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.  

Why I Am Taking My HAE Cure Idea to My Grave

Last year, as I was researching the hereditary angioedema (HAE) therapeutics landscape, I came across data in the C1 inhibitor gene regulation literature that gave me a proverbial heureka moment: if I could do ‘X’, then I would have a cure for HAE. Dazed by the excitement of being in possession of such an idea, I verified that the enabling technologies (genome editing and LNP delivery) were fit for purpose.

It basically seemed that all I had to do was to verify my approach in tissue culture and the rest would be ‘simple’ business development. In the vast majority of cases, HAE is caused by mutations in the C1 Inhibitor gene. It is a rare disease affecting close to 10000 patients in the US and EU. Nevertheless, it is a lucrative, currently ~5B market with therapeutics mostly replacing missing C1 inhibitor in the form of recombinant or purified C1 inhibitor or agents interfering with proteins involved in bradykinin generation or the bradykinin B2 receptor. 

It is getting a bit crowded, but there still is room for improvement to achieve the goal set by the HAE community: a life free from disease. Intellia’s NTLA-2002, a one-time CRISPR Cas9 therapeutic now in phase 3, achieves a 80-90% attack rate reduction by destroying prekallikrein gene activity. The efficacy is in line with competing therapeutics that need to be taken indefinitely and cost around half a million USD- annually. 

A good estimate for the price of NTLA-2002 would be $3M, meaning that after 6 years this convenient treatment would start saving the healthcare system serious money for a disease that gets diagnosed typically in the late teens or early 20s. Nevertheless, the financial market could not be less excited by NTLA-2002 as indicated by the lackluster response to its clinical data. In theory, NTLA-2002 could treat every HAE patient and rake in $30B in the process. Compare that to the annual $5B and growing market for the traditional forever treatment options which will cost the system $60B plus over the next 10 years alone…without even treating every patient and leaving treatment gaps due to insurance that like to drag their feet. 

We are talking here about cost alone, not even how a one-time treatment would benefit a HAE patient: less stress, both financial and emotional which in itself is a major risk factor for HAE attacks. Considering the lack of investor interest in NTLA-2002, I concluded that going from 80-90% attack rate reduction to 100% attack rate reduction following a one-time treatment, is not worth the time and financial risk. Disclosing the idea also does not make sense since it would destroy any IP value if the idea was picked by somebody else or conceived independently, further reducing chances it would ever get to patients. 

Just recently, Pfizer, a major player in hemophilia therapeutics, declined to bring a reasonably effective and safe hemophilia A one-time gene therapy discovered by Sangamo to market. Again, I ask myself, why would Pfizer ever want to destroy a lucrative market it participates in? I can only see how an outside company would want to disrupt such a rare disease market (hemophilia is bigger than HAE, so that situation could actually happen e.g. in the form of Regeneron). 

What about patients? I am slowly coming to accept that the best possible treatment for a disease will often not be the end-game in terms of therapeutic development. Sometimes it is pharma’s profit model, sometimes society that does not feel that a patient deserves better. Take for example a rich country like Singapore where patients do not have access to modern HAE medicines and where even women are still treated with the androgen sledgehammer. 

It seems we have arrived at a turning point where it has become hard to invest in best possible therapeutics in a resource-constrained environment where money gets diverted to the military and AI. Making matters worse for rare disease drug development, as international ties continue to deteriorate, the target population becomes even smaller.

The Lp(a)-Lowering Drug Race

Cardiovascular disease (CVD) remains a leading cause of death and morbidity worldwide.  According to the WHO, 18 million of the 68 million deaths annually can be attributed to it.  That's more than 1 in 4. This does not include those whose quality of life is severely impacted by it.  Despite the positive impact of LDLc-lowering drugs (statins, PCSK9 inhibitors), blood-lowering, anti-diabetic, and weight-loss medications, other risk factors remain to be addressed.

Among them is lipoprotein little a [Lp(a)], an independent and causal risk factor for cardiovascular disease.  Except for a few with extremely high Lp(a) who were lucky enough to be diagnosed, often after family members encountered cardiovascular events early in life, it has been largely met with a shrug in general medicine.  The dynamic, however, is quickly changing now as the PCSK9 outcomes trials are strongly supportive that lowering Lp(a) should be able to lower CVD risk (O'Donoghue et al 2019) and new strategies mostly centered around sequence-targeted therapeutics allow for robust Lp(a) reductions.

This week, Novartis disclosed that the eagerly anticipated results from the pivotal trial of the most advanced of these clinical candidates, the RNaseH antisense compound pelacarsen has been delayed.  It was originally projected to deliver results in 2025, but is now guided to read out in the first half of 2026.  As this has raised a few eyebrows, I will take the opportunity to provide an overview of the Lp(a) competitive landscape and the pharmaceutical companies involved.

    

Lp(a) is a genetic determinant of CVD risk independent of LDLc and ApoB100

Lp(a) is quite similar to LDL-cholesterol lipoprotein particle, but in addition to carrying one molecule of apolipoprotein B 100, cholesterol and phospholipids in its outer shell, there is a apolipoprotein (a) molecule attached to ApoB100 via a disulfide bond.  If LDL cholesterol is referred to as the ‘bad’ cholesterol, Lp(a) on a per particle basis is the lipoprotein supervillain.  It not only gets more easily through the blood vessel endothelial cell layer so that it becomes a substrate for plaque build up, it is quite inflammatory as a result of being a good carrier of oxidized phospholipids.  It is also thought that there may be a prothrombotic effect partly due to its similarity and evolutionary relationship with plasminogen.   

