Increasing RNA Editing Target Space Through Chemical Modification 5’ of Target A

Not all target adenines (A) are created equally.  It is well documented (Schneider et al. 2014; Picardi et al 2015) that sequence context plays an important role in rendering A ripe for editing. So to realize the full potential of the platform, it is important to optimize the editing oligos taking sequence context into account.  Chemical optimization will play a critical role here.

ProQR and Wave Life Sciences have demonstrated most advances in this regard and I have previously discussed the promising dZ modification at the orphan base that the Beal lab in collaboration with ProQR have developed.  Today, I will shine light on the recent chemical optimization work by the prolific Beal lab on improving editing when the base 5’ of the target A is an unfavored G (Doherty et al. 2022).

Funding for this work came from ProQR and the Rett Foundation.  Rett is a neurological genetic disorder where RNA Editing looks like a great option for many patients. As another aside, Dr. Beal holds equity in both ProQR (as do I) and CRISPR-based editing company Beam Therapeutics.

 

Structural analysis of guide-target RNA

Using a target RNA derived from the alpha-L-iduronidase (IDUA) gene, Doherty and colleagues first confirmed that when the base 5’ of the target A is a G, editing efficiency is very low when this G is Watson-Crick base-paired to C (known non-preferred context).  As observed before for ADAR deaminase domains (Schneider et al. 2014), the efficiency could be improved when C was replaced by G for full-length ADAR2,  or by either G or A for full-length ADAR 1 p110.

The Beal lab is the leading laboratory when it comes to structurally analyzing ADAR enzymes in complex with guide and target RNAs and then translating these insights to chemically optimized guide RNAs.  They therefore were interested in what was going on in this G-G context and found that the G 5’ of target A was in a syn conformation as opposed to the anti conformation seen in Watson-Crick interactions.

This conformation was stabilized by the 5’ G hydrogen-bonding with its Hoogsteen face to Ganti in the editing oligo.  As a result, the clash of the 2-amino group of the 5’ G base with an amino acid residue in the deaminase flipping loop was averted.  To wit, the flipping loop plays a critical role in editing as it facilitates the rate-limiting flipping of the target A out of the substrate duplex into the deaminase reaction pocket.

Still, there were apparent structural adjustments that had to be made by ADAR2 to accommodate G relative to what is observed in an ideal U:A context.  They therefore replaced G with various A- and G-derived modified nucleosides.  Interestingly, Gà3-deaza dA was found to result in the best editing efficiency, ~2-fold higher than a simple G.

Like G:G, the 5’G was in syn when paired to 3-deaza dA.  However, this conformation was additionally stabilized by not one, but two interactions of Gsyn with its phosphodiester backbone thus rationalizing the improved editing efficiency.

It is great to see the RNA Editing field make progress in elucidating the rules so that most theoretical target sites also become addressable ones with sufficient potency.  Certainly the bases around the to be edited A which are expected to interact with the deaminase domain of ADARs are a hotspot of such research activity.  For example, in this year’s Nature Biotech paper by Wave Life Sciences, an inosine (I) was taking the place of 3-deaza dA thereby doing away with the loop-clashing 2-amino group altogether.   It would have been nice to quickly add on a GalNAc and confirm the findings in animals.  I am guessing though that ProQR has done that experiment already.

RNA Editing to Generate Protective Variants

Last week, ProQR participated at its first investor conference after a half year hiatus following a failed binary clinical read-out of a antisense splice modulator and corporate re-organisation to solely focus on RNA Editing.

To raise awareness of the company as a serious contender in this exciting field, the CEO detailed the new corporate strategy of growing the company as a major future biotechnology company.  This will be based on foundational IP, know-how and cash-generating partnerships as well as a broad internal pipeline reflecting the numerous ways RNA Editing can bring unique differentiation to addressing disease.  At the EuroTIDES two days later, the company provided scientific data illustrating the types of possible applications.

What caught my attention here is using RNA Editing for generating protective variants.  Protective variants are genetic variants in the human population that make carriers less likely to develop a disease.  PCSK9 and ApoCIII are well known in the cardiovascular field for those and have yielded promising therapeutics as a result.  Protective variants can also be found in other areas such as infectious disease (e.g. CCR5 and HIV) and Alzheimer's.

Not least because the story surprised and intrigued me for its translational potential, I will illustrate protective variant generation using RNA Editing taking PCSK9 as an example. 

 

Mimicking a PCSK9 mutation causing drop in bad cholesterol

With Inclisiran, an RNAi trigger suppressing the expression of PCSK9, RNA Therapeutics have become a commercial reality in cardiovascular disease aimed at very large populations.  A key attraction of this agent is the infrequent, semiannual dosing regimen that clamps down bad LDL cholesterol by ~-50%.

50%, however, somewhat lags the efficacy of the competitive PCSK9 monoclonal antibodies (~-60%) which have to be given at least monthly.  The fact that more robust LDL cholesterol lowering should therefore be possible also for RNA Therapeutics, the race for the most effective PCSK9 in this class is yet to be decided. 

Finding better RNA knockdown agents may be one, certainly attainable strategy, although AstraZeneca has just given up on an RNaseH antisense oligo that had looked promising in that regard.  Using an entirely new mechanism another.

