Aptamer-Targeted RNAi Trigger Delivery

In honor of 25 years of aptamers, or better the SELEX process which underlies the discovery of aptamers, I thought it might be a good time to revisit aptamers for the delivery of RNAi Therapeutics.

Aptamers are nucleic acids that have been selected to preferentially recognize a target, usually a protein, via their 3-dimensional structure in analogy to how monoclonal antibodies recognize their targets.  Aptamers are showing most promise in therapeutic development for the targeting of extracellular proteins in the eye for applications like wet AMD and diabetic macular edema (see Fovista from Ophthotech). 

Its success for systemic applications has been much more modest, however, with short circulation times and unexpected adverse events in a recent phase III study (likely due to the PEG portion of the aptamer drug) largely accounting for it.

Aptamers have also been considered as cell-targeting agents for RNAi Therapeutics.  Early reports suggested efficacy in HIV and cancer models.  Skepticism around the on-target mechanism in these examples was considerable though largely due to questions around how they were supposed to escape the endosomes.

I also fell into the camp of doubters (and still have some reservations), but have adjusted my view to a more productive one after it became clear that IF you had highly productive endosomal uptake like ASGPR/GalNAc and a highly stabilized RNAi trigger, gene silencing is possible even without explicit endosomal release chemistry.

Time to try the next iteration: Aptamer-DPCs

As there may not be another ASGPR-type receptor in the body and to compensate for lower drug exposure compared to the liver, in the quest to make aptamer-delivered RNAi Therapeutics more robust, the new learnings of RNAi trigger stability are probably best applied within the context of DPC delivery technology by Arrowhead Research.

Accordingly, the perhaps 10x lower uptake in say PSMA-expressing prostate cancer cells will be compensated by adding the RNAi trigger-aptamer complex (as one or separately) to a masked endosomal release polymer.  In case that the target cell receptor is only abundant, but does not support productive endosomal uptake, another aptamer may target a second co-receptor on the same cell (akin to some bispecific antibodies, co-receptors in viral cell uptake).

Following endosomal uptake, the masking groups come off, endosomal permeability increased so that the RNAi trigger may escape into the cytoplasm.  In certain configurations, a Dicer substract-type RNAi trigger structure may simplify design and increase stability.

Dicerna Keeps Searching for Its Identity

Dicerna Pharmaceuticals recent move from Watertown to Cambridge is symbolic for its continued search for a place in the RNAi Therapeutics landscape.  Following some setbacks in its cancer and home-brew LNP efforts, the company now pins its hope on that it can compete head-on with Alnylam in the development of GalNAc-RNAi trigger conjugates for gene knockdown in the liver.

Oncology on hold

Like others in the field, confidence in its cancer program (DCR-MYC in phase I/II studies for solid cancers and HCC) seems to be low.  In the absence of clear-cut early development-stage cancer responses and confirmation of bona fide tumor-wide gene knockdown, cancer drug development remains a hit-and-usually-miss for the Oligonucleotide Therapeutics industry.

As a result, Dicerna seems to view their own mouse data with skepticism just as I myself have yet to see data supporting tumor penetration and bona fide knockdown in well-controlled studies.  The company has to be credited that it is now setting the bar for DCR-MYC quite high when clinical data from higher-dose cohorts is expected to emerge around year-end.  If DCR-MYC does not make the cut, Dicerna will likely cut its losses in cancer drug development and LNP research in general.

DCR-PH1 close call

Dicerna management was also surprisingly frank about their hesitations about the technical success of their most interesting current program, namely DCR-PH1 for the treatment of hyperoxaluria type I. 

After reviewing the latest non-human primate studies, it now appears that at least an 85% mRNA knockdown of the HAO-1 target gene will be required to see the key oxalate biomarkers ‘move’, and over 90% for more robust movement.  Based on rodent data, the company had thought that 75% might be sufficient.

In NHP studies of DCR-PH1, an 84% average peak knockdown was seen following a single dose of 0.3mg/kg of a Tekmira SNALP LNP formulation with 68% knockdown remaining at week 4.  0.3mg/kg seems to be the current well-tolerated upper dose of Tekmira’s LNP formulations and almost identical (protein) knockdowns were observed with 0.3mg/kg of Tekmira LNP-formulated ALN-TTR02. 

In clinical 3-weekly multi-dose studies of ALN-TTR02, this translated into sustained 80-85% target gene knockdowns.  This means that Dicerna now relies on the safety of DCR-PH1 to allow for doses of around 0.5mg/kg.  Not impossible, but probably a close call given the history of SNALP LNP and further exposes DCR-PH1 to competitive threats.

GalNAcs coming

Given the stage of their internal cancer and LNP efforts, Dicerna is now pinning its hopes on taking on Alnylam with GalNAc-RNAi trigger.  This is where Dicerna is currently investing most of its R&D efforts in.

It has now disclosed non-human primate data from those efforts, with 5 consecutive daily doses of 2.5mg/kg GalNAc-Dicer substrates resulting in ~70% knockdown of HAO-1 2-3 weeks after this loading dose.  Given the larger molecular size of the extended Dicer-substrates versus Tuschl-type siRNAs, this corresponds on a molar basis to ~1.5mg/kg of Alnylam’s GalNAc-siRNAs.  

This is somewhat less than what Alnylam presented for their PH1 program at OTS 2014 (ED80s in rodents of ~2.5mg/kg weekly) and Dicerna's GalNAcs would seem to require some further refinements to be competitive.

But in this case, they will end up with something that has little pharmacological distinction, is 3-4 years behind Alnylam, which in turn is not shy to put legal/IP pressure on its competition.

In my opinion, Dicerna management and Board need to put in quality time to find their true identity.

Disclosure: I am short DRNA as a relative valuation short for my ARWR long position. DRNA has a slightly larger market cap than ARWR, but ARWR has a distinguished, more mature DPC pipeline with ARC-520 and ARC-AAT two attractive candidates in the clinic whereas DRNA has nothing in the clinic it apparently has confidence in.  It's possible that both stocks are grossly undervalued, but relative valuation is one of my main RNAi investment methods and this is why I'm applying it here. Nothing personal.

Arrowhead Publishes SubQ Delivery Technology to Go Beyond the Liver

In late 2012, Arrowhead Research shocked the Oligonucleotide Therapeutics world when it presented spectacularly potent and prolonged gene knockdown data in non-human primates using a subcutaneously administered single-molecule Dynamic Polymer Conjugate (DPC) formulation.  This arguably represented the most elegant delivery technology at the time.  Moreover, also due to its small, but not too small size (10-20nm) and slight negative charge, it provided us with a glimpse into the future of systemic RNAi delivery for regulating genes beyond the liver.

It certainly got my full attention and made me invest almost 100% of my stock portfolio back then.

Unfortunately, despite the validation in non-human primates which suggested clinical readiness would not be far off, the subQ DPC technology has seemingly struggled to reach clinical/commercial maturity. Not only Arrowhead’s lead development candidate, ARC520 for HBV, but also its second development candidate, ARC-AAT for AAT-related liver disease, was still based on the intravenously administered two molecule DPC version.  Although the reasons for the delays were never really disclosed, a few comments here and there made it seem very likely that chemistry and manufacturing issues were behind the delay.

