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.