Lp(a) distribution in general population (from Varvel et al 2016)

Unlike LDLc, Lp(a) levels are largely genetically determined and cannot be modified by nutrition and exercise.  They stay more or less constant after 5 years of age.  Levels range widely within a population.  Whereas many have Lp(a) levels of 10mg/dL or less (~22nM), the distribution has a long tail with ~10% of the population, especially black people, who have more than 90mg/dL.  This puts them at ~1.4x and higher risk of suffering CVD events like CVD death, myocardial infarction or of developing calcific aortic valve and peripheral artery disease (Erqou et al 2009).  The increased risk is maintained after having suffered a first event with almost 2x the risk for persons in the top decile versus those below 10mg/dL (Madsen et al 2020).  This is relevant when thinking about using Lp(a) lowering as a strategy for primary or secondary CVD prevention.

Evidence that intervention can reduce CVD risk

Currently the best advice one can give to somebody with highly elevated familial Lp(a) is to address all the other modifiable CVD risk factors.  In terms of medication, this typically means LDLc-lowering by statins and, even better so, the newer PCSK9 inhibitors.

This is because PCSK9 inhibition reduces Lp(a) by 20-30% (Sabatine et al 2017; Schwartz et al 2018; Ray et al 2017) .  The high variability of the Lp(a) reduction between subjects allowed for the determination that in the FOURIER trial of Amgen’s evolocumab there was a 15% relative risk reduction for certain cardiovascular events for each 25nM Lp(a) lowering (~11.6mg/dL; O'Donoghue et al 2019).  Remarkably, those above the median Lp(a) at baseline (37nM) not only had the highest absolute Lp(a) reduction, they had a 23% lower CVD hazard ratio versus only 9% for those below the median.  Importantly, these findings were independent of the magnitude of LDL-c lowering in the same subjects  which was the same across the Lp(a) ranges.

This in my mind is the strongest evidence that not only life-long differences in Lp(a) have a meaningful impact on CVD risk, but even just 5 years of pharmaceutically lowering Lp(a) may have a big impact.

The Lp(a) competitive landscape

It is therefore not surprising that half a dozen or so notable Lp(a)-lowering efforts are in clinical development.  At the same time, medical societies like the EAS, EHA and AHa and KOLs are busy raising awareness around Lp(a).  Still, a lot of work remains to be done: less than 1% in the western medical systems have been tested, despite the fact that guidelines are increasingly recommending getting tested at least once in a lifetime. While universal numbers are hard to come by, reports from various health systems suggest that testing is finally gaining traction (e.g. Bhatia et al 2023).  The pump is thus getting primed for the successful roll-out of Lp(a)-lowering agents.

RNaseH pelacarsen (Ionis, Novartis)

Leading the pack is pelacarsen, originally discovered and developed by Ionis Pharmaceuticals and subsequently licensed to Novartis in 2019.  Pelacarsen is a subcutaneously administered GalNAc-ASO working by RNaseH-mediated knockdown of apo(a) mRNA.   It is currently in a global pivotal phase 3, Lp(a)HORIZON outcomes study evaluating its ability to reduce major cardiovascular events (MACE) in a secondary prevention setting in 8000+ patients with Lp(a) >70mg/dL.

The 80mg once monthly should achieve 80% Lp(a) lowering based on a phase IIb study in a similar population (Tsimikas et al 2020).  Safety and tolerability have been good.  This means an absolute reduction of ~55-60mg/dL for a patient at the lower end of the range, a number that has been estimated (Madsen et al 2020) to be enough for ~20% relative risk reduction.  

Given the uncertainties about the exact relationship between pharmaceutical Lp(a) lowering and risk reduction, Novartis conservatively added a second cut analyzing the effect of pelacarsen in the population with >90mg/dL of baseline Lp(a) for efficacy.  If the study hits in either cohort, it can be considered successful.

The study is an event-driven one, meaning that in this case 993 MACE need to accumulate.  The fact that the data monitoring committee (DMC) has apparently communicated to Novartis that the projected results are now due in 2026 instead of 2025 suggests that insufficient MACE events have occurred since study start in 2019 and completion of enrolment in mid-2022 (2 ½ years minimal follow-up).  

It is plausible that the slower-than-anticipated event rate has to do with a population that is highly controlled for other risk factors, especially LDLc, as I expect most to be on maximally tolerated statins and a good proportion on PCSK9 therapeutics, too.  More optimistically, slow event accrual could also be due to an extraordinary efficacy of pelacarsen.  I suspect, however, that the trial protocol provided for interim looks not only for futility (early looks), but also for efficacy at this late stage of the trial.  The most likely scenario therefore is that the DMC saw maybe 800 events and concluded that the study would benefit from running its full course.  At the very least, the study was not stopped for futility.  

Personally, 70mg/dL is too low a baseline Lp(a) to expect a strong MACE response.  I see Lp(a)-lowering to have most initial utility in the top Lp(a) decile (>90-100mg/dL) and it would therfore not surprise me if HORIZON only hit in that bracket.

RNAi olpasiran (Arrowhead, Amgen)

Hot on the heels of pelacarsen is olpasiran, an RNAi agent discovered by Arrowhead Pharmaceuticals, licensed to and now in development by Amgen.  This liver-targeted RNAi trigger commenced a pivotal phase 3 trial in another secondary prevention study for atherosclerotic cardiovascular disease in 2022 and has finished enrolling 7000+ subjects in just 1 ½ years.  This means that results from OCEAN(a) Outcomes could become available within a year of Lp(a)HORIZON in late 2026. 

The apparent enthusiasm by Amgen is encouraging since they, as the developer of PCSK9 antibody Repatha, are best positioned to understand the likely impact of modulating Lp(a) on CVD outcomes.  In addition, the efficacy of olpasiran trounces that of pelacarsen with a time-averaged Lp(a) reduction of more than 95% (vs -80%) despite of using quarterly instead of monthly subcutaneous dosing (O'Donoghue et al 2022).  The marked difference in efficacy will also allow for insights into whether, as is widely assumed now and as is the case for LDLc, it is the absolute Lp(a) lowering that matters or the relative percent lowering. 

