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.