Gentle Endosomal Escape to Expand Reach of Oligonucleotide Therapeutics

One of the attractive aspects of oligo-mediated ADAR RNA Editing is that it can piggyback on decades of oligo delivery R&D.  New breakthroughs in oligonucleotide delivery are therefore instantly applicable to and greatly enhance the value of the platform. 

In order to make extrahepatic target engagement of oligonucleotide therapeutics as robust as in the liver, various cell-targeting ligand conjugates are being attempted. This includes transferrin receptor-1 in the muscle or various lipid conjugates for the CNS.  The goal is to get highly stabilized oligonucleotides being taken up into the endolysosomal compartments of target cells from which they would somehow escape into the cytoplasm.

Increasing their release through the addition of endolysosomal disrupting agents is highly effective, but it may not be surprising that spilling the insides of endosomes and particularly lysosomes into the cytoplasm in addition to the intended oligonucleotides is a dangerous proposition.  This is illustrated by (not just) the experience with Arrowhead’s DPCs, but also various cationic polymers before it. 

Certain liposomal nanoparticle (LNPs) chemistries are better tolerated.  This may be related to how they release their payloads into the cytoplasm and which may involve complex lipid bilayer dynamics that provide oligonucleotides the opportunity to slip through.

Trapping Oligonucleotides in Metastable Endosomal Compartments

Trapping stabilized oligonucleotides in a similar metastable endosomal membrane environment is what Finicle and colleagues (in a collaboration with Ionis Pharmaceuticals) sought when inhibiting both endosomal recycling and lysosomal fusion with the synthetic sphingosine analog SH-BC-893.

Indeed, ‘893-treated tissue culture cells accumulated ~4 times the amount of a model phosphorothioate gapmer and there was a 9x oligonucleotide increase in the desired cytoplasmic-nuclear compartment.  This in turn lowered the IC50, the concentration of gapmer oligo necessary to inactivate 50% of target RNAs, by over 100 fold.  The IC50 of a model RNAi trigger was similarly lowered 20 fold.

As expected, crude endosomolytic agents proved extremely effective when added to tissue culture cells after the addition of oligonucleotides, but highly toxic.  And when compared to gentler oligo delivery enhancers reported before (AZD8055, 6BIO), ‘893 turned out to be significantly more effective.

Importantly, the sensitization benefit translated to the in vivo setting in a mouse model. Here, ‘893 was given orally around the time of subcutaneous oligonucleotide administration. 

While no gene silencing was really observed when a gapmer antisense oligo was hybridized to a cholesterol-conjugated complementary oligo (which was supposed to aid CNS delivery) and then subcutaneously administered, ‘893 led to noticeable, albeit still quite modest target engagement in some areas of the CNS.

This means that either the approach is optimized and ‘893 can be used in a subcutaneous oligo delivery setting when invasive intrathecal administration is prohibitive, or that ‘893 may be better suited for use along with intrathecal administration of oligos with the goal of achieving liver-like robust target engagements.  For highly severe and relatively rare diseases, the latter would be my preference.

To generalize the in vivo benefit of ‘893 following systemic oligo administration, greatly enhanced RNA silencing was seen in the mouse lung.  Again, I would have liked to see it compared to the benefit that ‘893 might bring to local, in this case aerosolized delivery.

 

Safety of Approach

That this endosomal kinetic entrapment strategy may have clinical legs is supported by the apparent safety of ‘893.  Indeed, this compound is already being considered for cancer and obesity-related indications.  So who knows, you may end up taking your oligos along with ‘893 and similarly acting agents that have anti-ageing activity.  And even if ‘893 should reveal negative side effects following chronic dosing, as a PK enhancer of oligonucleotides it may only have to be given infrequently, depending on how often the oligonucleotides need to be administered.

 

It is nice to see oligonucleotide deliveries making continued inroads throughout the body.  After the liver and CNS, the lung and muscle are becoming fertile hunting grounds for Oligonucleotide Therapeutics developers.  Because of its novelty and differentiation, RNA Editing will be busy with liver- and CNS-related applications alone for some time to come, but when there is robust clinical target engagement by oligos in other organs, RNA Editing can instantly jump on those opportunities.  Next up in terms of extrahepatic delivery is Arrowhead Pharmaceuticals with a number of early clinical readouts for RNAi gene silencing indications of the lung.