Finally, I like the study because it focuses on a population in the top decile of baseline Lp(a) (>200nMol, ca >90mg/dL) where genetically CVD risk increases exponentially.  I also would not be surprised if lowering Lp(a) well below the genetically defined 50mg/dL risk threshold brings further benefit in an interventional setting.  Doing everything to keep these streaky macrophages from bursting by depriving them of their oxidized fatty meals could be quite beneficial.

RNAi lepodisiran (Dicerna, Eli Lilly)

Two years behind olpasiran in terms of phase 3 initiation, but every bit as potent and even more long-lived in its Lp(a)-lowering activity is RNAi rival lepodisiran.  Lepodisiran is a Dicer-substrate RNAi molecule discovered by Dicerna (now Novo Nordisk) and developed by Eli Lilly.

Lp(a)-lowering by single dose of lepodisiran

In a single dose study, a large dose of 608mg, achieved time-averaged ~95% reduction in Lp(a) over a year.  A lower dose of 304mg effectively lowered Lp(a) by a peak ~98% in the first months before leveling off at around 90% at the half year mark (Nissen et al 2023).  

The ACCLAIM-Lp(a) ASCVD phase 3 study in 12500 subjects in secondary prevention and for those at high risk will test 3 semi-annual doses before moving on to annual dosing.  Though the dose has not been disclosed, I consider both 608mg and 304mg dosages possible with this dosing regime.  The choice will have probably been strongly informed by whether Eli Lilly believes the clinical benefit of going from 10nM to 5nM Lp(a) will balance out formulation, drug administration, and potentially safety issues coming with the high dose.

Besides Eli Lilly, Novo Nordisk, Amgen, and pretty much everybody with a stake in the lipid-related CVD market believe in the advantages of infrequent drug administration as evidenced by their investments in this area.  Novartis this quarter saw the PCSK9-targeting RNAi drug Leqvio (licensed from Alnylam) cross the $1B run-rate for the first time.  It is 284mg of canonical siRNA oligonucleotide administered basically semi-annually.  This demonstrates the shift from daily oral pills that most people will have stopped using after a year despite their need for life-long treatment.  This is understandable since most of them do not suffer acute symptoms. 

2-dose study of zerlasiran

This brings us to the final notable RNAi contender, zerlasiran by Silence Therapeutics.  In early trials, 300mg showed clear potential for maintaining Lp(a) reduction of more than 95% using quarterly dosing.  Being phase 3 ready, Silence Therapeutics stood to benefit the most from a timely Lp(a)HORIZON reveal by Novartis.  Not only may have they been able to adjust their Lp(a) target levels for their outcomes trial, a successful readout may also have been the trigger for a Big Pharma like Pfizer or Merck with no stake in Lp(a) yet to acquire zerlasiran, if not acquire Silence Therapeutics (market cap $300M) altogether.  

Life-long Lp(a)-lowering using CRISPR genome editing

If you like infrequent dosing, why stop at semi-annual or annual when there are life-long options going after the same apolipoprotein(a) target?  This includes base editor VERVE-301 by cardiovascular specialty company Verve Therapeutics which is partnered with Eli Lilly, but still in preclinical development.  Already in phase I is competitor CTX-320 by CRISPR Therapeutics using more traditional CRISPR Cas9 cleavage of apo(a).  Both use standard intravenous LNP delivery to liver hepatocytes.

Finally, in addition to RNAi and CRISPR, Eli Lilly is also developing an oral small molecule drug. Muvalaplin is designed to inhibit the interaction between ApoB100 and Apo(a) and thus the biogenesis of the Lp(a) particle.  Daily doses barely lower Lp(a) by 60% and as a small molecule there are concerns about hemodynamic effects given the similarities of apo(a) and plasminogen (Nicholls et al 2023).

Disclosure: I have been and am still positioning myself for HORIZON by investing across the Lp(a) landscape, including Silence Therapeutics, Verve Therapeutics and Ionis.  Focused Lp(a) testing companies would also fall into my interest area, so please pitch me ideas if you have some in this area.

Huntington’s Disease Therapeutics Breaking Through (Finally!)

Huntington’s is one of the large devastating monogenic diseases for which disease-modifying therapies have yet to materialize.  This is about to change as a combination of big data sciences (single cell sequencing and GWAS) together with good old molecular biology is unraveling the detailed mechanisms of this neurodegenerative disorder.  This provides an understanding for when and how to intervene.  New therapeutic modalities, in particular sequence-specific therapeutics like oligonucleotides and genome editing that have emerged in recent years will play an important role in this endeavor.

In this blog entry, I will try to summarize the research findings and how individual steps along the pathogenic pathway may be targeted.  Hold on, it will be an exciting one.

Somatic repeat expansion

Huntington’s Disease is linked to the expansion of the CAG triplet repeats found in exon 1 of the huntingtin gene.  If you inherit a copy of huntingtin from either parent that carries 40 repeats of CAG or more you will almost certainly develop symptoms of HD (manifest HD).  Symptoms intially involve emotional instability and cognitive deficits, progressing to include the famous movement abnormalities, ultimately culminating in life-shortening problems with basic physiological processes such as feeding and breathing.  These follow neurodegenerative processes which can be observed decades before symptom onset (Scahill et al, 2025), most notably severe striatal atrophy.  If you start with a number that is closer to 40 at birth, symptom onset may be in the early 60s, but if it is 50 or so, it will be more like at age 30.

