to the realization that it does not make sense to have two companies operating in the same city on the same RNAi delivery technology, while diverting money and attention on lawsuits fighting each other.

As you can see, despite the broad progress in delivering RNAi to many other organs and tissues, based on the available data, RNAi delivery to the liver (not only by SNALPs, but also an increasing number of competing technologies) appears to be the most advanced in terms of efficacy and should be one of the major drivers of the valuation of the RNAi (and also microRNA) Therapeutics space in the next 1-4 years by filling the clinical pipelines with high-quality, promising drug candidates.

2008: The Year of the Liver

It is true that next year could bring the first human proof-of-concept for an RNAi therapeutic. But since results from the experimental infection studies for ALN-RSV01 were originally expected in late 2007 and the results would be an overhang of work done in ’07, I find it more appropriate to pick a fresh candidate. It is also true that the lung may rival the liver in the number of programs moving into the clinic in ‘08 given recent data that suggest delivery of siRNAs to the lung by nebulizer to be fairly innocuous, and the fact that AstraZeneca and GSK should be busy working in that area following their agreements with Silence and Sirna Therapeutics, respectively.

Nevertheless, I feel that most systematic progress has been made in delivering RNAi to the liver with more progress likely to follow. There have now been a fair number of publications that clearly show target-specific RNAi knockdown in the liver, unlike maybe some of the lung studies where there were occasional data interpretation issues. Of course, in declaring 2008 ‘The Year of the Liver’, I should disclose that I am somewhat biased due to the hepatotropic focus of the laboratory that I work in.

For siRNA-mediated RNAi in the liver, I currently see three front-runners, although it is possible that among the considerable work that is ongoing and not yet published, others have reached a similar stage of technological maturity. These are the in-famous SNALPs, Mirus Bio’s “Dynamic PolyConjugates”, and MIT’s lipidoids. SNALPs, being worked on by Protiva, Tekmira, Alnylam, and Sirna/Merck, are probably 12-18 months ahead of the game and best characterized, whereas the data on PolyConjugates and lipidoids are very promising, but still somewhat spotty.

SNALPs, stable nucleic acid lipid particles, are set to enter the clinic in 2008 with programs in hypercholesterolemia and liver cancer (both Alnylam) and possibly a clinical candidate chosen by Tekmira. The hypercholesterolemia program is also an opportunity to get an early measure of therapeutic efficacy, possibly in ‘08. Protiva’s and Sirna/Merck’s intentions are less clear and I believe that Big Pharma often chooses to keep phase I programs secret for competitive reasons, so that it is theoretically possible that Sirna has already entered the clinic or is about to do so with the long-anticipated SNALP RNAi for Hepatitis C.

It is obvious that there were some delays in bringing SNALP RNAi to the clinic, and I believe that this is largely due to dosing and safety issues. Both of these concerns may come down to the propensity of SNALP liposomes to be taken up by immune cells such as Kupffer cells in the liver and plasmacytoid dendritic cells which a) function like a sink for the liposomes when they enter the liver so that SNALPs become available for entering the desired hepatocytes only after the sink is saturated, something that complicates dosing; and b) increases the risk of triggering unwanted cytokine responses. I am optimistic, however, that by varying the composition of SNALPs, safe and efficacious formulations may be found, particularly if the liposome uptake mechanism by the professional immune surveillance system were to be different from that by the hepatocytes, which I think is quite reasonable to assume. I therefore hope that the fact that so many scientists are working on SNALP RNAi is a sign of its promise rather than desperation. As such, the number of R&D staff at Tekmira has more than doubled from 17 to 39 in the year ending September 2006 largely due to work on SNALPs.

As I have written before, I am much taken by Mirus’ PolyConjugate work, although this is based on only a single paper published in the middle of ‘07 (see 24 July 07 Blog: “Mirus Scientists Publish Elegant Paper on Targeted siRNA Delivery to Hepatocytes”). The neat aspect of that work was that it showed that it should be possible to avoid the Kupffer cells in the liver and specifically target hepatocytes for knockdown with the help of carbohydrate ligands (I am curious whether similar targeting ligands would also work in the context of other formulations). As we know, this work is partnered with Pfizer, and I wonder whether the upcoming loss of exclusive marketing rights for their wonder-drug Lipitor will spur them into action here.

I am still waiting for more data on lipidoid-mediated RNAi which hopefully will become available soon as was indicated in the footnotes of an October 2007 paper on the effect of siRNA delivery on microRNA function. From both a business and scientific perspective, it will be interesting to see whether Alnylam may choose lipidoids over SNALPs for its first liver RNAi programs and the overlap of SNALP and lipidoid both in IP and manufacturing terms.

The liver may get further attention from the targeting of microRNA-122 for the treatment of hypercholesterolemia and Hepatitis C, and other programs on targeting certain microRNAs for the treatment of hepatocellular carcinoma. There are various programs by Santaris, Regulus and others that have progressed into larger animals and we may even see a first IND being filed for one of these applications by the end of 2008. Development in the antisense field, particularly the partnering of ISIS’ mipomersen, should also generate heightened awareness for RNA-based therapeutics of liver disease.