A Visit to ProQR

Yesterday, I had the pleasure and privilege to visit ProQR in Leiden (Netherlands).  Thank you, Sarah and Andy from IR, for making it happen! 

The goal of my visit was to see whether the people and the context in which it all happens are conducive to ProQR's delicate quest of growing, leading, and establishing the immensely valuable RNA Editing platform as a new therapeutic modality.  

It takes a special mix of enabled technology, intellectual property, and committed people to make this happen.  Luckily, it has been done before in biotech and is therefore achievable with the careful execution by skilled and highly motivated people. 

In addition to the people aspect, two issues were on top of my mind as they will likely determine shareholder return over the next 3-5 years: 

the editing oligonucleotide chemistry and the pipeline.

 

Chemistry and structure

Historically, ProQR has heavily relied on phosphorothioate (PS) backbone chemistry for their now discontinued cytoplasmic (CFTR) mRNA correction and RNaseH/steric blocking antisense projects.  Publications by ProQR and collaborators similarly reveal their use of this chemistry for ADAR RNA Editing.

While the selective use of the PS modification for editing oligo stabilization purposes should be acceptable similar to what is practiced and proven in the RNAi space, insisting on its broad use can have catastrophic consequences for platform development as Ionis Pharmaceuticals' antisense journey has shown. 

Even their wild success story, splice modulator SPINRAZA which has saved the lives of many babies with the spinal muscular atrophy mutation, suffers from PS-related side effects.   On a side note, the fact that SPINRAZA, but not other intrathecally administered RNAseH mechanism-based PS-ASO so far has proven to be an impactful drug, is partly due to it relying on a gain-of-function approach where low double-digit % target engagement can be sufficient.  And yes, ADAR Editing is primed for similar gain-of-function applications.

Obviously, ProQR could not reveal much about the chemistry and structure of their upcoming EONs for competitive and IP-related reasons. Still, they were able to communicate that they share my concerns and are aiming to minimize chemistry-related tox issues while retaining necessary oligonucleotide stability.  In particular, the 10+ oligo chemistry team that I saw hard at work and the background of their Chief Scientific Officer Gerard Platenburg make me hopeful that ProQR are indeed looking broadly for acceptable backbone chemistries.

On the structural side, again, ProQR could not say much in detail except that they believe the meat of the action in coming up with general EON design and optimization rules is where the oligonucleotide interacts with the ADAR deaminase domain and that there could be differences between different tissues.

When challenged with my observation that in many publications in the space often only 1-2%-type mRNA editing efficiencies are seen whereas I would prefer 10-20% efficiencies, the team was quick to point out their belief that the inherent ADAR editing capability is close to 100%.  I agree as such high ADAR efficiency can be observed in neuronal development and editing efficiencies north of 50% have been demonstrated with optimized candidates for more advanced alpha-1-antitrypsin candidates by ProQR and Wave Life Sciences.

ProQR does not believe it has to screen 1000+ oligos to find candidates of acceptable efficacy (off-targeting is not really a concern with ADAR Editing) as it is now routinely practiced in RNAi and RNaseH antisense drug development.  While this suggests that ProQR are confident about their funneling strategy, I would caution that since screening costs have come down dramatically over the last 2 decades, when it comes to finding the best possible development candidate for an indication you are excited about, this is a place where capital is well spent.

  

The pipeline

CEO Daniel de Boer understands the importance of maintaining momentum following the recent landmark deal with Eli Lilly until first clinical data, possibly in 2024 (my guess) can speak for itself.  Maintaining momentum is of critical importance because a strong currency (=share price) and investor base is instrumental for biotech success.  Providing additional preclinical data and pipeline visibility is also key in getting the attention of the industry for business development.

The next catalyst in the ProQR story is therefore the much anticipated revelation of its clinical pipeline.   Previously, ProQR guided this to occur by either year-end 2022 or early 2023.  Daniel was positive that such an event can be held in Q1 along with preclinical data in support.

