Eli Lilly Comes Back for More ADAR Editing

Barely two weeks after GSK took an exclusive license to the ADAR Editing industry’s leading pipeline asset (alpha-1-antitrypsin) and other ADAR targets from Wave Life Sciences, Eli Lilly and ADAR pure-play ProQR announced a major expansion of their RNA Editing partnership from a year ago.

Here, I will lay out what the deal says about the position of RNA Editing within the pharmaceutical industry, the dynamics within the RNA Editing space before going into some specifics of the deal itself.

 

ADAR Editing to exploit exploding genetic insight into disease

The deals were driven by the desire of large pharmaceutical companies to exploit their investments into understanding the genetic basis of disease by matching them with complementary therapeutic modalities. Not only is ADAR Editing as a genetic technology itself an obvious consideration for this challenge, unlike most other modalities which work by inhibiting pathways, ADAR Editing is amenable to gain-of-function and gene upregulation.

Similarly, RNA and genome editing (‘CRISPR’) are unique in their ability to generate protective variants, that is variants thought to protect a person from developing a disease such as cardiovascular disease or Alzheimer’s.

Target modulation versus all-or-nothing forever

Genome editing may have captured our imagination and overshadowed ADAR Editing for most of its existence, but for many applications its mechanism is imperfect or for technical reasons not applicable (yet).

The delivery challenge for large nucleic acids as in genome editing is, however, bigger than for ~30nt oligonucleotides which have emerged as the preferred strategy to mediate ADAR Editing.  It is therefore unsurprising that currently the greatest value of genome editing is in combination with cell therapeutics such as in immune oncology or for hematopoietic stem cell-based strategies where delivery happens ex vivo. For in vivo delivery strategies, liver hepatocytes will be its main target tissue and it is here where most of the competition between ADAR and genome Editing will play out.  

But also where there is such direct competition, there are often good reasons to not pursue a permanent change in the patient's DNA.  For example, permanently burning liver fat in an uncontrolled manner by turning thyroxin hormone receptor-beta into constitutively active versions could be dangerous.  This is where a daily (small molecule) pill or a quarterly administered oligonucleotide therapeutic like RNA Editing would be preferable.

 

In Eli Lilly’s words: ‘“We have been impressed with the progress to date with our partners at ProQR and have conviction that RNA editing can be an important alternative to other more permanent therapies.”

 

For other targets, a biallelic modification may not be tolerated by the cell.  It is for example hotly debated whether huntingtin in Huntington’s Disease should be fully ablated or whether residual huntingtin activity is important for safety reasons.

Short oligo-mediated RNA Editing is the way

It is no coincidence that GSK and Eli Lilly have partnered with the leading ADAR Editing companies that employ short oligonucleotides to both recognize target RNA and recruit endogenous ADARs.  In the earlier days of RNA Editing, a longer editing oligonucleotide expressed from DNA along with ADAR co-expression was predominantly practiced.  However, this approach not only suffers from the challenge of having to deliver larger cargos to target cells, ADAR overexpression is associated with widespread, likely intolerably high off-targeting. 

Clearly, Roche should have known about these fundamental challenges in 2021 when it sealed its collaboration with DNA-directed ADAR player Shape Therapeutics.  Roche must feel the intended approaches it is intending the technology for justify such delivery and would expect the collaboration to focus on reducing the level of off-targeting.

Eli Lilly and ProQR partnership

In September this year, when I got finally convinced to look more into the potential of ADAR RNA Editing, I could not believe how well positioned and cheap ProQR was:

ProQR had stumbled across an oligo-mediated method to achieve targeted RNA Editing about 10 years ago.  At the time I was extremely critical of the company because they pursued a form of mRNA repair (for cystic fibrosis) that in my book as an RNA molecular biologist did not exist.

Predictably, that project failed and I can imagine that failure and resulting skepticism of the technology led the company to explore other methods of repairing mRNA.  They obviously stumbled across ADAR Editing when virtually nobody, except for a few DNA-directed project teams was paying attention.  This means that their IP may be close to having gatekeeper potential for oligo-directed ADAR Editing.  

Another thing that I liked about ProQR during my research was that, unlike its closest competitor, Wave Life Sciences, ProQR is now fully exclusively focusing its cash towards the platform instead of having its budget being overwhelmed by expensive clinical trials on less exciting drug development prospects.