Other predictions for 2008:

1) Unpredicted delivery technologies
2) Pfizer finally makes its move
3) News on RNAi for wet AMD- good and/or bad?
4) Protiva-Tekmira dispute resolved (wishful thinking)

For a nice presentation on the powers of SNALPs by Protiva chief scientist Ian MacLachlan, please visit: http://mms.technologynetworks.net/RNAi06Presentations/maclachlan/Player.html

Mirus Scientists Publish Elegant Paper on Targeted siRNA Delivery to Hepatocytes

The ability to systemically administer siRNAs and functionally modulate gene expression in tissues of interest is considered by many an important step to opening up the therapeutic potential of RNAi to a wide range of diseases. The liver is arguably the best example where intravenously administered siRNAs have already been shown to potently knockdown target genes from mice to non-human primates.

The most promising delivery technologies so far (next to gene therapy vectors such as AAV which I will not discuss here) involve liposomes such as the SNALP particles which were first pioneered by Protiva scientists and subsequently became the subject of intense legal battles involving Protiva, Tekmira, Sirna Therapeutics, and Merck. While facilitating highly efficacious gene knockdown, toxicities such as liver enzyme elevations and non-linear relationships between knockdown and siRNA dosage have been repeatedly reported with these chemistries. While the toxicities were observed at relatively high dose levels, non-linear dose responses complicate the choice of the right dose for entering clinical trials.

One step in the right direction was taken when Daniel Anderson from the MIT reported earlier this year at the Keystone meeting the identification of a slightly different class of compounds which they termed “lipidoids”. Importantly, this class of chemistries appear to efficiently promote RNAi gene knockdown with little if any apparent toxicities and linear dose responses.

The just released paper by Rozema and colleagues from Mirus Bio Corporation (Rozema et al. PNAS Early Edition 24 July 2007: Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes) sheds light on some of the issues above and holds out a new paradigm for achieving safe and efficacious therapeutic RNAi knockdown in select cell populations of the liver. Rather than regarding drug delivery to the liver as a passive process given that the bulk of intravenously injected drugs will pass through it with a relatively high chance of entering resident cells there, the present approach involves attaching a simple galactose-derived ligand thereby actively targeting asialoglycoprotein receptors (ASGPr) displayed on hepatocytes.

Knockdown of ApoB100 and microscopic analysis of fluorescently tagged small double-stranded nucleic acids confirmed that hepatocytes were indeed efficiently transfected. Equally important, however, was their observation that Kupffer cells, a major population of macrophages in the liver that are implicated in many cases of drug-related liver toxicities, did not take up the siRNA mimicks, whereas non-ASGPr targeted particles were able to transfect surrounding cells, including Kupffer cells.

Taken together with the SNALP and lipidoid data, this study supports the hypothesis that Kupffer cells may act as a sink for certain siRNA formulations such as SNALPs, thereby not only causing non-linear dose responses, but also an immunogenic response and related toxicities. It also shows that it should be possible to design simple and small siRNA nanoparticles for RNAi delivery from relatively cheap materials. Although lipidoids appear to be a clinically viable technology already, it is comforting to know that alternative routes exist and gratifying to see almost daily improvements being made in the RNAi delivery field.

PS: An interesting aspect of this publication was data the authors obtained on ApoB knockdown. In addition to serving as a target for early proof-of-concept studies for systemic RNAi delivery, ApoB has been a favourite for treating hypercholesterolemia using both antisense and RNAi. ISIS Pharmaceuticals in particular has an anti-ApoB antisense compound in late phase II clinical trials. However, only a month ago, Alnylam reported that in their hands ApoB knockdown led to unacceptably high levels of fat accumulation in the liver (fatty liver phenotype), a finding that was confirmed in the present study. Moreover, both reports indicate that this was a siRNA sequence specific effect and was achieved using two different delivery methods. This physiologic response makes a lot of sense, since ApoB’s main role is in the export of cholesterol and triglycerides from the liver. Failure to export them should accumulate them in the liver.

This is a good example where RNAi can serve both as a target validation tool AND a platform for developing innovative drugs. Consequently, I fully support Alnylam’s decision to target PCSK9 in their hypercholesterolemia program instead, which is a genetically well validated target for reducing LDL-cholesterol and heart disease (see also Blogs from 6 May, 2007: “Preventing Heart Disease with RNAi Therapeutics”, and 9 May, 2007: “ISIS Copies Alnylam’s Heart Disease Strategy“). It is curious then that ISIS maintains and has published the absence of such a phenotype. For the sake of patients and stakeholders in ISIS, I can only hope they are right. The explanation for the apparent discrepancy? I cannot really offer a good one except to speculate on a fortuitous ISIS 301012 antisense off-target effect.