In addition to minimizing technical and biology risk, ProQR apparently also understands that market potential matters.  Indeed, RNA Editing is such a unique mechanism that it can either address new, significant markets with no real alternative treatment approaches or go head-to-head with other modalities as the most elegant and promising mechanism, for example alpha-1-antitrypsin or Huntington's disease (my thoughts, not indications ProQR mentioned). 

So don’t expect ultra-orphan gene mutation correction programs where patient populations are sliced down to a few hundred applicable patients and less.  The recent fate of numerous gene therapy companies hurtling towards bankruptcy despite having been able to produce promising clinical data for such ultraorphan indications show that at the moment this business model does not work.

In addition to market size and having a unique modality angle, minimizing delivery and biology risks are top selection criteria.  Expect the line-up to come with early biomarker opportunities to make go/no-go decisions as early in clinical development as possible. It therefore would not surprise me if most if not all the first-generation pipeline candidates targeted liver hepatocytes.  And why not, since the unique ADAR Editing mechanism, especially for gain-of-function provides differentiation in an otherwise relatively crowded target tissue.

Having said that, I would not mind an exciting CNS program either to gain some experience with this target organ as long as the pipeline and thus the fate of the company is not dominated by a single program.  Instead, the company should give itself ~3 shots on goal and if any one of them hits the target, the company would be wildly successful.

 

People and culture

It was the late-stage failure of an antisense oligonucleotide-based orphan drug program in ophthalmology that prompted ProQR to shift gears and become 100% committed to ADAR RNA Editing.  Along with it came a considerable reduction in workforce mainly affecting those working on the clinical programs.

The surviving employees and increasingly new additions, numbering 128 now and set to grow to ~150 by mid-2023 are lucky to inhabit a nicely refurbished headquarters that reflect a respect for nature (natural colors and materials, lots of plants) as well as good RNA science and biotech leadership.  It may be news to mRNA pioneer Katalin Kariko that a meeting room is named after her. Of course, the coolest room featuring sauna-type wooden benches is named in honor of the late founder of the orphan drug biotech model and ProQR mentor, Henri Termeer.

The space offers plenty opportunities for flexible work and informal meeting and exchange to help build the cohesion among the workforce and provide the dynamic work environment necessary to overcome the challenges faced by new therapeutic modalities.

One concern that I had before visiting ProQR was that not being in a major biotech hub, a Leiden-based biotech may find it difficult to attract top international talent.  But the ~100 scientists, largely from all over Europe, will feel right at home in this small university town setting where the quality of the coffee and toasties matter more than living close to say an internationally acclaimed opera house.  Being next to Amsterdam and Schiphol airport are equally important in this regard, but also to make it easier for Board members, advisors, and collaborators visiting the company.   

Management seems to get along each other well with almost all of them going back to around the founding in 2012.  To wit, ProQR was born as an initiative by Daniel de Boer in his quest to find a good drug for his child that was diagnosed with a rare disease.  This quest led him to a meeting with fellow Dutchman Henri Termeer- the rest is history.  

While Daniel will have learned a lot in his 10+ years of biotech leadership and has proven to be a very capable businessman, it is good to know that he is supported by a leadership team with deep experience in the biotech business (Rene Beukema, corporate development) and oligonucleotide chemistry and therapeutics  (Gerard Platenburg). 

Bart Klein (SVP Axiomer) deserves a special shout-out for his pioneering role in oligo-guided ADAR RNA Editing. 

I vividly remember an encounter with Bart at a scientific meeting, possibly the 2017 OTS meeting in Bordeaux if not the 2014 ESCDD meeing at a Dutch seaside place.  Anyhow, he excitedly urged me to have a look at ProQR’s cool technology. I unfortunately hesitated and showed myself closed-minded as my view of the company at the time was clouded by their CFTR program that was built on the flawed notion, probably a PCR artefact, of a cytoplasmic mRNA cut-and-paste repair mechanism.  Although this program dominated the valuation and public attention of ProQR at this early time in oligo-guided RNA Editing, it was this little side project that, in truly visionary fashion excited him most.  Today, this belief is the reason why what commanded maybe 20% of the company mindshare up until the early 2022 sepofarsen failure is the reason why ProQR is still alive and kicking and may very well be the foundation of one of those stunning biotech comebacks.

Nothing is certain in biotech, but here is a story with a high probability of success.