As such I was confident that regardless of the neglect by the public markets, the pharmaceutical industry which has gotten used to the concept of Oligonucleotide Therapeutics would be attracted by the notion of piggybacking on existing oligonucleotide delivery and chemistry progress to open up unique new genetic drug target space with RNA Editing.

So after initially buying access to 5 liver and CNS-related targets for $20M in upfront cash and an equity investment of $30M for a little over $7.5 per share, Eli is paying now $60M in upfront and making a $15M equity investment at $1.6 per share for 5 more targets.  The focus this time has shifted from the liver to central and peripheral nervous system applications, and the protective variant concept was highlighted in the press release.  Lilly now owns approx. 17% of ProQR. 

Each target comes with $250M in potential development and commercialization milestones and given the early stage of the collaboration, royalties from commercial sales are capped at single digit percentages.

Another perk for ProQR is that it may access Eli Lilly oligonucleotide know-how and technology under the collaboration.  Eli Lilly has a rather long history of developing antisense and RNAi molecules.  ProQR values such external input a lot and as such partnership deals had been limited to those companies with a substantial investment in oligonucleotide therapeutics. And that Eli Lilly happens to be the partner again speaks to the apparent success of the ongoing collaboration.

ProQR stated in the conference call that Eli Lilly has an option to gain access to a final 5 targets for $50M and that the companies would be incentivized for this to happen in 2023.

But since the enterprise value (market cap minus cash on hand) of ProQR is barely $50M, why not just attempt to acquire this gem after the 6-month standstill expires this summer?  But before that will be the JP Morgan Conference in January, a biotech dating lovefest where new relationships are formed and deals are born.  Oligonucleotide powerhouses Alnylam and Ionis, or Big Pharma, this time perhaps of the European flavor would be my candidates playing interference with Eli Lilly.

GSK Partners with Wave Life Sciences for Access to RNA Editing

This week, we have seen further confirmation of the increasingly recognized value within the pharmaceutical industry of Oligonucleotide Therapeutics in general, and RNA Editing in particular.

In a landmark deal, GSK obtained an exclusive license from Wave Life Sciences to the RNA Editing industry’s lead, albeit still preclinical WVE-006 development candidate for the treatment of alpha-1-antitrypsin disease.  In addition, GSK has the right to evaluate Wave’s oligonucleotide platform (editing, splice modulation, and RNAi/ASO silencing modalities) to then advance up to 8 programs into development.

In return, Wave will receive $120M in upfront cash, another $50M in an equity investment, and the potential to earn up to $3.3B in development and commercial milestones in addition to royalties on drug sales.  Because of its more advanced stage in development, WVE-006 stands to earn relatively more in milestones ($525M) and royalties (tiered double-digit, up to the high teens).

While I view Wave doing this deal largely to feed its voracious appetite for cash to feed what I consider to be less exciting clinical work in Huntington’s (ASO knockdown) and Duchenne muscular dystrophy (exon skipping), GSK will bring its genetics-based target insights to the collaboration table so that Wave could advance up to 3 related programs that it would wholly own.

Seeing the AATD program go to GSK was a disappointment to me at first.  Ultimately, I thought that this program would end up shouldering the weight of Wave’s market cap as the company’s lead program once the current clinical pipeline will meet its expected fate.  However, during the discussion of the deal the company’s CEO Paul Bolno made it clear that not only is GSK much better suited to advance ‘006 especially with regards to its lung-related endpoints, it is RNA Editing and gene upregulation that Wave considers the most valuable elements of its PRISM oligonucleotide platform and that it wants to maintain control over.

Gene upregulation can be achieved by either masking destabilizing sequences in an (m)RNA by antisense oligonucleotide, or by using RNA Editing to disrupt those or slightly change the protein to make it more stable.

After the 2021 deals between Shape Therapeutics andRoche (neuroscience, DNA-directed RNA Editing) and ProQR and Eli Lilly, this marks the third such Big Pharma deal in the ADAR sector.  It is reminiscent of the 2004-5 phase when Big Pharma started to take note of RNAi through a few measured investments.  

Expect the noise and excitement to grow over 2023 as RNA Editing approaches the clinic.  But unlike RNAi, a lot of delivery work has already been undertaken so that the trajectory of RNA Editing should be smoother from a technology point of view.  Only yesterday, Avidity Biosciences reported on the  expansion of the targetable tissue universe to the muscle and Arrowhead Pharmaceuticals is about to report important data on targeting RNAi to the lung.

