Alnylam Second-Generation GalNAc Chemistry ~5x More Potent

The GalNAc-RNAi trigger strategy pioneered by Alnylam and Arrowhead Research has opened up new opportunities for RNAi Therapeutics, partly due to the fact that they may be administered subcutaneously (note: for Arrowhead that means the single molecule DPC which is not yet in clinical development).  Although the first such product candidate, ALN-TTRsc, looks like it could be a decent drug for a severe disease such as TTR amyloidosis, there is room for improvement both in terms of efficacy (--> injection volumes) and tolerability/safety (liver enzyme elevations, skin reactions).

It is therefore no surprise that Alnylam keeps stressing the fact that it has improved upon ALN-TTRsc, now referring to the original GalNAc chemistry as ‘standard chemistry’ (STC) and the improved version as ‘enhanced stabilization chemistry’ (ESC).  By inter- and extrapolating data from various model systems and for various target genes, the company has come up with the notion that ESC ‘has the potential’ to be around 50x more potent than STC (IR departments know that investors will be blind to qualifiers like ‘has the potential’).

I love it when maths meets biology.

These numbers games, of course, make little pharmacological sense, mostly due to the fact that the same delivery chemistry can result in disparate knockdown efficacies just due to sequence and target gene differences.  In addition, concluding anything about a dose response from a ~25% knockdown in a single-dose, single dose level phase I study (--> ALN-AT3) is impossible.  In RNAi, a 25% knockdown can be achieved with homeopathic drug levels and does not inform at which drug concentrations more robust >50% knockdowns will be observed.

Apparently, Alnylam is seeing it the same way and probably has received the same criticism from other sources.  It has now provided on two recent occasions much more informative datasets on the relative potencies of STC versus ESC.

At the Cantonese Nucleic Acids Forum (CNAF) in Guangzhou, China, in early November, Dr. Manoharan revealed that if you turn the STC of ALN-TTRsc into an ESC, the gain is a 5x in potency.  Consistent with this 5x notion is the Nair et al. paper that published 2 days ago in JACS where the same exercise for an siRNA sequence against the murine transthyretin gene resulted in the same 5x improved potency.

Of importance to the RNAi community, the enhanced metabolic stability was achieved by the use of phosphorothioate bonds at the 5’ ends of both the guide and passenger strands, while the 3’ ends are protected in both generations by phosphorothioates in the overhang (guide strand) and the GalNAc ligand (passenger strand), respectively.  I would not necessarily have predicted that phosphorothioates were tolerated at the guide 5’ end and this could be all the material difference there is between STC and ESC.

All eyes are now on the ALN-AT3 phase I data presentation at the upcoming ASH meeting next Monday (abstract here).  To wit, in part A of that study, Alnylam reported a ~25% mean peak knockdown for the 0.03mg/kg starting dose in healthy volunteers earlier this year (single dose).  Although there was no dose response data and they had failed to reach the maximum allowable AT3 knockdown of 40%, part A was deemed a success with the study proceeding into part B in hemophilia patients for further dose escalation and repeat dosing.  First data from that part is to be revealed.  

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.

Alnylam GalNAc Improvements Incremental, but Likely Enough to Beat PCSK9 Antibodies

Over the last week, Alnylam presented pre-clinical data for their new development candidates for the treatment of hypercholesterolemia (ALN-PCSsc) and liver disease related to forms of alpha-1 antitrypsin deficiency (ALN-AAT).  These candidates are based on second-generation GalNAc-conjugate chemistry that the company is now dubbing ESC (Enhanced Stabilization Chemistry).  They involve the increased use of chemical nucleic acid modifications for stability with attendant improvements in knockdown potency and duration over first-generation GalNAc conjugates such as ALN-TTRsc.  ALN-TTRsc is the lead GalNAc candidate and currently in phase II development.

Borderline first-generation GalNAcs

In a phase I study of ALN-TTRsc, potent knockdowns were achieved with about:

-60% knockdown at 2.5mg/kg;

-80% knockdown at 5.0mg/kg (ED80), and

-90% knockdown at 10.0mg/kg.

At the risk of insulting medical geneticists for oversimplifying, assume that an RNAi technology that can safely achieve an 80% target gene knockdown provides for a solid platform.  In the case where a subcutaneous route of administration is desired and/or necessary, this should ideally also fit into a 1ml injection volume which in the case of GalNAc conjugates would correspond to a 2.5mg/kg dose.