Newer findings (Handsaker et al, 2025), show that having a huntingtin copy with 40 CAG repeats per se is not toxic to a given cell.  But having 36 (incomplete disease penetrance), and especially 40 repeats or more is essential for stage 1 of pathogenesis: somatic repeat expansion.  Genome-wide association studies (GWAS) have highlighted the critical role of the mismatch DNA repair (MMR) in facilitating such repeat expansion, also in post-mitotic cells in the CNS (Gem-HD, 2015).  It is possible that as the transcription bubble in the triplet repeat DNA is formed during huntingtin mRNA transcription, chances of the DNA bubble collapsing out of register increases exponentially with repeat length.  The consequently misaligned DNA strands would then be recognized by MMR and haphazardly resolved, in most cases by adding a CAG triplet on both strands. 

The reason why symptom onset can differ by decades depending on starting CAG numbers is because repeat expansion itself is length-dependent and the initial expansion is quite slow until it picks up steam later (Handsaker et al, 2025).  It is when repeat numbers reach ~150 in a given striatal projection neuron (aka medium spiny neuron), toxic substance X (see discussion below) gets produced and the cell starts to suffer.

The exact timing of manifest HD for a given CAG repeat number, however, also varies substantially and depends on additional factors, many of them genetic.  Genome-wide association studies (GWAS) looking at those differences have clearly shown that proteins in the mismatch DNA repair pathway mediate repeat expansion (MSH3, FAN1, MLH1 etc) and have critically contributed to the mechanistic model of somatic repeat expansion outlined above (Gem-HD, 2015; Gem-HD, 2019).  

An obvious therapeutic strategy therefore would be to inhibit repeat expansion by targeting DNA repair or by cutting down its size, if not get it below the 36 risk threshold using CRISPR genome editing (e.g. by twin prime editing).  A number of companies among them Ionis, Biogen, Rgenta, Atalanta, LoQus23, Prime Medicine are attempting just that.  Just binding DNA with zinc fingers has also been shown to inhibit expansion (Mathews et al, 2024).  This strategy would also be able to simultaneously reduce the generation of toxic factor X. 

DNA repair could in theory be targeted by small molecules.  There is, however, perhaps unsurprisingly ample evidence that doing so indiscriminately throughout the body may be too risky a strategy.  For example, early research of somatic repeat expansion in HD showed that knockout mice of the MMR protein MSH2 develop cancer (Wheeler et al, 2003), and while having heterzygous mutations in MMR genes slows HTT repeat expansion, it causes Lynch Syndrome, the most common genetic predisposition for cancer (Skarping et al, 2024).

More targeted approaches are therefore called for.  These may include delivery specifically to the cells most affected by repeat expansion (i.e. the striatal projection neurons) or at least limit delivery to the post-mitotic and therefore less cancer-prone CNS using oligonucleotides, mRNA and/or DNA (by AAV, LNP).  Alternatively, one could identify and copy rare missense or exon-skipping genetic modifier mutations in the human population that are protective against repeat expansion without predisposing to cancer.  This could involve CRISPR prime or base editing, or non genome-editing ADAR RNA editing.   

Toxic Factor X

Somatically expanded CAG repeats in the huntingtin gene DNA per se, however, is not what causes cellular toxicity.  This is illustrated by GWAS hits that disentangle somatic expansion from age of disease onset and disease duration (GEM-HD, 2019).  Furthermore, while CAG repeat length at birth strongly correlates with age at onset of disease and death, other genetic factors determine the duration between onset of disease and death (Keum et al, 2016).  Finally, while the MSH2 knockout mice mentioned earlier (Wheeler et al, 2003) did not exhibit CAG expansion, they still showed some striatal-specific huntingtin inclusions characteristic of HD, albeit at much reduced levels compared to wild-type mice.

Historically, HD was thought of as a 'polyglutamine' disease, meaning that it is extended polyglutamine stretch that is toxic to a cell, either by forming inclusions causing cellular stress or by inappropriately interacting with other proteins.  In fact, polyglutamine inclusions are characteristic of disease pathology as they are found in the cells most affected by HTT. Interestingly, these inclusions are best detected by antibodies directed at the N-terminus of HTT encoded by exon 1.

However, this polyglutamine-only model is seriously challenged by the observation that other CAG expansion diseases carry extensions in untranslated UTR elements and by the fact that mutations in HTT exon 1 itself that extend the stretch of glutamine by non-CAG synonymous codons do not increase inherent toxicity and may in fact delay disease onset and death.

In addition to protein, differences in HTT expansion should affect RNA structure and RNA polymerase II transcription.  Toxic RNA is thought to be at the root of other repeat expansion diseases such as type I myotonic dystrophy as they by sequester important RNA-binding proteins away from their normal functions.  Intriguingly, HTT RNA clusters in a repeat length-dependent manner around the huntingtin gene locus and these clusters appear to be seeded by exon 1 RNA (Ly et al, 2022).

Exon 1 mRNA

In the 90s it was found that merely expressing a fragment of the human huntingtin in mice comprising exon 1 makes for a good mouse model of HD (Mangiarini et al, 1996).  The R6/2 line is widely used to this day, also because it models highly aggressive disease pathogenesis.  Little did the authors of that paper probably suspect that the highly toxic exon 1-only huntingtin is not only generated in a length-dependent manner in mice (Sathasivam et al, 2013)...it is also expressed in patients (Neueder et al, 2017).  In fact, there is (yet to be published) talk (Huntington's Study Group 2024, Wexler Symposium 2024) that exon 1 mRNA production is tied to somatic repeat expansion. 