Back to the Future

Last week, Arrowhead Research finally published a paper showing that single molecule DPC is still alive and kicking (Rozema et al.2015) and is progressing towards clinical application.  In essence, the new single-molecule subQ DPC prototype comprises of a membrane-active polymer which has been masked from premature cytotoxic interactions by pegylation and cell-targeting ligands that are added via protease-sensitive bonds; as before, the highly modified/stabilized RNAi triggers are appended by disulfide chemistry. 

The DPC is made in a 4-step process followed by a purification step to remove unwanted side-products and reactants.  The latter step is apparently important when going into primates.

The new old DPCs are thus distinguished from the intravenous version not only in that it combines the RNAi trigger and endolysosomal release polymer in a single molecule, but most importantly by the nature of its triggered release mechanism.  Whereas in the former DPC generations triggered release was dependent on changes in pH such as they occur when a DPC is endosomally taken up, they are now responsive to the presence of certain proteases in lysosomes. 

pH-dependent formulations apparently suffered from instabilities both in the body and during storage.  This was adequate for targeting genes in the liver because of the ready access of macromolecules in the circulation to this organ following intravenous administration, but not when the DPC first has to reach the circulation from the subcutaneous space and when less well accessible target organs are the ultimate destination.

Accordingly, non-liver single-molecule DPCs of the latest publication had impressive circulation half-times of the intact, protected molecule of 11 hours.  Similarly, such DPCs are stable for at least a year both in solution and when lyophilized. 

The extra-hepatic potential thus facilitated by increased stability now needs to be demonstrated by finding suitable targeting ligands and I’m sure Arrowhead has been busy working on that.  It should be noted that for target tissues where high concentrations comparable to the liver are unlikely to be achieved following systemic delivery, the extra kick that comes from an explicit release chemistry could provide a critical advantage over competing approaches.  These include simple conjugates of the GalNAc-type and probably also self-delivering RNAi trigger chemistries which incorporate ‘milder’ release chemistries (like lipid tails).

Knockdown lasting for weeks and months

The most impressive demonstration of the single molecule DPC performance in the Rozema paper came from the primate studies.  Here, a single administration of 0.5mg/kg 2’-O-methyl/F-modified RNAi trigger led to a highly potent knockdown (peak knockdown >95%) of liver expressed Factor VII with >80% knockdown of 2 and 4 months following subcutaneous and intravenous administration, respectively.

Following the 2012 delays and some uncertainties around what was really new and old in the recent publication, I am somewhat hesitant to declare that subQ DPC is now fully de-risked and ready-to-go.  In that regard, it would be helpful to learn more about the tox profile of the new molecules and related to that which polymers will be eventually used (e.g. 2-molecule with melittin-like peptide, a polyacrylate in the publication).

Nevertheless, since Arrowhead has said that the new 2015 development candidate may be from the subQ line of DPCs (or if not going after a extra-hepatic target) one would think that the most important challenges have now been overcome.

Alnylam’s Scientifically Dishonest GalNAc Claims

Arrowhead Research, as the successor of Mirus Bio, can regard itself as the father of GalNAc-conjugated RNAi delivery.  Accordingly, in 2007, Rozema and colleagues published a seminal paper in which a multivalent polymer-conjugated GalNAc construct was utilized for the hepatocyte-specific delivery of RNAi gene silencing.

In 2015, Alnylam likes to be recognized as the inventor of GalNAc-oligonucleotide Therapeutics, with competitors like Isis Pharmaceuticals and Solstice Biologics playing the roles of copy-cats, and Arrowhead Research failing to get much mention at all.

This, however, is as noted in the introduction far from the truth, and a recent paper on ‘sequentially assembled’ GalNAc-RNAi triggers by Alnylam (Matsuda and colleagues, 2015) is yet another example for how they would like to re-write history to suit their (IP) goals.  History repeating itself you might think after all we’ve been through with SNALP LNP.

Matsuda re-discovering Rozema

Alnylam likes to laugh off Arrowhead’s GalNAc approach by claiming that you need a magic triantennary GalNAc ligand design with highly specific geometry to achieve tight ASGPR target receptor binding and subsequent cellular internalization.  By contrast, Arrowhead Research would be only using monovalent GalNAc which are known, in isolation, to be much poorer ASGPR binders.

The existence of the triantennary design obviously has not evaded Arrowhead Research.  Nevertheless, they have opted for monovalent GalNAcs most likely for their chemical simplicity and therefore reduced cost of goods.

The apparently high cost of triantennary GalNAc synthesis was acknowledged in the Matsuda paper and was said to be the motivation for testing RNAi triggers in which instead of a single triantennary ligand, monovalent GalNAcs were distributed along the RNAi triggers.

Short story short, having GalNAcs conjugated on 3 sequential nucleotides or every other nucleotide did not impact potency much compared to the triantennary 'parent' design.  In other words, the benefit from multivalent binding can be achieved by bringing monovalent GalNAcs together in space.

This, of course, is the same principle behind the Arrowhead approach, where GalNAcs are added to the free amines along a polymer/peptide (an RNAi trigger is just another polymer).  In the case of the melittin-like peptide, I have highlighted the basic amino acids to which GalNAcs are expected to be conjugated:

NH-LIGAILKVLATGLPTLISWIKNKRKQ-COOH

As you can see, towards the C-terminus (right hand side) of the peptide, there is a cluster of 4 positively charged amino acids that is expected to generate a multivalent ASGPR binder (note that 3 and 4 GalNAcs have similar binding affinities).

Shockingly, while masquerading as the inventors of GalNAc Oligonucleotide Therapeutics with statements like these…

‘The triantennary GalNAc ligand was subsequently used for hepatocyte-specific delivery of antisense oligonucleotides and short interfering ribonucleic neutrals (siRNNs) in mice, and anti-microRNA therapeutics in humans, confirming the value of the parent trivalent design.’

…they failed to even cite the Rozema paper and went on to say that now (i.e. for the first time) they were going to test the hypothesis that sequential monovalent GalNAcs could do the same job.  This obviously is a clear case of willful scientific dishonesty in their campaign to re-write GalNAc history.

Silence Therapeutics not even a pimple

While the Matsuda paper is geared towards claiming the sequential GalNAc assembly idea and is an affront to Arrowhead Research, it is also a reminder that Silence Therapeutics has been similarly treated with disregard in Alnylam’s ‘invention’ of ‘enhanced’ GalNAc-siRNAs. 

This is because (like Arrowhead Research actually), Alnylam, at least in essentially all RNAi trigger examples in the Matsuda paper, uses the AtuRNAi trigger design, US patents of which claim 2’-O-methylation every other base with a staggered pattern as it regards the annealed guide and passenger strands.

It therefore looks more and more like Alnylam will have to approach Silence Therapeutics for a license sometime before enhanced GalNAc-siRNAs hit the market (at least 2 by 2020 according to Alnylam’s 2020 guidance), if not ALN-TTRsc already (~2017-8).  If Alnylam will have to approach Arrowhead Research for a license regarding GalNAcs, I do not know, but given Alnylam’s noise, worth paying attention to the intricacies of the various IP estates. 

Arrowhead Acquires 30 Alnylam Exclusive, Priority Target Picks and Plus More from Novartis

Arrowhead Research keeps mopping up the billions of Big Pharma dollars spent on RNAi Therapeutics R&D and IP.  After acquiring the Roche assets for dimes on the dollar in 2011, heralding Arrowhead Research becoming a real biotech company, it is now Novartis’ turn to give their RNAi assets to dedicated RNAi hands.