There is some controversy around acceptable injection volumes and as often is the case, increased standards are applied to RNAi Therapeutics.  I say this because drugs and drug candidates such as expected mega-blockbuster PCSK9 antibody from Amgen, AMG-145, has been administered at 2ml volumes.  In fact, to achieve once-every-4-week dosing, 6ml (3x2ml) have been administered (see Giugliano et al. 2012).

In light of this, ALN-TTRsc has failed the 1ml test, but 80% are certainly possible with this first-generation GalNAc chemistry.  There is therefore room, and in some cases a competitive need (àcompetition with more potent delivery technologies such as Tekmira’s SNALP LNPs and Arrowhead’s single molecule DPCs) for improvements in GalNAc conjugation technology.    

Second-generation data could indicate progress

In agreement with this, Alnylam is now advertising the ESC second-generation GalNAc and has presented critical non-human primate data for their new development candidates ALN-PCSsc and ALN-AAT.  Non-human primate data are ‘critical’ because the RNAi knockdown observed in monkeys typically closely predicts what will be seen in humans based on the experiences with ALN-PCS (SNALP), ALN-TTR (SNALP), and ALN-TTRsc. 

In interpreting the newly presented data, it should be noted that in the case of ALN-TTRsc, an 80% knockdown was already observed at 2.5mg/kg (and 5mg/kg) in non-human primates, but that this shifted to 5mg/kg in humans.  Numerically a relatively small difference, in practical terms an important one.   

ALN-PCSsc achieved 80% PCSK9 target gene knockdowns at 2mg/kg in a weekly multi-dose study in non-human primates.  This resulted in a highly competitive 60% LDLc lowering in the absence of statins.  Similarly impressive in light of the monoclonal antibody competition was that 80% PCSK9 knockdown and 50-60% LDLc reductions were achieved and sustained for over 3 months (!) when a single dose of 10mg/kg (2x2ml) was given.  Compare this to 57% LDLc lowering with AMG145 in a once-every-4 week regimen in the MENDEL-2 phase III study of AMG145:

-ALN-PCSsc (RNAi): 50-60% LDLc reduction, 2x2ml subcutaneous, once-every-3-months

-AMG145 (monoclonal antibody): 57% LDLc reduction, 3x2ml subcutaneous, once-every-4-weeks (phase III MENDEL-2 monotherapy study)

(Yes, I do keep an eye on cash-rich, sub-$2B market cap The Medicines Company, Alnylam’s licensee for ALN-PCSsc for this reason).

Before I get carried away with all the advantages of the RNAi platform over monoclonal antibodies for even extracellular targets such as PCSK9, in terms of GalNAc improvements, these knockdown results are very much in line with what was seen for TTR in non-human primates. 

The same applies for ALN-AAT where a single dose of 3mg/kg translated into a 60% knockdown which, based on TTR and PCSK9, will likely translate into a weekly multi-dose ED80 of 2.0-2.5mg/kg in non-human primates.

However, with the caveat of different half-lives for different target genes, the durability of the PCSK9 knockdown is quite impressive and more than what is typically seen with e.g. SNALP LNPs which have historically utilized minimally modified RNAi triggers.  Such triggers may be turned over more rapidly in liver cells.

Similarly, the RNAi trigger sequence in the TTR development candidate is an extraordinarily potent one with low single-digit picomolar EC50 potency in tissue culture.  It is therefore possible that equivalent animal potencies have now been achieved with less potent sequences meaning that the underlying delivery technology has improved.

It’s been fascinating to watch the various delivery platforms, and indeed RNaseH antisense, compete over the years and pushing each other to new heights.  In this context, I am anxiously waiting for Arrowhead’s presentation at next week’s TIDES meeting with the intriguing title:  “Next Generation Dynamic Polyconjugates for siRNA Delivery in vivo,”

Market commentary: I remain mostly on the sidelines.  Years ago, these scientific data would have made me jump head-first into the market with me buying all I could in the opening minutes.  These days, however, I view them as ensuring the long-term value of RNAi stocks, but fail to see how they will support share prices in the current, growth-to-value rotating trading environment for more than a trading week or so.  I am therefore speculating on a capitulation event before some of the important clinical data read-outs roll in.