If you now consider that exon 1 is extremely toxic on its own, gets generated in a repeat number-dependent manner, and that the characteristic N-terminal huntingtin inclusions may not represent cleavage fragments of full-length huntingtin and that exon 1 mRNA may seed huntingtin RNA clusters, a compelling model emerges whereby once CAG somatic repeat expansion has reached a certain length, highly toxic exon 1 mRNA/protein gets produced and triggers manifest HD.  This would also explain why the duration of manifest HD is independent of the original repeat length.

Mechanistically, it is possible that the expanded CAGs can impact the dynamics at which RNA polymerase II moves along the huntingtin gene which may allow for cryptic polyadenylation sites in huntingtin intron 1 to be recognized.  The formation of complex R-loop structures between nascent RNA with extended CAGs and equally CAG-extended DNA may play a role here.   

Therapeutic Strategies Aimed at Toxic RNA/Protein

There are a number of obvious modalities you could deploy to target the toxic agent in HD.  These include RNaseH antisense, RNAi and genome editing for HTT knockdown, steric blocking antisense oligonucleotides to inhibit intron 1 polyadenylation site usage.  Genome editing and ADAR RNA editing may also be used to interrupt the stretch of CAGs.  Disrupting even just 10% or so of 150 CAG repeats in a given RNA or protein could have a huge impact on its toxic property.

While many of such agents are in preclinical development, some have reached the clinic already.  Unfortunately, straightforward lowering of total, full-length  huntingtin by RNaseH antisense (ASO) by Ionis and Roche failed to show benefit in a pivotal clinical trial (McColgan NEJM, 2023).  In fact, the trial with tominersen was stopped early for toxicity as it may have done more harm than good as the therapy seemed to drive ventricular expansion along with striatal atrophy.  While some argue that this shows the perils of lowering wild-type along with mutant huntingtin, this not only is inconsistent with human genetics showing no such effect in people with heterozygous null mutations in the huntingtin gene.  If HD was caused by huntingtin insufficiency, there would be many more mutations aside from CAG repeat expansions that associated with HD.  More likely, toxicity stemming from the heavily phosphorothioated oligonucleotide backbone led to increased death of already stressed striatal cells.  

But sure, there should be no harm in targeting exclusively or predominantly mutant huntingtin as does Wave Life Sciences with their RNaseH antisense oligos targeting naturally occurring variations (SNPs) in the HTT gene correlating with the mutation.   

Both strategies, however, fail to address highly toxic exon 1-only mRNA/protein as they are targeted downstream of exon 1.  This is where UniQure’s DNA-directed RNAi and Alnylam’s synthetic oligonucleotide RNAi that target huntingtin in exon 1 itself come in.  

In fact, the clinically more advanced of the two (Alnylam's ALN-HTT02 has just entered phase I), UniQure's AMT-130 has generated data suggesting almost disease stabilization as measured on the widely used cUHDRS scale along with stabilization, if not lowering of neurofilament, a biomarker for CNS injury over time.  The data was so compelling when compared to external control in a propensity weighted manner that the FDA and UniQure are now working on expeditiously bringing the intrastriatally administered AAV therapy to the market possibly in 2026 already.   

At this stage, I should disclose that I have started to invest in UniQure.  Being first-to-market with a disease-modifying drug for HD would obviously be enormously lucrative for a well-financed company with an $800M market cap.  While their intrastriatal approach makes it difficult to tell how much huntingtin knockdown is achieved, also on a single cellular level, and how much is therapeutically impactful, the increasingly promising clinical evidence and them probably pursuing the most impactful target increases my confidence in their success.  Alignment with the FDA on the statistical analysis of their ongoing study which would form the basis for an accelerated approval along with upcoming 3-year follow-up data in the next 6 months will be key.

Please let me know in the comments below, if you come across evidence disproving the exon 1 hypothesis.  I will keep looking, but have yet to find it.

Longer-term, I am excited by the prospect of exon 1-targeting therapeutics with agents slowing somatic repeat expansion, an area seeing significant investment.  Since somatic expansion is a stochastic, asynchronous process and overlap generation of toxic RNA/protein, combining the two mechanisms will likely be most impactful. 

 

 

Genome Editing for Hereditary Angioedema Promises a Life Free from Disease Burden

Intellia Therapeutics is about to unveil phase II data on NTLA-2002 at the end of the month.  NTLA-2002 aims to be a one-time treatment for hereditary angioedema (HAE). Prior phase I data looked intriguing with all 10 subjects becoming essentially attack free in the months following (1-time) drug administration and safety findings limited to around the time of treatment.

Still, the study involved too few patients for too short time to clearly define the commercial product in terms of dose, safety, and efficacy (including durability).

 

HAE is a rare disease with an estimated incidence of 1:10000 to 1:50000 depending on geography. These angioedema attacks are characterized by tissue swelling due to the influx of liquid into tissues through leaky vasculature as a result of bradykinin generation.  With few exceptions, they occur in people due to their genetic lack of C1 esterase inhibitor function.   HAE must be differentiated from allergic edema and as such is not responsive to antihistamines or corticosteroids.

In addition to outwardly visible soft tissue swelling, e.g. of the lips and hands, it can also involve internal systems like the abdomen and lead to excruciating pain.  Suffocation from laryngeal edema is probably the most feared disease manifestation.  It is therefore not surprising that many patients become depressed, stop engaging in normal daily activities to avoid anything that may set off attacks, including stressful situations, physical activity and injury (including surgery). 

Rapid progress in the understanding of the biology behind HAE has resulted in a number of treatments for on-demand and prophylactic uses and significantly improved the outlook for patients.  Still, there is widespread dissatisfaction with the incomplete protection and requirement for regular drug administration of current prophylactic treatments while the utility, convenience, side effects and even embarrassment that come from taking on-demand drugs is being questioned.

Switching between drugs and treatment approach is common and with costs often on the order of $500,000 per annum, access can be a real issue causing additional anxiety.