What Arrowhead bought

For $10M and $25M in cash and stock, respectively (representing a dilution of ~5%), Arrowhead research acquired

1)      new Novartis RNAi trigger chemistry that the company claims to fall outside competing RNAi trigger IP (thus avoiding milstone and royalty obligations);

2)      intriguing new RNAi trigger chemistry that supposedly enhances RISC RNAi effector loading of RNAi triggers in the cytoplasm and which  could enhance the potency and duration of gene silencing; and

3)      the RNAi-related IP rights that Novartis acquired from Alnylam in 2005, most notably the 30 target picks.

Arrowhead in the house

10 years ago, Novartis made headlines by getting access to 30 exclusive target picks under Alnylam RNAi trigger IP.  For the privilege of picking targets not only ahead of Alnylam, but also excluding Alnylam from these targets, Novartis paid $10M in cash and made a $58M equity investment at a 16% premium to the ALNY trading price back then (so say $20M overall), plus the usual biotech milestone (up to $700M) and royalty obligations.

At the time, Alnylam was criticized for selling much of the farm.  This is because 30 target picks might have been too much given the state of RNAi delivery technology at the time.  Archrival Sirna Therapeutics gloated that it would never enter into such broad sweeping deals and consequently started to win business from other Big Pharma names, culminating in the $1B acquisition by Merck in 2006.

Novartis had time until October 2010 to officially nominate its target picks. At the time, SNALP LNP delivery to the liver was the only game in town for clinically relevant RNAi delivery.  Therefore, if Novartis had any brains, it would have spent some of the picks on the juiciest liver targets in addition to their oncology dreams.  

Usually, I don’t give much credit to the critical thinking ability of Big Pharma, but given that a number of Novartis RNAi folks came from Sirna Therapeutics and had worked on liver targets such asHBV early on, it is a good assumption that, yes, a few targets are aimed at the liver.

Note also that Alnylam never entered the HCV drug development race, instead pointing to their unwillingness to compete with its microRNA joint venture Regulus Therapeutics for the target.  I never bought that argument and instead suspected that Novartis was on HCV.

This, of course, adds an interesting facet to the somewhat uneasy relationship between Arrowhead Research and Alnylam and how today's deal impacts Alnylam’s 3 STAr franchises, namely viral hepatitis, cardiometabolic, and orphan diseases.

Value of Novartis RNAi assets in the eye of the beholder

I fully expect the usual suspects to spin today’s news as Arrowhead Research (once again!!) acquiring assets that a Big Pharma had put on hold (in the case of Novartis in early 2014) and nobody else allegedly wanted.  This may be partly true given that Novartis did not appear to be successful at developing strong RNAi delivery technologies.  So the Novartis RNAi assets in isolation may not have been worth that much.

Arrowhead, however, is in a different position given that its DPC delivery technology is being validated in the clinic.  I expect the ARC-AAT results towards the end of the year to remove any doubt about that.  Moreover, Arrowhead is on track to commit its subQ DPC version into clinical development, instantly increasing the value of any cardiometabolic targets that Novartis may have picked.

This illustrates that for Arrowhead Research today was about expanding its RNAi trigger IP leverage in addition to increasing its chances of finding the best possible RNAi trigger against a given target from its broad stable of RNAi trigger structures and chemistries (usiRNAs, Dicer-substrates, canonical), and finally adding a unique RNAi pharmacology trick to its toolbox.  All of this to be married with its DPC delivery technology so that the result would be worth far more than the sum of its parts.

Today, we have only glimpsed part of the strategic and technological importance of the deal. Stay tuned as the movie unfolds.  Kudos to Arrowhead Research for making the bold, but mostly right strategic decisions.

The Tide May Have Turned for ARWR

Arrowhead Research emerged as a major RNAi Therapeutics player due to its- at least publicly- single-minded focus on HBV.  During this time (early 2013-early 2014) it saw a meteoric rise in its stock by more than 10-fold.

Management got so caught up by their own campaign of pushing Arrowhead Research as an HBV stock that they set themselves up for failure by setting overly ambitious goals for that program. 

As a result, the stock plummeted almost as rapidly as it had risen first by the Fed-induced biotech sell-off in spring 2014, and especially after first clinical results (see here and here) of ARC520 in HBV-infected patients did not live up to the hyped-up expectations.  90% HBsAg knockdowns had been the stated goal for a single-dose 2mg/kg.  This was despite preclinical studies which suggested that more than 2mg/kg of the endosomolytic DPC component was needed to achieve such robust knockdowns.

While my jaws certainly dropped in disbelief when I heard this, in my mind this has to be chalked up to a lack of full understanding of their company's own technology rather than gross misconduct.

…but for me it has always been subQ, subQ, subQ, extrahepatic

While I very much liked the fact that Arrowhead Research was at the very cutting edge of the ‘HBV-The-Next-HCV'  wave, what originally got me all fired up about Arrowhead Research was an OTS presentation in late 2012 where they presented impressive (robust and long-lasting) knockdown in non-human primates with a subcutaneous, most likely single-molecule version of their DPC delivery technology.  Knockdown that was more potent than anything out there (Alnylam GalNAc-STC at the time) combined with the convenience of subcutaneous instead of intravenous administration.  The latter is practiced with their more advanced two-molecule DPC version underlying ARC520 and ARC-AAT in the clinic already.

Single-molecule DPCs should also be the foundation for reaching tissues beyond the liver, making the transition back to single-molecule DPC all the more valuable.  Given that the liver has been solved for oligonucleotide therapeutics with Alnylam’s and ISIS’ GalNAcs, opening up new tissues to RNAi is obviously all the more attractive.

It is unclear what held the company back from taking the non-human primate achievements almost 3 years ago into the clinic.  Scale-up manufacturing issues rank highest on my list of possibilities.

Company guides for 2015 IND for either subQ liver or extrahepatic i.v. candidate  

During this week’s Q4 earnings conference call, the company indicated that they have finally achieved long-awaited technological breakthroughs so that we can now expect them to file an IND for either a liver target using for the first time a subcutaneous DPC formulation or an IND for an extra-hepatic target. 

Correction/clarification (2 Feb 2015): The company contacted me to clarify that what they said was that they will file an IND in 2015, and in addition to that, nominate a new development candidate that will either be extrahepatic or a subQ liver candidate.

Interestingly, if the extrahepatic program should make it to the finish line first, it would still be administered intravenously, which leads me to believe that it is a target in the kidney which I consider the only other obvious target tissue amenable to 2-molecule DPC.  If the target cell is not the proximal tubule cell, it would suggest that Arrowhead has identified a GalNAc-ASGPR-type ligand-receptor pair for the kidney.

ARWR 2015 playbook

Be it as it may, the prospect of both a highly competitive delivery technology for the liver and the availability of a new target tissue makes this a highly attractive re-entry point into ARWR.  At $6+ down from the mid $20s not even a year ago and with almost half of its valuation in cash, I do not see much downside from the 3-4mg/kg results of ARC520 to be reported in Q2 2015. 

