Follow-Up to Simplified GalNAc-RNAi Trigger Discussion

Just days after the Matsuda et al. publication on simplified, non-triantennary GalNAc-RNAi triggers (the subject of the previous blog entry), another related paper from Alnylam came out (Rajeev et al. 2015). 

The Rajeev et al. publication further supports that monomeric GalNAc designs as pioneered by Arrowhead Research for the use in RNAi Therapeutics and others (e.g. Matulic-Adamic et al., 2002) before for non-RNAi Oligo Therapeutics applications allow for effective ASGPR receptor recognition and subsequent gene silencing.  

The main difference to the Matsuda paper was that instead of GalNAc-modified nucleotide monomers, non-nucleosidic GalNAc monomers were employed (note: the Matulic-Adamic et al. also explored both nucleoside- and non-nucleoside-based monomeric clustering GalNAc strategies). They, however, were also appended to the 3’ end of the passenger strand as part of the phosphoramidite-based RNA synthesis.

The use of non-nucleosidic phosphoramidite monomers further simplifies the chemistry demands and thus cost of goods of GalNAc-siRNAs.

Ironically, not only does this paper also fail to cite the seminal Arrowhead and Matulic-Adamic research, this significant effort to get away from the apparently costly triantennary design is also at odds with Alnylam’s claims in July 2014 that a patent covering bi- and triantennary conjugates designs ‘broadly cover[ed] conjugate-based delivery of RNA Therapeutics’.

Obviously, if non-bi- and triantennary designs work, then one cannot consider the issued patent to broadly cover conjugate-RNAi triggers.

It will now be interesting to see how broad the claims will be that the patent offices will grant related to the simplified designs.  The re-writing of history effort obviously is not aimed at impressing investors or enabling the field, but part of a GalNAc IP landgrab effort, ideally resulting in the issuance of claims covering any monomeric GalNAc designs.

The one thing that I would like to give Alnylam credit for is that they will put significant resources behind the technologies they consider promising- whether they invented, own, or have a license to them or not- and thus advance RNAi Therapeutics as a field.

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.