Enter NTLA-2002

NTLA-2002 promises to address all these challenges. 

That’s a bold statement, but the phase I clinical data provide hints that this could become reality, commercially in 2027 if you are living in the US or even earlier if you decided to and qualified for the global pivotal clinical trial that has just commenced.

NTLA-2002 is a CRISPR-based genome editing treatment that permanently and selectively disrupts the KLB1 gene in liver hepatocytes.  KLB1 codes for plasma prekallikrein (PKK) which is a critical component in the kallikrein-kinin inflammatory system.  Importantly, there are people that naturally lack plasma PKK (Fletcher’s syndrome) or another key component, Factor XII, and these appear to be perfectly healthy.  They are typically only identified by coincidence following routine lab tube tests showing prolonged activated partial thromboplastin time without an actual negative impact on bleeding or otherwise in the natural context.  Deleting KLB1 in the liver should therefore be safe. 

NTLA-2002 comprises of a messenger and guide RNAs entrapped in liposomal nanoparticles (LNPs) similar to the covid mRNA vaccines. Following one-time intravenous administration, these traffic to the liver hepatocytes where the guide RNA directs the CRISPR Cas9 enzyme to disrupt the KLB1 gene. 

Such a formulation already exists commercially in the form of ONPATTRO.  ONPATTRO is an RNAi therapy that is administered every 3 weeks.  Similar to the phase 1 study of NTLA-2002 (Longhurst et al 2024), transient immune suppression (corticosteroids, antihistamines) is practiced around the i.v. administration.  The great thing about NTLA-2002 is that the side effects from this procedure are well recognized and managed and would only present once- if at all.

As mentioned above, all10 subjects in the dose-ranging phase I study of NTLA-2002 have essentially become attack free once the medicine had a chance to do its work, destroy hepatocyte KLB1 and normalize the kallikrein-kinin-system.  This indicates that after the optimal dose has been found, a good number of HAE patients may go on with life and forget about their disease!  So patients may not only enjoy the best efficacy of any HAE treatment, they would only have to take it once.

The phase II study results will more than triple the number of patients that have been (hopefully successfully) exposed to NTLA-2002 and thus provide a good basis for the dose chosen to undergo confirmatory phase 3 testing.  Based on recent statements, the company will select the 50mg dose which in the phase I study gave -88% plasma PKK reduction, a level well above the -45% reduction where HAE attack rates start to drop off precipitously (Riedel et al 2024).   

For those that continue to experience a rare attack, it may be possible to give another dose of NTLA-2002.  Data from another (monthly administered) antisense oligonucleotide therapeutic (donidalorsen) targeting the same PKK gene indicate that attack rate reduction and PKK inhibition are tightly correlated (Riedel et al 2024).  It is therefore conceivable that once plasma PKK drops below a critical concentration in the blood, swelling attacks are virtually eliminated.  A presentation at the upcoming ACAAI meeting by the sponsor of donidalorsen, Ionis Pharmaceuticals, should further shed light on this.    

Medical innovation is costly and NTLA-2002 which promises to be the second approved CRISPR genome editing therapeutic following Casgevy for sickle cell disease will be no different.  Given the rarity of HAE, it would not be surprising if it cost 2 to 3 million USD for treating a patient.  This compares to other medications in the space, including Cinryze or Takzhyro, which often carry annual price tags exceeding $500,000.  Sure, healthcare administrators are likely to throw up their hands and have sticker shock, but simple math shows a one-time treatment to be a real money saver for patients that have decades of disease-free lives in front of them. 

Disclosure: this is not medical advice. Always talk to your HAE specialist doctor first before making a treatment decision.

The Nucleic Acid Therapeutics Race to Revolutionize Alpha-1-Antitrypsin

Alpha-1-antitrypsin disease (AATD) is caused by mutations in the SERPINA1 gene coding for alpha-1-antitrypsin (AAT).  There are an estimated 100000 alpha-1 patients in the US alone, making it a rare, but not ultra-rare disease.

Correcting these mutations, replacing AAT through gene therapy, or inhibiting the particularly pathogenic Z-allele is subject to the efforts of a number of nucleic acid-based drug developers, most notably Arrowhead Pharmaceuticals (RNAi), Wave Life Sciences and Korro Bio (RNA Editing) as well as CRISPR genome editing companies Beam Therapeutics (Base Editing) and Intellia Therapeutics (CRISPR Cas9 and targeted gene insertion). 

The protein name is somewhat misleading as it’s main function is to antagonize neutrophil elastase activity in the lung. Insufficient AAT activity can lead to lung injury during pulmonary stress, especially respiratory infections or smoking.  Historically, lung disease has been addressed by smoking cessation and preventing lung infection (e.g. through vaccination). 

In those patients that do progress to symptomatic lung disease, the standard of care still remains weekly infusions with plasma-derived (!) alpha-1-antitrypsin.  Although the evidence of benefit is substandard, partly the result of having a ‘good-enough’ therapy approved decades ago on simple biochemical measures, this is now a $1B+ market and growing with the increased identification of this genetic form of chronic obstructive pulmonary disease (COPD).  Growing awareness of the liver aspect of the genetic condition also contributes to better patient identification.

Achieving 50-60% of normal AAT activity is expected to be therapeutic based on human genetics.   

As lung health and longevity has been improving in alpha-1, it is estimated that 15% of adult patients develop the serious condition of liver cirrhosis.  There is also an infant form AAT liver disease manifesting in ~10% with alpha-1 mutations that can result in cirrhosis and the need for liver transplantation.  Although this is less well understood than the adult form, it is likely to involve excessive accumulation of alpha-1-antitrypsin in the liver.  These patients typically carry the Z allele on both chromosomes (piZZ).  This allele is particularly prevalent among the European and US AATD populations (~90% of 235k worldwide).  As true AAT null mutations are rare, piZZ is by far the most prevalent genotype of patients presenting with either lung or liver disease. 