Personally, I expect an 80-90% knockdown at 4mg/kg, but since I have no idea how the market would react to an 80% knockdown, the results are a coin toss to me, but with a somewhat larger upside (up to $14) than downside (down to $5) from here.

If the stock trades down, but somewhat dependent on the safety data, it may be an opportunity to snap up ARWR for the ARC-AAT phase I results coming up by the end of the year.  I consider ARC-AAT a very robust program with increased knockdown potency compared to ARC520 and much less ambiguity around what an X% knockdown means.

 

Right now, Arrowhead Research is an ARC520-only story and that should change once ARC-AAT becomes recognized as a medically and commercially very attractive product candidate (e.g. an orphan indication with an estimated 100.000 patient population in the US alone).  And I am convinced that I'm not the only investor to recognize subQ and extra-hepatic as the ultimate value drivers for ARWR all of which could propel the stock back to its 2014 highs over the next year.

Antibody-RNAi Trigger Conjugates Show Signs of Life

Despite jettisoning RNAi Therapeutics 4 years ago, the more innovative arm of Roche, Genentech, has continued to dabble in the technology.  In particular, it has been interested in applying its monoclonal antibody know-how, including antibody-drug conjugates (ADCs) to the delivery of RNAi Therapeutics.  A recent publication by Cuellar and colleagues provides insights into these efforts.

THIOMABs for the creation of drug-like conjugates

While antibodies have a relatively long history in the delivery of RNAi Therapeutics, some of the early findings were generally quite difficult to replicate.  Part of the problem may have been the fact the early efforts involved structurally ill-defined non-covalent protein-nucleic acid complexes held together by charge-charge interactions.

To get around this issue, Genentech applied their THIOMAB platform which allows for the covalent addition of therapeutic payloads at defined cysteine residues.  This process yielded THIOMAB-siRNA conjugates with one, or more often two RNAi triggers per monoclonal antibody.  To help visualize them, also for pharmacokinetic considerations, think of the monoclonal antibody part being ~13x bigger/heavier than each RNAi trigger.

Of note, the RNAi triggers were partially (~50% of residues) modified with 2’-O-methyls and 2’-F for stability (siSTABLE from Dharmacon).

Not all receptors are created equal

One of the reasons why I am picking out this paper for discussion is the thoroughness of the research presented.  For example, the Genentech researchers selected not just one, but seven distinct cell surface receptors for which various THIOMAB-siRNA were created which in turn were be tested in a number of settings.  The receptors were partly chosen to capture a range of cellular trafficking behaviors such as rapid lysosomal uptake, recycling receptors, and slow-turnover receptors.

Importantly, the research validated a critical rationale for the use of antibody-RNAi trigger conjugates namely uptake of the RNAi triggers that is dependent on the presence of the cognate receptor and covalent linkage to the antibody.

For all the conjugates (ARCs), the bulk was shown to accumulate in lysosomes regardless of presumed uptake kinetics.  Interestingly, despite the seemingly shared uptake pathway, if not dynamics, only two of the seven receptors were associated with gene silencing (TENB2 and NaPi2b, but not e.g. Her2). 

The degree of silencing was much weaker compared to when the same conjugates were lipofected with ~50% silencing starting to be seen at 10nM.  This was followed by a shallow dose-response plateauing at 70-80% silencing around 500nM.  Based on the data presented, it seems that limiting amounts of receptors were responsible for this. 

Selective accumulation in mouse tumor model

A highlight of the publication to me was the investigation of THIOMAB-siRNAs in a mouse tumor model.  Although you may consider a ~30% silencing (i.e. 70% expression of normal remaining) when you cherrypick tumor areas of most efficient delivery a somewhat disappointing outcome as was reported in this case, important lessons can be learned from that.

Firstly, THIOMAB-siRNAs only accumulated in the tumor when the tumor expressed the cognate receptor.  This is unlike nanoparticulate RNAi delivery to tumors which relies on a passive process of accumulation (the EPR effect).  This opens up the prospect of RNAi drugs with ‘cleaner’ delivery profiles with an increased margin of safety.

Selective accumulation for these conjugates also suggests that the ~180kDa macromolecules were able to relatively rapidly exchange between tumor interstitium and blood circulation despite their size.  Consequently, other conjugates in a similar size range or below, including Arrowhead’s DPCs, should be amenable to such ‘active targeting’ as well.

Nevertheless, despite this apparent agility, imaging techniques showed that tumor delivery was largely restricted to areas next to the vasculature.  This limitation in fact is what is also seen with nanoparticles which rely on the EPR effect.  So while the active targeting capability is an important step forward, tumor penetration issues remain to be solved.

Path forward

Despite the arguably underwhelming in vitro and in vivo knockdown results if you just look at the numbers, I am optimistic that the studies have further supported that systemic RNAi delivery can be applied beyond the liver, vascular endothelial cells, certain cells in the kidney and phagocytes.

What is missing in that particular piece of research making the conjugates that make it into the cells count.  Given the lysosomal accumulation, an obvious strategy to unlocking the true potential of ARCs would be to apply endosomal escape chemistries, especially masked chemistries such as in the DPCs by Arrowhead Research (actually specifically referenced by the authors).

In almost the same vein, the sparse chemical modification used mean that potency improvements will be gained if heavier modification is applied even without adding endosomal escape chemistries.  This is also because some of the data are consistent with a model whereby the THIOMAB-siRNA conjugates get broken apart in late endolysosomal compartments and it is from that population of freed RNAi triggers that escape into the cytoplasm may occur (note: this does not exclude spontaneous endolysosomal rupture as an additional escape pathway).  If RNAi triggers were made metabolically more stable through more extensive modification, the amount of RNAi triggers available for such escape would obviously be larger.

All that is lacking then is the issue of tumor penetration.  I could imagine, however, that some self-delivering and/or lipophilic strategies could be quite useful here.  The Genentech researchers actually speculate that the RNAi trigger may have been the culprit in limiting the claimed natural ability of monoclonal antibodies to more deeply penetrate tumors.  This suggests that charge masking may overcome this limitation.  

I am open to this idea, but based on my understanding of the literature, tumor penetration is a challenging issue for basically all therapeutic modalities, monoclonal antibodies and 'even' small molecules, so let's not blame double-strandedness for all pharmacologic problems.

With interest in RNA Therapeutics growing by the day, expect lines of delivery research such as this one to be picked up and pushed to the next level.

PS: for antibody-mediated RNA delivery, Avidity NanoMedicines and the Roche-ISIS collaboration on CNS delivery should also be worth watching.

January RNA Therapeutics Deal Frenzy Kicked Off

As is now tradition for Januaries in RNA Therapeutics, expect this month to be filled with mouthwatering business development announcements.

Without a doubt, privately held messenger RNA Therapeutics company Moderna Therapeutics stole the show today by announcing a $450M financing involving 'Viking Global Investors LP, Invus, RA Capital Management, and Wellington Management Company, LLP, as well as existing investors AstraZeneca and Alexion Pharmaceuticals'.

Biggest private financing in biotech history EVER.

With $800M in cash, close to a billion USD raised since starting up 2 years ago or so, the biggest challenge will be how to efficiently put the capital to good use and move into the clinic in the not-too-distant future.  Before that happens, we should be seeing one of the hottest biotech IPOs in 2015.

