Increasing the Reach of ADAR Therapeutics by Protein Structure Prediction

'Only' being able to convert an ‘A’ to an ‘I’ when genome editing can seemingly re-write the code at will had been seen as a major limitation of the technology.  Examples such as the large and underserved market in correcting the piZZ genotype in alpha-1-antitrypsin disease using A-->I RNA editing were the exception.  With the help of artificial intelligence-enhanced structure prediction tools, however, the financial incentive to pursue genetic disease should increase again.  Indeed, ProQR’s first Rett Syndrome candidate already points in this direction.

Addressing the p.R270X mutation

Rett Syndrome is an X-linked dominant genetic haploinsufficiency neurological disorder caused by mutations in the MeCP2 protein leading to decreased functional activity as a master regulator of gene expression and neuronal development.  There are many ways to cause the loss of activity of a protein, so mutations can typically be found throughout an affected haploinsufficiency gene.

ProQR’s 1st candidate for Rett Syndrome addresses the p.R270X mutation where the arginine codon CGA at position 270 in the protein is mutated to the stop codon UGA, thus leading to a truncated protein that is also destabilized at the mRNA level via the NMD pathway.  Luckily, the CGA codon contains an actionable ‘A’ and although editing it to a G-like Inosine would not restore the wild-type protein, it happens that mice with tryptophan-coding UGG at position 270 behave like rescued wild-type mice.

Beyond p.R270X

It is well established that a second mutation can modify, if not rescue a disease caused by a first mutation.  At a protein level, this may be due to functional restoration by coaxing the protein back to its original functional structure.  

Rett Syndrome affects around 50,000 females in the US and EU of which ~3,500 would be due to p.R270X.  Point mutations across the MeCP2 gene overall account for 60% of cases. Such numbers are too low for one to expect to come across compensatory mutations in the MeCP2 from population genetics.  So instead of relying on serendipity one may systematically ask whether the structural pathological change resulting from a given point mutation or group of point mutations (characterized for example by destabilization of the DNA-binding domain of MeCP2) could be reverted back towards wild-type if one ADRA edited one of the ~350-400 Adenosines in the MeCP2 mRNA based on AI-powered structure prediction tools like AlphaFold.

This could then be verified by a cellular functional assay of MeCP2 as a master epigenetic regulator.  Regarding approval and clinical trial population, a label may just say that a patient with a very rare mutation that could not be confirmed in clinical trials due to low subject numbers may still qualify for the drug as long as such an assay supported it.  This path has already been trodden, for example with Vertex’ Cystic Fibrosis drugs.

AI increases IP value around platform drug modalities

Artificial intelligence is an amazing development.  The above strategy is just one example of how it may decrease future drug development cost and efficiency, thus increasing the attractiveness of going after rare diseases again.  I would not be surprised therefore if structure prediction may help to address the most common missense mutation in Rett Syndrome, p.R106W, which affects 3x the patient number of p.R270X.  

There are, of course, many more applications also to RNA editing such as chemistry based on structure prediction around the A to be edited when paired to the editing oligo. 

AI, however, is also a scary development for people like me.  When I came across this concept, it seemed like Gemini had it all figured out already.  I fully expect that in a year’s time, I will have to re-think the point of blogging full-stop when AI can tell for itself which are the more or less valuable concepts.  One thing is sure, AI will open the flood-gates for therapeutic strategies leveraging genetic platform technologies like ADAR, RNAi, antisense, CRISPR and gene therapy.  Investing in companies with fresh, gate-keeping IP in these technologies could therefore more valuable than ever.  It is therefore possible that sooner than later I will be watching all this from the beach as a passive investor in these platforms.  

Wave Life Sciences to Focus RNA Editing on Gene Upregulation

Yesterday, oligonucleotide therapeutics developer Wave Life Sciences provided a high-level preview on how it will deploy its RNA Editing technology.  Accordingly, modulating protein-protein interactions and, even more so, increasing gene expression will be the declared mechanisms of action of development candidates following its lead candidate WVE-006 for alpha-1-antitrypsin disease (AATD).