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.

Big Pharma Investments in RNA Editing

When it comes to new platform technologies, investors generally like to see their belief validated by large pharmaceutical companies.  In addition to confirming the soundness of the scientific approach, in times when access to capital is constrained, such partnerships also provide an important financing source.

In RNA Editing, Venture Capital certainly has taken the charge (and risk) by investing close to $600M in Series As and Bs spread between Korro Bio, Shape Therapeutics, EdiGene and ADARx (the last two are not pure-plays) largely in 2020-1.  There have, however, been two notable Big Pharma deals that materialized in the second half of 2021.

 

Shape Therapeutics-Roche

In August 2021, Shape Therapeutics announced its first Big Pharma partnership.  Shape apparently has been working on the DNA-directed expression of editing RNAs harnessing endogenous ADARs, especially in the CNS.  Their favourite delivery vehicle is AAV viral delivery.

It is an interesting approach, since despite of going through the trouble of gene therapy-type delivery, they choose not to bring exogenous ADARs along for the ride.  This makes sense since overexpression of ADARs is linked to widespread off-targeting and the molecular size of ADAR may be a vector capacity issue, too.  As it would have involved essentially naturally occurring ADARs (plus/minus a few optimizing mutations), the cost in terms of immunogenicity though may have been tolerable.  This is in stark contrast to genome editing technologies like CRISPR where, because of delivery in the CNS, you would likely have to deal with the extended expression of entirely foreign proteins.

Shape and Roche will tackle a number of neuronal diseases together, likely Alzheimer’s, Parkinson’s and more rare indications like Rett Syndrome.  Of note, Roche has suffered a major setback in oligonucleotide-based neurodegenerative drug development when efficacy and tox issues derailed a late-stage Huntington’s disease drug candidate based on the intrathecal administration of phosphorothioate antisense molecules.  So for them opting for AAV-based expression of targeting RNAs is worth taking note of.

Rett Syndrome is a truly intriguing indication highlighting a few of the unique advantages of RNA Editing.  Rett Syndrome affects ~1 in 10-15k female births.  It is a severe, early onset neurodevelopmental disorder caused by too little MeCP2 expression due to mostly spontaneous (as opposed to inherited) mutations.  Nevertheless, persons suffering from this X-linked gene condition can still live into their 40s and 50s- with severe disabilities. There are no drugs approved specifically addressing Rett Syndrome. 

Rett Syndrome would seem like an ideal candidate for the development of gene therapy.  What makes, however, gene therapy particularly challenging in this setting is that while too little of the master epigenetic regulator that MeCP2 is gives you Rett Syndrome, too much of it is neurotoxic.  Add X chromosome inactivation mosaicism into the mix and the therapeutic window of MeCP2 expression narrows dramatically:

for each (neuronal) cell just enough to give you MeCP2 function, but not more, certainly not >2x normal MeCP2 expression.

As a technology that does not change the rate of gene transcription, RNA Editing is ideally suited for Rett Syndrome and it is estimated that 40-50% of cases can be addressed by the technology.  The downside is that in order to address all of those mutations, similar to Duchenne’s and exon skipping, a number of RNA editing molecules would have to be developed.

 

ProQR-Eli Lilly

A month following the Shape deal, ProQR announced a partnership with Eli Lilly for up to 5 targets in the liver and CNS. This was accompanied by a $20M upfront consideration and a $30M equity investment.

Unlike Shape, ProQR (pronounced ‘Procure’) is pursuing a more traditional approach to drug development in the form of synthetic oligonucleotides for A-to-I editing.  Eli Lilly has shown great commitment to RNA Therapeutics for a while now with for example two RNAi compounds licensed from Dicerna (now part of Novo Nordisk) in clinical development for two cardiometabolic indications and a recent whopping $700M investment into a Genetic Medicine research site for RNA- and DNA-based drug development.  In the CNS, Eli Lilly will be interested in applying the new platform to the usual suspects including Alzheimer’s and pain.

 

As RNA Editing is moving into the clinic (Wave Life Sciences, alpha-1-antitrypsin) and more people hear about the platform and come up with great ideas of where to apply it, but also as oligonucleotide therapeutics more and more becomes part of the mainstream pharma mindset, I expect additional Big Pharma deals to materialize soon.