Before Moderna announced their financing, ISIS Pharmaceuticals was the talk of the day with their GI-related autoimmune deal with Johnson&Johnson.  Not only does the early development deal provide ISIS with another nice junk of upfront cash, this time $35M, and a gazillion in potential biotech milestone payments and royalties, jn practical terms I expect the research to be particularly useful to ISIS as it develops oral delivery for systemic applications (think lipid franchise for which the new CEO will be officially enthroned tomorrow).  

I believe that the deal was primarily driven by JNJ's interest in the space following the whopping $710M upfront deal by Celgene last year when it acquired rights to an antisense compound developed by oligonucleotide therapeutics nobody Nogra Pharma for IBD applications.  The high upfront payment indicates that competitive bidding was involved and it is likely that JNJ lost out.

Remember, Big Pharma moves in herds.

The good news for JNJ is that if the first-generation phosphorothioate DNA is indeed working by an on-target mechanism (which I doubt), then a competing compound employing much more advanced chemistry and design by ISIS Pharmaceuticals will be infinitely more potent and beat Celgene in the market.  And for $35M and $800M in milestones if everything goes according to plan, it's a comparative steal.

For ISIS Pharmaceuticals it is just another step to broaden their expertise and applications of their platform.  Moreover, the upfront payment will help the company to remain cashflow neutral to slightly positive until 2017, at which time I expect drug sales to be finally taking off.

Stabilizing RNAi Triggers against Cytoplasmic Degradation Pays Dividends

In describing the preliminary phase IIa results of ARC520 for HBV, Arrowhead Research noted that the duration of gene silencing (2 months and more) was surprisingly extended in Man compared to the preclinical experiences in rodents and non-human primates.  Alnylam hasnoticed the same with its GalNAc-siRNA conjugates, especially the highly modified ESC version. 

The extended gene silencing activities, of course, bode very well for RNAi Therapeutics in general when in the early days (~2002-2003) I was a bit apprehensive when gene silencing in my transfections of cancer cell lines persisted for only 2-3 days (as we now know largely due to their rapid cell division).  To maximize the duration of gene silencing, thereby opening up RNAi Therapeutics to new applications and increasing its competitive profile, it is important to understand the factors underlying it.

Alnylam explained the differences to the preclinical experience because rodent and monkey hepatocytes seem to have a more hostile, degradative cytosol compared to human hepatocytes (hypothesis 1).  In one experiment, only 6% full-length ESC-GalNAc-siRNA remained after a given time in rodent and monkey cytosolic extracts while in human liver cytosol extracts more than 60% persisted.

This, however, was only a correlation and I have considered it equally likely that the difference in gene silencing duration might be a function of more stable RISC complexes in humans (hypothesis 2) or increased stability in the endo-lysosomal compartment (hypothesis 3).  Especially for GalNAc-siRNAs, I would think that the reason that it works in the first place is due to them being able to accumulate in endo-lysosomes from which they only get released in the wake of natural vesicle membrane turnover.  So chemical stability here would be a critical factor since the endo-lysosomal compartment is known to be highly degradative.

DPC and SNALP: two endosomolytic technologies with different durations of gene silencing

While I still consider that endo-lysosomal stability of the naked RNAi trigger is critical for approaches like GalNAc-siRNA conjugates, the new DPC-enabled ARC520 results strongly indicate that another critical factor lies downstream of endo-lysosomes.  This is because in the DPC approach which involves strong endosomolytic activities that should activate soon after endocytic uptake, the risk of the RNAi trigger being degraded in the endo-lysosomes should be low.  Similarly, there should be little contribution to gene silencing from RNAi triggers that get released into the cytoplasm in a delayed fashion.

SNALP is another delivery technology where the RNAi triggers that become active in gene silencing get released into the cytoplasm soon after endocytic uptake.  However, while clinical data supporting 3-4 week dosing frequencies have been obtained with SNALPs (e.g. ALN-TTR02), the silencing does not appear to be as extended as with DPCs.  So given that one marked difference of the payloads used with SNALPs and DPCs is the modest degree of chemical modification historically used with SNALPs, this, too, points towards cytosolic stability of the RNAi trigger being important for the duration of gene silencing.  Parenthetically, it also suggests that Tekmira may want to similarly explore heavily modified RNAi triggers while being mindful not to step on the McSwiggen patent toes of Alnylam.

RISC-optimized ultra-stable single-strand RNAi triggers

In the case of traditional double-stranded RNAi triggers as e.g. used with DPCs, the stabilized RNAi triggers get used up over time as they are recruited into RNAi effector complex RISC.  Part of this process involves their unwinding into single-strand RNAs with the guide strand being retained.   It is known that once used, a 'normal' guide strand (or microRNA) is not recycled into another RISC complex and will likely suffer metabolic destruction once the protein components of RISC have become degraded as part of natural protein turnover.  And even if the guide strand had been stabilized, because a standard single-strand molecule that had relied on being part of a double-strand structure for RISC recognition, old age will eventually catch up here, too. 

What a waste after all this effort of getting the RNAi trigger into the cytoplasm.  So why not take a cue from the single-strand RNAi practitioners who optimize single-strand RNAi triggers also based on being able to be recognized by RISC?  If a corresponding dsRNA contained corresponding recognition elements, then the guide strand could contribute to another round of gene silencing, thus extending and enhancing knockdown.  On the other hand, the lessons learned from stabilized dsRNAi triggers should also benefit the single-strand RNAi approach as increased cytosolic stability should also increase their duration of activity: RISC-optimized ultra-stable single-strand RNAi triggers.

Leveraging New RNAi Trigger Chemistries for Gene Knockdown in Phagocytic (and Other) Cells

Much of the achievement of solid RNAi gene knockdowns in hepatocytes (liver) by non-LNP means (Arrowhead DPCs and Alnylam’s 2^nd gen GalNAc-siRNAs) has involved the use of heavy modifications that render the RNAi triggers highly stable. This is because nucleic acids that are not protected by the delivery chemistry itself would otherwise be subject to rapid degradation in extracellular body fluids.

In the case of Alnylam’s GalNAcs, they can even function in the absence of an explicit endosomal release chemistry.  Moreover, GalNAcs and DPCs have shown more sustained gene knockdowns than is achieved with LNP delivery which historically has relied on minimally modified RNAi triggers. 

All that is required is receptor-mediated uptake into the endosomal-lysosomal pathway which is an immensely degradative environment (esp. the lysosomes).

Considering the extended duration of silencing and degradative environment, it seems as if the RNAi triggers have to be able to survive for long enough in late endosomes/lysosomes so that when they get an opportunity to escape by as yet undefined mechanism(s), they are still there ready for gene silencing action.

Such chemistry progress may also be particularly useful for RNAi gene silencing in phagocytic cells of the immune system.  This is because there have been multiple reports, especially concerning the use of LNPs (e.g. Novobrantseva et al.)  where some, but not the very robust, hepatocyte-type of gene knockdown have been obtained.  It is the ability to confidently achieve robust knockdowns that opens the gate to a flood of therapeutic applications, and this is why pushing borderline-technologies over the edge is so tremendously valuable.

Uptake into phacocytes is usually not the problem as these have evolved to scavenge for foreign particles and macromolecules.  In fact, phagocytic uptake is often a nuisance in RNAi delivery both because it may cause off-target toxicity and because it can make pharmacology less predictable.