The Z alpha-1 antitrypsin variant protein misfolds, aggregates and gets stuck in the endoplasmic reticulum of the liver.  This results in liver inflammation and progressive injury (cirrhosis, hepatocellular carcinoma/HCC).  A small fraction (10-15%) of Z-AAT does get exported, but even if it makes it to the lung it is less functional and may even exacerbate lung inflammation due to its propensity to precipitate.

Removing Z-AAT is expected to halt and even reverse liver disease based on human genetics (1 Z allele not sufficient to cause disease) and preclinical animal models.

Here I will discuss some of the more promising, new innovative approaches utilizing a number of nucleic acid therapeutics modalities to address the lung and/or liver complications of AATD.  It also explains the following rank order of the approaches in terms of promise for AAT lung and liver disease.

1.    RNAi for A1AT-related liver disease (Arrowhead Pharmaceuticals and Takeda)

While innovation in the lung space of AATD has stalled, the disease attracted fresh attention in the pharmaceutical industry about a decade ago for its liver-related complications as (piZZ) alpha-1 patients get older and increasingly suffer from liver failure and HCC.  This time also saw the rise of new therapeutic modalities such as RNAi. 

The RNAi Therapeutic ARO-AAT by Arrowhead Pharmaceuticals has demonstrated almost complete elimination of the highly expressed gene in phase I and II clinical trials.  In preclinical models, this has been shown to reduce existing liver Z-AAT aggregates and inflammation over time.

In a small (n=25), 2:1 ARO-AAT/placebo randomized phase II study in Z-AAT patients with liver fibrosis, there was a robust -68% reduction in liver AAT globule burden.  At 52 weeks, this translated to 50% of subjects on ARO-AAT having a 1 or more point improvement in Metavir fibrosis (scale: 0/no fibrosis to 4/cirrhosis).  Due to the small study size and the 3/8 responses (38%) in the placebo group, this did not reach statistical significance.   Another small open-label study demonstrated a similar 50% fibrosis response.

Importantly, RNAi knockdown of Z-AAT did not worsen pulmonary health over 1 year in the controlled (no smoking etc) trial setting.  This may be partly due to Z-AAT being functionally impaired anyway and may do more harm than good in the lung. 

A longer 2 year 160 patient pivotal phase 3 study with F2-4 disease is underway to statistically confirm the benefit of ARO-AAT (aka TAK-999) on liver fibrosis (in F2+3 patients).  Results from that study can be expected in early 2026. 

2 years should be plenty of time for liver globules to turn over.  And as the liver is good in regenerating once you take the fibrogenic trigger away (see HCV, NASH etc)- as long as the liver is not in a late-stage cirrhotic stage already- this should translate into a fibrosis benefit.

For AAT liver disease, Arrowhead enjoys at least a 5 year headstart over the non-RNAi competition which has yet to enter the clinic.  Dicerna, now a Novo Nordisk company, has also been developing an RNAi trigger (belcesiran) for AAT which is in a phase II and has demonstrated -77% knockdown following a single dose.  

In terms of efficacy, Arrowhead’s RNAi approach with it’s almost complete elimination of AAT in the liver appears to be substantially superior over the competition with non-RNAi approaches struggling to get to -70-75% Z-AAT reductions.  This assumes that more knockdown is correlated with efficacy or at least the time for efficacy to manifest.  Based on human genetics, the equivalent of life-long treatment, where subjects with just one Z allele have not much added risk of developing liver disease, this may not be necessary.

 

2.    CRISPR Cas9 for Liver Disease 

The non-RNAi approach in development for AAT liver disease that is likely the next most efficacious one is CRISPR Cas9 DNA cleavage as developed by Intellia Therapeutics.  Intellia has already demonstrated 90%-type gene knockout efficiencies in hepatocytes for TTR amyloidosis and hereditary angioedema in the clinic, so expectations are for similar target engagement in the AAT program. Cas9-targeted DNA cleavage is certainly a powerful tool to downregulate gene expression.

The question, however, is whether you would want to risk disabling a gene permanently when you have non-permanent alternatives like RNAi that are at least equally efficacious.   Similar to transthyretin in TTR amyloidosis, alpha-1-antitrypsin is a highly expressed gene in the liver that has important functions in human biology and health.  TTR for example is a carrier of thyroxin and retinol binding protein (à vitamin A) while AAT is involved in the homeostasis of protease activity for example during infection in the lung and probably has similar homeostatic functions in other tissues. 

Side effects from the long-term ablation of AAT may only manifest after years and may then require life-long supplementation (vitamin A, thyroxin), or after certain stress situations (non-genetic COPD etc).  To my knowledge, unlike for say PCSK9, human genetics does not support that lifelong absence of AAT is ideal.  Similarly, regulatory agencies will be worried about cancer resulting from genome re-arrangements rearing their ugly heads 10 years or so down the line following on- or off-target DNA cleavage in and outside liver cells (including germline).

In my opinion, unless genome editing can demonstrate clear efficacy advantages over otherwise quite safe and non-onerous, non-permanent alternatives, it is only with the accumulation of long-term safety data that we will be able to tell whether CRISPR Cas9 can be used more broadly.

 

3.    RNA Editing for Lung and Liver Disease (Wave Life Sciences, Korro Bio)

When it comes to addressing AATD lung disease, my favorite approach is via RNA Editing with an exciting candidate by Wave Life Sciences (WVE-001) about to enter the clinic.  Korro Bio aims to enter a competing RNA Editing candidate into the clinic in late 2024. 