At the Core of Highly Active RNA Editing Oligos

 Increasing the inherent potency of ADAR guide RNAs (AgRNAs) through chemical and structural means is critical for the success of RNA Editing. For activity, the AgRNAs need to pair with the elements around the target Adenosine (A) and activate resident cellular ADAR enzymes.

There are 3 ADAR enzymes that are relevant for therapeutic RNA Editing: 2 isoforms of ADAR1 (p110, and the longer p150) and ADAR 2.

ADARs fundamentally comprise of N-terminal double-stranded RNA binding domains (dsRBDs) and a C-terminal deaminase domain which itself can also recognize structural features of the double-strand RNA elements around the target 'A'.  

ADAR1 p110 is fairly uniformly expressed in the nucleus across tissues (possibly least in muscle; Picardi et al), but appears somewhat less effective in oligo-guided RNA editing compared to its interferon-induced cytoplasmic bigger brother p150.  Of note, the lung, one of the initial attractive target organs for this new therapeutic modality is an exception as it naturally expresses more p150 than p110.  Finally, ADAR 2 (mostly nuclear) is most highly expressed in the CNS and also quite effective in oligo-mediated RNA editing.

ADAR expression levels are important as more ADAR means more effective RNA Editing.  It is therefore important to use clever chemistry in the design of AgRNAs to make the most of what ADAR is present in a cell. 

ADAR structure

A breakthrough step in that direction was the structural elucidation by the Beal laboratory at UC Davis of hADAR2 in complex with its dsRNA substrate (Matthews et al 2016).  Although the structures of the hADAR1 isoforms have not been solved yet, the high sequence similarity between the ADARs as well as mutation experiments with hADAR1 (Park et al. 2020) suggest that the deamination reaction is highly similar between the enzymes.  Chemical strategies successful for one enzyme should therefore be directly applicable to the other one.

Having said this, it remains an open question whether one ultimately ought to tailor AgRNAs depending on which ADAR is most relevant in a given target tissue and disease setting.

After capturing the structure of human ADAR2 with substrate dsRNA in which the target 'A' (actually a more easily trapped nucleoside analogue replacement 8-azanebularine) is flipped out of the double-strand into the catalytic deaminase pocket of ADAR2 ready for deamination, Matthews and colleagues noted that this rate-limiting step was apparently facilitated by hydrogen bonding between a glutamate residue of ADAR2 with the base originally opposite target A.  This base is also referred to as the orphan base at this stage of the reaction and cytosine is thought to be preferred at this position.

The structure provides an explanation for the base preference as the nitrogen 3 (N3) in the cytosine base ring can hydrogen bond with the acidic side chain of ADAR2 glutamate 488.  This functions as a firm handshake thus displacing and keeping target A out of the double-strand and pushed into the deaminase pocket.

This model also neatly explains the 60x increased activity of a hADAR2 mutant in which glutamate 488 has been replaced with a glutamine amino acid residue as the amide group of glutamine very happily provides a hydrogen for hydrogen bond formation.

Cytidine analogs

While co-delivery of (engineered) ADAR was strongly considered in the early ADAR RNA Editing days, it suffered from widespread off-targeting and delivery challenges.  So to still take advantage of this structural insight for improving therapeutic RNA Editing efficiency, the Beal group, this time in collaboration with Dutch RNA Editing pure-play ProQR investigated whether nucleoside analogues, in particular cytidine analogues with improved hydrogen donating ability could similarly enhance AàI editing (Doherty and colleagues 2021).

I can only imagine the excitement in the lab when it was found that, indeed, cytidine analogues Benner’s base Z and pseudoisoC could increase the rate of deamination.  Importantly, it was shown for Benner’s base Z that this benefit translated to improved AàI editing in living cells.

It should be noted that the deoxy forms of the nucleoside analogues were used as this is known to be tolerated in the orphan position and should make the AgRNAs more stable. 

Of interest, a bulkier adenosine analogue that should be a ready hydrogen donor impaired deamination in the test tube.  Curiously, Wave Life Sciences successfully used this 8-oxodA in their efficacious alpha-1-antitrypsine AgRNA in the recent Nature Biotech paper discussed in this blog (Monian et al 2022).  It remains to be seen whether the discrepancy is explained by sequence and modification context or whether the in vitro deamination assay is not always predictive of performance in cells.

Regardless, 8-oxodA is one of the analogues covered in the patent applications by UC Davis and ProQR (WO 2020/25237641).

Disclosure: I own shares in ProQR.