In the case of untargeted nanoparticles such as LNPs, phagocytosis is the likely uptake mechanism.  Similar to endosomal uptake, phagosomal uptake involves the fusion with lysosomal compartments meaning that phagosomal contents are also exposed to a highly hostile environment.  Since the normal endosomal escape chemistries and mechanisms do not appear to be very effective in phagosomes, one strategy besides of possibly tailoring existing mechanisms to the phagosomal environment (e.g. lipid pKas) is to simply use the same ultra-stable RNAi trigger chemistries that are showing promise in the liver.

It is also possible that ligand-targeted conjugate approaches will be useful here, whether they enter the cells via phagocytic mechanisms or not.  In fact, Arrowhead Research (then Mirus Bio) in their seminal publication on DPCs (Rozema et al.) have demonstrated efficient uptake of DPC-conjugates into liver phagocytes (Kupffer cells) by using mannose as the targeting ligand (they haven’t tested and/or shown the corresponding RNAi knockdown though and this may relate to their using much less nucleic acid modification back in 2007). 

Since the mannose receptor is expressed on phagocytes throughout the body and not just in the liver, more extended circulation times promises the application of this conjugate-targeting strategy more generally.

Similar principles may apply to the targeting of other ‘frontier tissues’ for RNAi delivery.  However, given the degradative and differing nature of phagocytosis which means that other release mechanisms are not readily applied to this process, RNAi in phagocytes should particularly benefit from the new RNAi trigger chemistry developments.  I look forward to seeing the results.

ISIS Pharmaceutical Reveals Dynamic PolyConjugate Efforts

When Arrowhead Research made a splash 1 ½ years ago at the Boston OTS meeting in late October 2012 with impressive subQ DPC knockdown efficacy in monkeys, there was a large number of meeting participants crowding the speaker after his presentation.  Although I sat a few rows away, the most eager questioner was an employee from ISIS Pharmaceuticals who managed to beat the Merck representative to the podium!

There were times when I believed that the interest of large companies in the technology of small companies boded well for partnership potential.  It has become clear to me, however, that while this may be true to some extent, the first instinct by the large guys is to get close to the innovators so that they can appropriate as much of the technology as possible without paying a dime.

While I have long shelved Merck into that category and DPCs will now be a research priority at Alnylam following their acquisition of Sirna/Merck, a now published patent application by ISIS Pharmaceuticals on melittin-based, GalNAc-targeted single-stranded antisense delivery (ssRNAi and conventional ASO) demonstrates that ISIS ticks no different.

With a priority date of WO 2014/089146 A1 of December 4, 2012, this poor employee and collaborating patent agent had to stitch together a patent application in just a month after returning home from the conference.  Unsurprisingly, the patent application lacks any actual experimental data to support the 'ínvention'.

I don’t want to be too cynical about this as this is how the industry and the patent system function.  It is an important lesson for small companies though that they should not get overly excited about interest they generate for their technology and they should only engage in deeper relationships once they have established that the larger party is not just trying to steal their technology by pretending to collaborate with them.

It is this unwillingness to share and attendant mistrust that in my experience impedes much business development that makes great sense on paper.  This ultimately delays timelines, leads to litigation, and makes the pie smaller for everybody.

And for the future of gene knockdown in the liver…if ~5mg per week may be possible for GalNAc-targeted gen 2.5 RNaseH ASOs, one can only wonder what a melittin-type escape mechanism would add to its potency, and safety. 

Comment on Seeking Alpha bear article on ISIS Pharmaceuticals

Last night, a detailed bear article on ISIS came out that shook the market in after-hours trading.  In summary, the thesis rests on the poor safety profile, especially immunogenicity of gen 2.0-based KYNAMRO (2’MOE gapmer) and that this is likely to translate to all other gen 2.0 drugs based on shared chemical composition and the (in my opinion idiotic) claim by the CEO of ISIS that KYNAMRO is a success and is representative for gen 2.0.  Talk about shooting yourself in the foot.

While I agree that the KYNAMRO data have raised legitimate questions around the safety of gen 2.0 phosphorothioate oligonucleotides, the bear article conveniently ignores the abundant clinical data that have emerged since for a number of other antisense drugs based on gen 2.0 chemistry.  These support the claim by ISIS that through improved screening, they are now able to better weed out the sequences that will likely prove immunogenic in the clinic. 

Importantly, in oligonucleotide therapeutics in general, while nucleic acid chemistry has a great influence on whether a molecule is immunogenic, it is the exact sequence composition that ultimately decides whether this is actually the case.

This is illustrated by the recent phase II clinical results for ApoCIII (no discontinuations noted), Factor XI, and GCGR.  Not only were robust gene knockdowns achieved, in sharp contrast to KYNAMRO, the company claimed that they were no flu-like symptoms, chills and other symptoms indicative of the immunostimulatory potential of oligonucleotides.  By contrast, about 1/3 in the KYNAMRO studies exhibited such events, with even higher numbers in the open-label extension phase.

Granted, given that these were 13-week studies, it is impossible to disprove the thesis that things are bound to get worse over the long-run.  However, no flu-like symptoms versus 1/3 of patients exhibiting flu-like symptoms in studies of comparable duration is a dramatic difference and allows one to extrapolate that the safety of the follow-on drugs will similarly be greatly superior to KYNAMRO over time.

While I had been tempted to let the Seeking Alpha article by Dr. Anonymous pass as raising legitimate concerns, the blatant failure to mention the more recent experience with gen 2.0 and the after-hours action last night makes this article suspect and outright useless given the lack of new information or insight.

Game-Changing Potency Improvement for 2-Molecule DPC by Arrowhead

At the TIDES meeting last month, Arrowhead Research presented advancements with its intravenous 2-molecule DPC delivery approach for gene silencing in the liver. By adding a protease-sensitive hydrophilic extension to the endosomolytic peptide (MLP), the company has improved both the safety of the delivery approach, and even more importantly so, the potency of 2-molecule DPC. 

The new formulation can be expected to mediate deep knockdown at the 2mg/kg endosomolytic peptide dosage at which Arrowhead Research stopped their dose-escalation for the HBV product candidate ARC520 which is based on the original 2-molecule DPC chemistry.  The reason why I have come to be on the fence regarding the future of ARC520, especially in a competitive environment, is that I consider 2mg/kg insufficient to mediate the multi-log HbsAg knockdowns that might be desirable.  If they had increased the dose to 3mg/kg, or better 4mg/kg, I would have a much more positive outlook on ARC520 (maybe this is still in the cards pending the outcome of the phase IIa result in Q3).

It is important to keep in mind that with DPC, it is the amount of endosomolytic escape agent that is rate-limiting for knockdown potency and dose-limiting for safety. The amount of (otherwise very safe) RNAi trigger by contrast is in vast excess, just to make sure that no potency is wasted. With first-generation DPC 2.0 (2-molecule), it was from around 2mg/kg for both Factor VII and HBV mRNAs that target knockdown started to take off. The fact that the same can be observed for various target genes supports the notion that as long as you have a fairly potent RNAi trigger, it is the amount of endosomolytic escape agent that determines that depth of the knockdown.

There was no reason for Arrowhead Research not to explore higher doses if they had no trepidations about the safety profile. Somewhat contrary to this notion, the company had claimed seeing no preclinical tox at up to 10mg/kg.