Intriguingly, the sequencing of the PCSK9 gene in an individual with conspicuously low LDL cholesterol in Canadian Quebec province (Mayne et al. 2011) revealed that a Q152H variant in the heterozygous (!) state could lower circulating PCSK9 levels by ~-80% compared to the average, non-related population. This translated to a 60-70% LDLc lowering.  Subsequent cell culture experiments confirmed the causality of this mutation in regulating LDL-receptor levels via PCSK9 expression.  Curiously, while it was initially assumed that the mutation inhibited autocatalytic cleavage of pro-PCSK9, these experiments suggested that such mutant PCSK9s can also inhibit PCSK9 cleavage and subsequent export out of the cells in trans.  This dominant negative effect explains why the mutation in the heterozygous state could have such an outsized effect on serum PCSK9 and LDLc levels.

While other protective PCSK9 variants had been found before, this one was intriguing enough for ProQR to follow up on.  As histidine (H) is unlikely to be unique in messing up PCSK9 processing, ProQR chose QàR editing at the 152 site, possibly because glutamines cannot be converted into histidine via AàI editing, but also possibly because they suspected that the more highly charged arginine (R) residue could be even more impactful.

Impactful it was.  Even at only modest 25% editing (which certainly can, and has to be improved upon for further development), cleaved and secreted PCSK9 in cell culture was reduced by roughly -90%.   

It is important for the RNA Editing space to continue to advertise what the technology can do.  Because RNA Editing is not very useful as a genetic tool for general, academic molecular biology, the onus is on the companies to raise the awareness as some of the best translational ideas may come from scientists and physicians that have yet to hear about the technology.

Ionis Widens Its Modality Horizons

Over the weekend, blue chip antisense oligonucleotide company Ionis and genome editing competitor Intellia presented data on targeting prekallikrein (PKK) for treating hereditary hemeangioedema (Ionis donidalorsen here, Intellia NTLA-2002 here).

Using CRISPR Cas9 endonucleolytic disruption of the KLKB1 gene coding for PKK following LNP delivery, Intellia came out as the apparent winner in this showdown.  Not only did they demonstrate more pronounced PKK inhibition, but also more consistent elimination of debilitating attacks characteristic of the disease.  Moreover, by exploring less frequent antisense oligonucleotide administrations despite suboptimal low -60% knockdown, Ionis indicates that it is worried about the safety and tolerability profile of donidalorsen. 

Whether reversible approaches like antisense and RNAi or irreversible approaches like CRISPR gene disruption will ultimately prevail in the HAE race remains to be seen and will likely be decided by the safety of suppressing PKK expression over the long-term. If there is an overshoot of CRISPR-mediated gene disruption that would e.g. result in blood clotting abnormalities, even for a subset of patients, the field would be wide open for reversible methods.  

Ionis invests in genome editing

But whether that will be antisense remains to be seen.  Especially for targets in the liver, RNAi currently clearly rules the land for gene knockdown: highly potent, titratable and reversible knockdown with 5 years counting without a notable setback, especially related to off-target toxicity.  By contrast, Ionis is being held back by persistent safety issues as it has been beating a dead horse with its phosphorothioate-based backbone chemistry although it appears to be finally weaning itself off with chemistries such as the Mspa backbone.

So it is probably the hope of leap-frogging the RNAi competition by adopting genome editing as Ionis today announced that it was partnering with CRISPR genome editing company Metagenomi.  The HAE data comparison could not have come at a more opportune time.  

One declared aim of the investment in genome editing is life-cycle management of existing franchises.  In the liver, these franchises (TTR amyloidosis, ApoC3, PCSK9 etc) are currently and in the foreseeable future being dominated by RNAi despite Ionis’ heavy investments, so it clearly makes sense to amortize its investments in disease-specific market research, commercial infrastructure and clinical trial experience to accelerate the success of a more promising approach. 

TTR amyloidosis is a great example where even GalNAc-conjugated follow-on antisense compounds are unlikely to challenge Alnylam’s suite of RNAi triggers.  Also due to this dominance, it makes less sense for Ionis to develop an RNAi competitor drug despite its access and now actual adoption of this modality for targets in the muscle.  But as TTR shows, other genome editing companies are already competing for some of these targets so it won’t be all that simple trying to leap-frog RNAi and Alnylam like that.

 

The rise of the multi-modality oligonucleotide therapeutics companies

After straight-forward antisense for gene knockdown and then splice modulation, with the recent adoption of RNAi and genome editing, Ionis is rapidly expanding its oligonucleotide modality toolbox.

In fact, it is becoming a little bit like smaller competitor Wave Life Sciences which has been practicing all types of antisense (knockdown, splice modulation, more recently RNA editing) and RNAi using a bewildering mix of chemistries.  Not only are they burning through cash as if there was no recession and inflation problem, I never liked that because clinical failure after failure (esp. minute target engagements at best) suggest that the company is stretching itself too thin.

By comparison, Ionis, with $2 billion in cash and a more experienced and bigger operation is a different beast altogether and may be able to pull it off, at least on a technical level.  However, instead of spending $80M in upfront alone on a modality that is somewhat further removed from its traditional chemistries (longer mRNAs, LNP delivery for CRISPR), it could have much more synergistically leveraged its investments in chemistry and delivery by investing that same amount in the ripe-for-the-picking RNA editing.  Accordingly, $80M is more than the market cap of my currently favourite RNA editing investment, ProQR.

I’m sure the opportunity to expand druggable targets and indications by applying existing delivery technologies and chemistry know-how by adopting RNA editing is not lost on RNAi players such as Alnylam and especially Arrowhead Pharmaceuticals.  Arrowhead in particular, having scooped up the RNAi assets of Novartis and Roche for peanuts has demonstrated an ability to recognize and act on similar opportunities.