Landmark Chemical Modification Study Shows RNA Editing Ready for the Clinic

At this stage, providing investors and the pharmaceutical industry with a clear line of sight that RNA Editing can be readily translated from concept into therapeutic reality is key to unlocking the next step-up in valuation.

A landmark study in March earlier this year by scientists from Wave Life Sciences (Monian et al, Nature Biotech) on chemically modifying ADAR guide RNA oligos (I will abbreviate them from now on AgRNAs due to missing consensus nomenclature) should go a long way in this regard.  It shows that applying a plethora of standard oligonucleotide stabilization chemistries (e.g. PS, PN backbones, 2’-O-methyl-, 2’-F-ribose) which are critical to enabling delivery and desirable durability do not compromise endogenous ADAR enzyme activity.

In fact, backbone stabilization for example via phosphorothioates, especially when in the SP stereopure conformation can actually greatly increase activity.  In a luciferase model system, editing activity of a fully (stereorandom) PS-modified AgRNA was 10x that of a corresponding AgRNA with an unmodified PO backbone.

Accordingly, when GalNAc-conjugated AgRNAs were tested in non-human primates, ~40% editing rates were observed for at least 2 months.  For this, a loading dose of 5mg/kg per day for 5 days was used.  This is on the higher end of what should be clinically acceptable, but as we know from experience with RNAi, what GalNAc works in non-human primates works even better in humans.

Illustrating the value of further refined chemical optimization of high-value candidates, impressive ~70% mRNA editing efficiencies were seen for a AgRNA against mutant SERPINA1 in primary mouse hepatocytes resulting in a concomitant increase in corrected protein.  SERPINA1 is also the target for Wave’s and possibly the industry’s first clinical RNAEditing program and addresses alpha-1-antitrypsin liver and lung disease.

What piqued my interest was that this AgRNA involved a 8-oxo-deoxyadenosine mismatch base opposite the adenine to be modified and a nearby inosine.  What this means will be addressed in my next blog entry...

So congratulations Wave Life Sciences on this study, but also they will admit that the study still only scratches the surface of what gains in potency will be possible with more detailed structure-activity studies.

RNA Editing Emerging as a Broadly Applicable Oligonucleotide Therapeutics Modality

This feels like RNAi all over again. 20 Years after my life-altering journey in RNAi Therapeutics started, I can’t shake a similar sensation for the almost boundless therapeutic opportunities around RNA Editing.

RNA Editing in our context refers to the directed change from Adenine (A) to Inosine (I) in an RNA molecule with ‘I’ being read as a ‘G’ by the molecular machineries inside a cell.  This process harnesses endogenous ADAR enzymes (Adenine Deaminases Acting on RNA) which recognize certain double-stranded RNA features upon which nearby ‘A’s are converted to ‘I’s.  These recruiting features can be created in a sequence-directed manner through the interaction of exogenously applied oligonucleotides with complementarity to cellular target RNA, typically mRNA.

Other types of RNA editing, for example ‘C’ to ‘U’ are also being explored, but as a readily sequence adaptable platform, ‘A’ to ‘I’ excites me the most right now and appears ready for prime time.

 

Boundless therapeutic opportunities

When I was first pitched with the concept of RNA Editing, I was skeptical.  I merely saw it as a gene therapy alternative for ultra orphan applications aiming to revert pathogenic ‘A’s into wildtype or less pathogenic ‘G’s. 

Even fairly common genetic diseases like Duchenne muscular dystrophy and cystic fibrosis for example can be caused by hundreds of different point mutations and it would seem overly cumbersome to develop different oligos to address unique mutations of individual patients.

Also, how would it compete here with genome editing technologies like CRISPR that aim for one-time treatments to achieve the same?

It was during a recent trip back to Stanford when I voiced such concerns as Billy Li remarked in passing that RNA Editing was not limited to such genetic therapies in the traditional sense, but could also be used more widely to modulate wildtype mRNAs to influence processes such as signaling pathways for diseases involving much larger target populations.

Billy Li is an associate professor at Stanford University and a leading researcher on ADAR molecular biology and genetics.

To influence signaling pathways, you may for example target RNA Editing to abolish phosphorylation sites or other amino acids critical for protein-protein interaction.  Often, this will have gain-of-function effects such that editing only a fraction of the target RNAs may result in dramatic upregulation of a pathway.

Tunability and temporary modulation as opposed to the binary nature of genome editing is another attraction of RNA Editing.  You can easily see for example that having a signaling pathway permanently fully switched ON could pose a safety issue.