Nevertheless, the thought process behind the improved 2-molecule DPC version leads me to believe that safety has been a concern. This is because the reason for adding the hydrophilic extension to the endosomolytic backbone was to limit non-specific, potentially toxic interactions with membranes outside the endosomes. This minimization of non-specific membrane interactions was confirmed in a test tube membrane interaction assay.

In a second step, Arrowhead scientists sought to get rid of the hydrophilic extension in the endosome so that it would not inhibit membrane disruption there. For this, the hydrophilic extension was rendered susceptible to cleavage by proteases that are found only in endosomes. With this trick, they regained the potency of the original DPC 2.0 version, but with the improved safety feature.

Going one step further and truly making lemonade out of lemons, they replaced PEG as the hydrophilic extension with more of GalNAc targeting ligand: whereas DPC 2.0 exhibited 40% and >90% gene silencing at 1 and 3mg/kg endosomolytic agent, respectively, the improved version DPC 2.1 achieved a >90% silencing at 1mg/kg already.

Given the increase in potency, even if the safety profile was not enhanced with DPC2.1, a conservative assumption, a 2mg/kg dose should now facilitate very potent gene silencing in the liver. This bodes particularly well for the 2^nd development program that Arrowhead will disclose this month. As a follow-up to ARC520 in HBV...?  I believe the company would be better advised to wait for a single molecule, subcutaneous version, the ultimate future of DPC.

Question of the day: why does adding more GalNAc to an already GalNAc-targeted molecule enhance gene silencing?  Could it be that they added a trivalent GalNAc (like Alnylam), and not more monovalent GalNAc as on the endosomolytic backbone?

Dicerna Should Focus on Conjugate Delivery

Barely 3 months a public company, Dicerna has yet to emerge as a distinct RNAi Therapeutics player similar to the likes of Alnylam (à subQ GalNAc), Arrowhead Research (receptor-targeted DPCs), and Tekmira (àhigh potency liposomal RNAi).  Without leveraging the potential advantages of its Dicer-substrate RNAi triggers on which it had been founded, Dicerna will be remembered by the market as not much more than an IP workaround play.

Currently, Dicerna is utilizing liposomal delivery for its lead programs in cancer and orphan disease primary hyperoxaluria 1 (PH1), a highly versatile approach, but a niche that might be better left to Tekmira.  At least for the greater good of RNAi Therapeutics investment dollars.

Instead, Dicerna should compete more directly with both Alnylam and Arrowhead in conjugate delivery.  This is because Dicer substrates allow for the liberation of the active small interfering RNA from the conjugated ligand and/or polymer backbone by Dicer directly chewing off the conjugate.  By contrast, for conjugates like current GalNAc-siRNAs or DPCs, removal of the ligand/backbone has to be accomplished via more complex or difficult-to-work-with linker chemistries including protease-sensitive linkers or disulfides.  Such removal is usually necessary to allow for efficient incorporation into the RNAi effector complex RISC. 

In fact, it may be this chemical attachment challenge that has delayed Arrowhead’s single-molecule DPCs which I am so keen seeing progress into the clinic.

Of course, similar to adopting liposomal delivery, becoming expert in conjugate delivery probably won’t happen overnight and requires the hiring of appropriate talent.  Competition for such talent ought to be fierce with both Arrowhead and and particularly hometown rival Alnylam well cashed up and even antisense company ISIS entering the field.

We have now well crossed the point that RNAi Therapeutics is ready to churn out drugs.  If each of the players were to focus on its core strengths, it would ensure that the reach of the technology will grow in a most efficient manner.  

GalNAc 2.0 with Greatly Improved Single-dose Efficacy and Duration

Last night, Alnylam kicked off a week of what promises to be exciting disclosures about continued progress in therapeutic gene silencing of genes expressed in the liver.  In a presentation by oligonucleotide star chemist Mutiah Manoharan at TIDES, the company provided a more thorough chemistry and pharmacology background behind the apparent improvements of the GalNAc delivery platform (Enhanced Stabilization Chemistry).

Journey along a hostile environment

The improvements are based on the observation that the 5’ ends of both the guide and passenger strands are subject to degradation by 5’-3’ exonucleases.  These may act at various stages during the relatively long journey of a subcutaneously administered RNAi trigger-conjugate: in the subcutaneous space, the circulation and lymphatics, and finally along the nuclease-rich endosomal/lysosomal uptake pathway in the target cell itself. 

Accordingly, by adding undisclosed chemical modifications to the 5’ (but also 3’) termini of the RNAi trigger strands, ~5 times the amount of RNAi trigger reaches the liver, and 10-100x RNAi trigger is found in the liver over time compared to first-generation chemistry as exemplified by ALN-TTRsc.  This means that single digit microgram per gram liver tissue can now be achieved at steady-state.  For comparison, gen 2.0 and 2.5 RNaseH ASOs (--> ISIS) depend for activity on ~100-300 microgram per gram liver tissue steady-state concentrations of phosphorothioated oligonucleotides. 

There obviously is a balance between maintaining high drug concentrations for efficacy and avoiding excessive concentrations for fear of causing inflammation and subsequent tissue scarring.  In that regard, Alnylam reports a wide therapeutic index, including in non-human primates which, laudably, were generally extensively used in these studies.

Great benefit for single-dose efficacy and duration

The new pharmacological profile is somewhat counter to a critical advantage of the RNAi platform over single-strand RNaseH technology: achieving great and sustained efficacy with minimal tissue exposure.  

Mechanistically, this fundamental capacity is explained by the fact that once loaded onto the RNAi effector complex, RISC, the duration of RNAi trigger activity in non- or very slowly dividing tissues such as the liver is largely limited by the slow (weeks) natural turnover of RISC.  By contrast, although RNaseH is a catalytic mechanism, too, no such sustained holding on to the antisense oligonucleotide is known for RNaseH such that the guide oligonucleotide has to be constantly available.

According to this model, an important determinant for the efficacy and very feasibility of traditional RNAi approaches is the size of the unloaded pool of RISC during the short period of time that an otherwise unstable RNAi trigger is available.  By contrast, unstable RNAi triggers are ill suited to take advantage of newly synthesized RISC complexes as part of natural RISC protein turnover.   

This is where GalNAc2.0 comes in: by extending the presence of the RNAi trigger, RNAi triggers can now also be loaded into newly synthesized RISC, thus extending the duration of gene silencing by replenishing the pool of RISC that gets lost during its turnover.  As discussed last week, in the case of ALN-PCSsc for the treatment of hypercholesterolemia, GalNAC2.0 can achieve sustained potent gene silencing of PCSK9 for 2-3 months following a single dose compared to only days/weeks with the old chemistry.  Moreover, when it comes to single-dosing schedules, GalNAc2.0 is also vastly (~10x)  in terms of maximal knockdown potency compared to GalNAc1.0 which relies on a loading dose schedule (5x daily injections) for efficient loading of free RISC.

ESC less transformational in multi-dose regimens

Somewhat lost in Alnylam’s press release was the fact that for multi-dosing, the benefit of GalNAc2.0 is less dramatic in terms of the amount of RNAi trigger required to achieve say a 80% target gene knockdown.  For example, for TTR, the ED80 with weekly GalNAc1.0 in non-human primates was ~2.5mg/kg, the same as that now reported for the ED80 with a GalNAc2.0-chemistry improved version in rodents.