WVE-006 was recently licensed to GSK and should be the first RNA Editing candidate to enter clinical development later this year.  A big milestone for the field.   WVE-006 corrects a common single nucleotide mutation in the alpha-1-antitrypsin gene, Z-AAT, that causes both liver and lung manifestations of AATD. Z-AAT is retained in liver hepatocytes to cause cellular stress instead of being secreted to do its job and protect the lung.  As such, WVE-006 can be considered both a mutation corrector and gene function booster.

 

Mutations often scattered across genes

More often than not, however, mutations causing rare genetic diseases are scattered across a gene and precision genetic medicines targeting small segments of a gene at a time may thus only address a subset of patients.  A prime example is Duchenne Muscular Dystrophy where even exon 51 skipping which is the approach with the largest addressable patients still only serves 11-13% of the overall DMD population.

                                DMD patient segmentation according to skipped exon (from Wave Life Sciences presentation)

A very interesting indication for ADAR RNA Editing is Rett Syndrome (affects 1 in 10000 girls by age 12 in the US).  Here as well are the mutations scattered across the MeCP2 gene.  Almost half of those would be addressable by RNA Editing (including eliminating stop codons), but each individual target would be quite small.

So instead of targeting the specific mutations, ADAR Editing may also be used to screen all adenines in the MeCP2 transcript to identify those that lead to an increase in protein abundance and thus function either by stabilizing the resulting mRNA or by increasing MeCP2 stability.  While this approach would not apply to Rett Syndrome caused by 2 null mutations on the X chromosomes, a say 3x increase in activity of the chromatin CpG-binding protein may be enough to alleviate disease in a large fraction of Rett Syndrome patients with MeCP2 versions having reduced activity.  Or consider mutant CFTR proteins in cystic fibrosis with reduced channel activity. Increase the abundance of those CFTR mutant proteins and it should increase the overall desired activity.

The screening approach would also facilitate finding potent RNA editing oligos due to the flexibility and increase in targeting space as opposed to having to optimize the editing oligo around a small defined target site.

 

mRNA technology

Wave Life Sciences likened the gene upregulation approach as a simpler version of mRNA therapeutic technology.  Simpler, because it does not involve the delivery of long mRNAs which necessitates the use of LNPs and similar larger nanoparticle formulations due to mRNA stability requirements.  By contrast, RNA editing can be mediated by oligos ~30 nucleotides in length, short enough to be amenable to conjugation and oligo chemistry strategies already applied in RNaseH and splice modulation ASO and RNAi.

Smaller also means better tissue penetration and delivery to more target tissues.

Moreover, meaningful expression from an mRNA only occurs in short bursts so that the frequency of repeat administration is dictated by protein half-life.  Meanwhile, the administration frequency for oligo-mediated editing, due to the longer persistence of highly stabilized oligos, can be expected to be in the weeks and months.

It should be noted though that RNA editing would essentially upregulate what is already present in the cell (with the exception of the one editing change), whereas mRNA therapeutics in sensu strictu can generate entirely new proteins.

RNA editing would also not be the first oligonucleotide approach to mRNA upregulation.  RNA activation, the targeting of promoter-proximal regions using RNAi-type double-strand RNAs, and the targeting of upstream 5’ UTR mRNA elements with steric blocking antisense molecules as developed by Ionis Pharmaceuticals are competing approaches.  These, however, have so far either lacked the robustness or the flexibility in terms of sequence choice that AàI editing should afford.  

 

Now more than ever in biotechnology, companies need to carefully tease out the unique, differentiating advantages of a platform technology when selecting an indication.  RNA Editing leaders ProQR and Wave Life Sciences are in the fortunate position that they can apply the new biotech paradigm starting with their first RNA Editing candidates.  Biotech is ripe for a reboot and RNA Editing should have every ambition to be part of it.

 

Disclosure: I own both ProQR and Wave Life Sciences shares, though ProQR considerably more. 

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.