In addition to changing the coding potential of a target mRNA, RNA Editing can also be used for altering RNA processing sites, especially those regulating splicing, RNA stability and transport.

 

Ready to shine

While it has taken RNAi Therapeutics roughly 15 years from the early start-up phase to having its first drug approved for clinical use, the timeline for realizing the therapeutic potential of RNA Editing should be shortened. 

In particular, much has been learned about the delivery of oligonucleotides such that related oligonucleotide stabilization and conjugation chemistries can be rapidly translated to RNA Editing.  First clinical trials will likely be for applications in the liver and CNS, followed by the eye and lung.   

Similarly, the modification toolbox and the dramatically lowered cost of large-scale oligo synthesis and screening can be exploited to improve targeting specificity and to identify highly active RNA Editing oligos.

   

Investment opportunities

I get my adrenaline kick when science and the stock market come together.  After seeing RNAi Therapeutics mature into a widely accepted therapeutic modality, I had increasingly turned my stock market trading to biotech in general.  While this allowed me to learn much about the regulatory and late-stage aspects of drug development and marketing, I have come to miss the passion I felt while diving into the molecular biology and competitive dynamics of RNAi as it was still developing.

I know many of you are biotech investors and feel the same.  So while RNA Editing investment opportunities are not limited to the stock market, this blog will shine particular light on publicly traded biotechs in the field. 

For full disclosure, I have started a position in ProQR (ticker: PRQR; market cap: $62M), to my knowledge the only public pure-play RNA Editing biotech.  After a failed trial involving antisense oligo-mediated splice modulation it now trades at ~50% below cash suggesting that all that was left is an empty biotech shell.  It couldn't be further from the truth as ProQRians have been working on RNA Editing for almost a decade and now find themselves leading the charge of a hot new biotech platform.  Along the way, ProQR has been working on potentially critical IP and it is also for this reason that Eli Lilly has partnered with it on up to 5 RNA editing programs.  ProQR has been in a quiet period.  The next catalyst will be the announcement of more concrete development timelines and programs.  While we are waiting, the projected 2026 cash runway and partnering potential provides the company and investors with a nice cushion in these turbulent times.

Wave Life Sciences (ticker WVE; market cap: $320M) may be first into the clinic with alpha-1-antitrypsin, but as a most diverse oligonucleotide therapeutics company it is burning through its cash much more rapidly, spending it on less promising antisense knockdown and exon skipping programs.  For RNA Editing, I like it for their deep expertise in oligonucleotide chemistry and their editing efficiencies appear leading.

Stoke Therapeutics (ticker STOK; market cap $520M) is another ticker to watch as it uses oligonucleotides in more general for gain-of-function purposes.

Reuters Article on Novavax: a Rebuttal

 [disclosure: I own shares in Novavax]

The protein-based vaccine by Novavax is about to become the gold standard covid vaccine: proven to be highly efficacious and better tolerated than existing options with 3-shot and ease of multiplexing making it ideally suited for fighting variants along with flu in the future.

Unfortunately, Nuvaxovid (aka Covovax) has not been widely available as manufacturing a protein is not as straightforward as making an mRNA-LNP particle.  Still, with partners such as the Serum Institute of India and SK Bioscience now making commercial-grade product, approvals are being granted globally and the broad roll-out is on its way (Indonesia since last December; South Korea from February 14; EU and Australia from February 21 etc).

Curiously, while there is little controversy about the suitability as a covid vaccine, it has been under constant attack from media articles trying to tarnish the image of this company and vaccine.  Politico and Reuters in particular stand out by publishing harmful articles seemingly whenever the stock price is gathering momentum.

Today, I will exemplify just how bad this has become by dissecting the latest hit piece by Reuters paragraph by paragraph.

Rebuttal: On January 10 at the JP Morgan Healthcare conference, the CEO of Novavax Stan Erck told the audience that the EU had orderd 27 million doses for Q1 2022.  According to an Indian government site that tracks vaccine exports, Novavax' main manufacturing partner the Serum Institute of India (SII) has shipped 26 million doses into the EU during January.  These will have to be cleared following batch testing and checking the paperwork.   Unless the Indian government laboratories (CDL Kasauli) have passed and shipped bad product, it is unlikely the the SDS-PAGE and similar standard techniques will find something substantially different.