Disclosure: I own >2% of the company stock. This blog post is not meant as a stock recommendation, but to provide greater visibility and transparency of ProQR and ADAR RNA Editing.

Reconsidering RNA Editing for Alpha-1-Antitrypsin Disease

Alpha-1-antitrypsin (AAT) is the lead program in the RNA Editing pipeline with Wave Life Sciences recently licensing its candidate to GSK.  It is safe to assume that a number of other companies are working on similarly developing RNA Editing drugs in the AAT area as well.  Developments in the competitive environment using other platforms such as gene knockdown (RNAi, ASO) and genome editing are therefore of high interest.

Arrowhead Pharmaceuticals SEQUOIA data

This week, Arrowhead Pharmaceuticals and partner Takeda revealed that its RNAi candidate for alpha-1-antitrypsin-related liver disease worked as intended in the SEQUOIA phase II study and over time should translate into a measurable benefit in terms of developing severe liver disease.

After 52 weeks of treatment, mutant Z-AAT was knocked down by a solid -94%.  This in turn led to a 2/3 decrease in the more inert aggregated form of alpha-1-antitrypsin.  It is the aggregated AAT that is thought to cause cellular stress, followed by a cycle of apoptosis and hepatocyte regeneration, ultimately leading to inflammation and fibrosis, if not liver failure in ~20% of subjects homozygous for the Z-allele of alpha-1-antitrypin.  

Indeed, ARO-AAT/TAK-999 achieved a striking benefit of reducing portal inflammation (see above graph) which should be highly predictive of a benefit on liver fibrosis progression.  Although a high 50% of trial subjects on ARO-AAT experienced a reduction in fibrosis, this was not statistically significant due to small patient numbers.  A larger and longer (2-4 years) registrational study is therefore planned to confirm the benefit and gain regulatory approval.

RNAi unsurpassed in reducing Z-AAT, but RNA Editing may be more elegant

If the degree of reducing the insulting Z-AAT was all that mattered, RNAi would be difficult to beat. ADAR RNA Editing and genome editing for example would have to overcome the limitation that target sequences are limited to a very specific area around the mutation whereas RNAi has the entire mRNA to play with.

But looking more closely, the name of the game is actually inhibiting aggregation.  It is now that I increasingly appreciate the elegance and power of the RNA Editing approach   which fundamentally rests on the genetic observation that heterozygous carriers of alpha-1-antitrypsin disease have a much reduced risk of developing related liver disease, if an increased risk at all.  These people carry one 'Z' allele and one normal ‘M’ allele. 

I had not paid much attention to this, thinking that mechanistically this is simply due to a concentration effect of 50% less Z-AAT polymerizing less well and having a protective genotype that manifests after decades of life does not necessarily mean that converting a patient already suffering from liver disease to such a genotype will necessarily translate to the fastest possible treatment benefit.  RNAi with its highly potent Z-AAT reduction may be faster.

It is now that it dawns on me that there is likely more to the MZ genotype benefit.  Looking at the Wave Life Sciences mouse preclinical data on its RNA Editing candidate, it always irritated me that whereas the mRNA editing efficiency was ~50%, the increase in circulating AAT was 7x.  Since only ~15% of Z-AAT normally gets out of the liver, this basically means that the 50% of M-AAT allows essentially ALL of the AAT, including Z-AAT to get out of the liver.  This is consistent with the ratio of M-AAT/Z-AAT in circulation reflecting the 50% mRNA editing efficiency in hepatocytes.

It therefore appears that the M-AAT (and potentially non-wildtype variants generated by RNA Editing) functions to poison the Z-AAT aggregation chain reaction, keeps Z-AAT in solution and fit for secretion into circulation.  What is more, unlike RNAi, this has the additional benefit of also addressing the lung manifestations due to insufficient levels of circulating AAT and for which alpha-1-antitrypsin disease is better known.  RNAi by contrast may have to rely on other developments in improving the management of alpha-1 lung disease as it reduces circulating AAT even further.

In light of the above, as a shareholder I am now changing my mind and support ProQR with its industry-leading IP and know-how to enter the exciting alpha-1 race to generate a best-in-class RNA Editing drug.  We should soon find out when ProQR will reveal its line-up of initial pipeline candidates.

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.