This confusion was not helped by the fact that direct comparisons between GalNAc1.0 and 2.0 were only shown for single-dose studies or by the fact the efficacy summary slide compares GalNAc1.0 for TTR with GalNAc2.0 for PCSK9.

Therefore, when the goal is to enhance the target product profile of your RNAi therapeutic by minimizing the frequency of subcutaneous administration (e.g. PCSK9 in light of the monoclonal antibody competition), then GalNAc2.0 certainly represents a very valuable advance, albeit at the cost of (still) relatively large injection volumes (10mg/kgà 4ml).  However, when it comes to the maximal potency against a given target gene, similar results may be obtained with GalNAc1.0 with possibly an improved safety profile.

Of course, more potent and at least equally sustained efficacy following subcutaneous administration may be achieved by Arrowhead's single-molecule DPCs.  If and when they can finally be translated into the clinic, is an important and open question.  I hope we see more data on that this week, also from the TIDES.

Further Possibilities for Arrowhead Phase I Dose Extension

Last night’s speculations on the reason for the phase I dose extension of the HBV RNAi phase I trial by Arrowhead Research was actually positive: they are tackling the potency issue presented by the 2mg/kg dose. 

In addition to further increase dose, it could also involve prolonging drug infusion times which we know, based on Alnylam’s GalNAc data, could optimize hepatocyte uptake by the GalNAc-targeted DPCs.

Regardless, you have got to question why extending beyond 2mg/kg had not been part of the original plan and why the study extension has not been publicly discussed, e.g. in the latest conference call.   

Extending the study to further potency is the most rose-colored scenario that one can draw.  The other scenario would have to do with safety concerns, possibly raised by regulators.  As I had discussed before, for DPC, especially the 2 molecule version, the tox-limiting element is the melittin-like peptide.  Melittin is derived from bee venom and although the MLP is not the identical sequence as melittin, there are theoretical concerns around allergic reactions.  In general, having peptides involved raises a new set of immune issues, especially when you require 2mg/kg of them.

Of note, one of the exclusion criteria for the phase IIa HongKong trial is excluding those with a history of allergy to bee venom, indicating that this has been an issue with regulators:

·         Has a history of allergy to bee venom or history of hypersensitivity reaction requiring an emergency visit to a physician or hospital and/or requirement for treatment with steroids and/or epinephrine.

So how about adding transient immune suppressant to the mix- e.g. an anti-histamine?  Nothing spectacular, a safety precaution, but once again highlighting the benefit of Arrowhead Research making advances with the single-molecule version, also for applications outside the liver (see today’s positive news around Endocyte and folate targeting for cancer as just one example of where such research could be directed at).

Potency matters, and wouldn’t it be ironic that as Tekmira is weaning itself off immune suppression (à dose-intensive TKM-EBOLA trial), on the back of developing more potent formulations, Arrowhead Research, not known as a public supporter of liposomal RNAi delivery, is adopting such?  

Arrowhead Research Patent Application Shows Ample Experience with Triantennary GalNAc-siRNAs

[Warning: this blog entry is not about to discuss a very recent development, but rather is intended to compare the liver gene knockdown technologies by Arrowhead Research and Alnylam based on a review of the patent literature; for the non-technical folks, a mention of Novartis towards the end might be of interest]. 

It has become clear that the DPC technology by Arrowhead Research, especially their 2-molecule-version used in ARC520 for chronic HepB, and Alnylam’s GalNAc-siRNAs share a number of features.  Based on patent application by Arrowhead Research that published last summer, there is evidence that the company has ample first-hand experience with the platform used by Alnylam.  Importantly, the data show that the addition of an endosomal release agent greatly increases the potency of GalNAc-siRNAs.

Large increase in potency with endosomal release polymers

Previously, I had speculated that simple GalNAc-siRNA conjugates as advertised by Alnylam have insufficient potency.  Accordingly, patent application US2012/0136042A1 by Alnylam showed that whereas simple GalNAc-siRNA conjugates had no or very little knockdown activity, the addition of a lipidic pharmacokinetic modulator with some endosomal release activity such as cholesterol allowed for more robust activity (see first image below).

Demonstrating the superiority of DPC delivery technology for gene knockdown in the liver, at least in terms of potency, the patent application by Arrowhead Research shows that even so, the activity of a GalNac-lipid-siRNA pales in comparison to its use along an endosomal release polymer: no knockdown with GalNAc-palmitoyl-siRNA alone, but an 80% knockdown when given together with the polymer (see table).

Note that a range of lipids, including cholesterol were evaluated in that patent application.  Also note that the preferred GalNAc-conjugation was the same triantennary GalNAc structure as used in Alnylam’s programs.  

Intellectual property consideration

While the Alnylam patent application claims priority to sometime in 2007, the Arrowhead patent application claims priority to sometime in 2010.  Given that the data suggest that Arrowhead should have chosen the triantennary GalNAc-cholesterol-siRNA backbone for ARC520 for maximum potency with the 2-molecule DPC approach, instead of the cholesterol-siRNA that they eventually chose, it is possible that IP concerns played a role in that decision. 

Another, non-exclusive explanation might have been manufacturing cost concerns which should favor simple cholesterol conjugates over triantennary GalNAcs.  This would also be justified in that it is the toxicity from the endosomal release peptide and not the RNAi trigger that is expected to be rate-limiting in terms of toxicity.  In other words, to compensate for the inferior potency of cholesterol-siRNA along the release polymer, you just give more of it.

In any case, given the overlapping research activities of the two companies as evidenced by the patent applications, possibly partly the result of the former Alnylam-Roche partnership, it will be interesting to follow the patent prosecutions to find out to which extent the patents by Arrowhead Research could impair the freedom-to-operate and novelty of Alnylam’s platform, both with regard to GalNAc3-siRNAs and GalNAc3-lipid-siRNAs.

An interesting player in this convoluted situation is Novartis.  Assuming Novartis has access to GalNAc-siRNAs from Alnylam, they might be able to combine them with the endosomal release polymers from Arrowhead Research for optimal DPC2.0 knockdown activity (of course, that assumes they take some sort of license from Arrowhead Research).  It’s time for Novartis to show their RNAi delivery hand anyway lest they suffer the same fate as their peers' with their RNAi investments losing all of their value.

Single-molecule subQ DPC

The potentially convoluted IP situation is another, albeit secondary reason, why I greatly look forward forward to Arrowhead Research adopting for their upcoming development candidates the new old single-molecule DPC technology for which they had shown very impressive non-human primate data at last year’s OTS meeting. 

Because the GalNAc residues in the single-molecule DPCs are distributed along the peptide, there is no need for a triantennary GalNAc cluster for similar hepatocyte targeting potency.  Moreover, PK modulation can be achieved by modifying the polymer without the need for direct modification of the RNAi trigger.

And with regard to ARC520- don’t get me wrong. The intravenously administered ARC520 is still an exciting candidate with good activity, it’s just not as potent as it could have been.  I therefore look forward to seeing a second-generation candidate enter clinical development once clinical proof-of-concept for the immune reactivation hypothesis has been formally obtained.  Such a candidate would have much increased potency (at least 10x) and could be administered subcutaneously.  A high-quality problem to have.