Regarding the Philippines situation, the vaccine is likely to be shipped in March once the 3-step process before roll-out has been satisfied:

1) approval (EUA on Nov 17, 2021)

2) HTAC recommendation for inclusion in national vaccination plan and DOH financing (29 December, 2021)

3) supply negotiations (ongoing)

Before that has occurred, Novavax and Serum will not send vaccine to places where it cannot be immediately used as they have only begun to turn stockpiled antigen and adjuvant into fully formulated products (~150M monthly capacity for SII sites alone).

The 10 million doses here likely refer to the 10 million doses that Novavax on January 10 confirmed have been shipped to Indonesia where they have been used already.  The doses are also shown on the Indian government export site.  Reuters, however, knows that it purposefully omitted that in fact 38 million doses have been shipped and that the balance (28 million doses) are currently undergoing batch release- some of which, as we will see later in the Reuters very own article, have actually been released for shipping.

That’s the normal process.  It’s great that Reuters acknowledges here that additional doses outside the 10 million in Indonesia are sitting in places like the EU ready to go out to healthcare providers once the paperwork is finished.  In Germany, many states are still planning with a February 21 start of vaccinations.  In Korea, SK Bioscience has today received batch clearance of the first batch of 840000 doses with vaccinations starting after Chinese New Year.

  

Standard cut-and-paste paragraph by Reuters, sadly adopted by the wider press, to ridicule the company. Which other company the size of Novavax 2 years ago has achieved what they did?  How about them filling in a major vaccine gap on the order of 2 billion doses, especially in countries that either cannot afford or do not have the cold chain required for mRNAs?

‘Tiny company’...well yes, if you consider Novavax 2 years ago on the brink of bankruptcy (biotech is a very high risk-high rewards business), but a company that will  soon hire their employee No 2000 is actually not so tiny in biotech terms.

But, of course, this is just part of Reuters ridiculing the company.

The only new information here as the broad roll-out is about to commence is that ‘individual vaccine batches’ have been cleared already in the EU.  Great to hear!  You are starting to contradict yourselves, Reuters.

Ominous mystery country will be revealed later in article. A bait for the reader to stay with article (spoiler alert: no, it's not the US or the EU).

 

80 million doses of the 1.1 billion doses promised to COVAX in Q1 as the company starts turning antigen + adjuvant into product and rich countries incentivizing the company to prioritize them.  Between Serum, SK Bioscience, Novavax' own sites that they had to set up and various other partners, a monthly run-rate of 200 million doses can be expected in the following quarters and should be able to satisfy the COVAX order.

 

 

Stan Erck said on January 10 that 10 million doses have been shipped in accordance with Indian government data.  An ‘Indonesian official who declined to be named’ apparently is only aware of the first November 26 shipment of 137500 doses, but not the subsequent December 98 million doses.  I guess fact-checking is optional for Reuters ‘journalists’.

But don't take it from Stan, but see it on the Indian government site or the United Nations OCHA January 5 Indonesia status update for yourself.

As I had noted before, Novavax and Serum will only send vaccine to countries that are ready to vaccinate.  The Philippines is not yet as it still needs to conclude its supply negotiations.

 

OK, if the Philippines regret having purchased other unused vaccine then by all means try and find reasons to renegotiate. We have all been there, done that.

 

Yes, I also had hoped that the vaccine would become available earlier than that after the December 20, 2021 approval, but the messaging in Germany has been February 21 for the first vaccinations and many states are making appointments accordingly.  Some are planning for February 28 and only a few early March.  ‘Early March’ would therefore indeed represent a delay.  We will see. Next up is South Korea next week.

 There is nothing factually wrong with this final paragraph, but it is symptomatic for Reuters to keep focusing on the hard work and challenges of bringing the first global protein-based covid vaccine to market.  Instead, it should have seized the opportunity- even if it was a critical article- to raise awareness of the unique opportunity for global health in front of us:

A better tolerated and efficacious covid vaccine without proven safety risks such as myocarditis and anaphylaxis.

As I look forward to getting my Novavax booster*, it is time to acknowledge the achievements of this tiny company where big ones such as Sanofi have failed.

*1st shot JNJ...rather severe chills, night sweats, headache, fever from 8-36 hours following vaccination; 2nd shot Biontech: better, but muscle ache around upper arm first, then entire body and general malaise 8-30 hours post vaccination. Now waiting for Novavax to reduce my chances of another 'bad day'.