However, Korro Bio candidate would likely have to be relatively frequently (weekly) administered via intravenous infusion and has no obvious efficacy advantage over WVE-001 and LNP-related toxicities to be expected (infusion reactions, triggering of innate immunity etc).  As a second mover with an inferior therapeutic profile, I am struggling to understand Korro Bio’s business rationale here (also discussed here).  

As discussed on this blog, RNA Editing in AATD aims to convert the pathogenic piZ allele into the healthy M allele.  Based on human genetics where MZ carriers are protected from both liver and lung disease, a 50% AàI editing rate may suffice for both indications.  Wave Life Sciences has been able to meet that bar in preclinical studies.  Given the newness of RNA Editing, results from the first clinical study for this modality utilizing Wave’s subcutaneously administered oligonucleotide chemistry will still have to show just how efficacious and sustained (in terms of dosing frequency) their approach will prove to be in humans. 

A significant uncertainty is how long it will take for even robust >60% editing to translate into actual clinical benefit. Unlike for the liver where the natural turnover of pathogenic globules will likely be rate-limiting for liver health improvement to manifest, generating healthy, wild-type alpha-1-antitrypsin should be immediately beneficial to the lung.   Identifying a patient population with still relatively healthy lungs but rapid functional decline should therefore be most promising for a clinical trial.

For this, Wave Life Science has recruited pulmonary disease powerhouse GSK through a out-licensing as setting a new standard of care will not be as easy as simply demonstrating AAT blood biomarker as the incumbents had gotten away with.  The fact that plasma-derived products from the stone age of biotechnology still dominate what today has grown into a blockbuster market shows how difficult it has been to find new therapeutic strategies worth investing in.  As the plasma-derived products have been unable to demonstrate a clear therapeutic benefit in controlled studies, this would likely involve running a relatively large and long-term clinical study being very mindful of stratifying the diverse patient population. 

The reason why RNA Editing could represent a meaningful improvement to the standard of care in AAT lung disease is that it promises to offer more sustained, tonic amounts of AAT instead of the spiked pharmacokinetics from weekly infused AAT.  Moreover, because RNA Editing converts mutant to wild-type AAT that is transcribed from the native gene under physiological controls (e.g. promoter activity), adverse effects from supraphysiological levels of AAT should not be observed.  

Wave Life Sciences claims that RNA Editing can not only address lung AATD, but also its liver manifestations.  To find out, GSK would have to run a separate study and hope that clinical success is not linear with Z-AAT reduction. Because if this were the case, RNAi would likely win out over RNA Editing not only in terms of knockdown efficacy, but also dosing frequency.  There is some preclinical evidence, however, that wildtype AAT may aid cellular export of Z-AAT, so a 50% mRNA conversion may reduce intracellular Z-AAT more than 50%.  Possibly worth a competitive gamble.

 

4.    Base Editing for Lung and Liver Disease (Beam Therapeutics)

Similar to Wave Life Sciences, Beam Therapeutics aims to correct the piZZ mutation, but this time applying CRISPR genome base editing for permanent correction.  Unlike traditional CRISPR Cas9, Beam’s CRISPR proteins are unable to make double-strand. Instead, a base-editing enzyme is tethered to the Cas protein which is guided to the target location by the guide RNA.  In this case, the base editor is an Adenine Base Editor converting proximal adenines into guanine.

Unlike RNA Editing where you can precisely determine the editing site through secondary fit and oligonucleotide modifications with essentially no off-target editing, tethered base editors will act on what is close by.  Most problematic for the Beam program appears to be a bystander adenine that is 2 bases away from the targeted adenine such that for each desired single edit you seem to get equal amounts of double-edited AAT mRNAs resulting in alpha-1 antitrypsin  that is significantly less functional.

This also raises that question on how many more unidentified off-target base edits there are.  The genome in the nucleus is not linear, but dynamic with tertiary interactions between chromosomes, within a chromosome and transcriptional hubs bringing multiple genes into close vicinity.  So imagine a crowded nucleus with thousands of CRISPR-tethered base editors floating around looking for adenine substrates.  It will sure be interesting how regulatory agencies around the world will be looking at this as well as the risk of germline editing following LNP delivery.

Base editing efficiencies are slightly less than RNA Editing in preclinical models with the added caveat around the double edits.

Beam intends to file for clinical trial applications in Q1 2024.   

  

5.    Integrational Gene Therapy for Lung Disease (Intellia Therapeutics)

Finally, in addition to gene demolition using Cas9 for liver disease, Intellia is advancing a separate CRISPR-mediated gene insertional approach for AAT lung disease.  Here, an LNP delivers Cas9 mRNA-gRNA to open up DNA downstream of the albumin promoter, highly active in hepatocytes.  An AAV carrying AAT cDNA gets administered alongside so that a certain fraction gets incorporated as the DNA damage repair machinery attempts to mend the lesion.

Preclinical non-human primate data support that marked expression can thus be achieved as the albumin promoter now drives transcription of AAT-cDNA.  It is noteworthy that albumin drop-in has gone a bit out of fashion in the genome editing field after Sangamo Biosciences had similarly claimed high preclinical expression for hemophilia and lysosomal storage disease almost a decade ago.  Strangely, as so often has been the case with Sangamo, this could not be reproduced at all in the clinic (barely detectable levels if any at all).  Intellia is using a curious inverted repeat design as their drop-in cassette and believes this to be a game-changer in the approach.  They may have done this to double their chances that the insertion happens in the correct orientation, but as a former molecular biologist trained in RNA polymerase II transcription and RNAi this looks like inviting trouble to me.

In any case, a surprising pivot by Intellia for AAT lung disease and we will have to see how this approach can thread the needle of achieving substantial, but not exaggerated amounts of AAT….over the long-term…and with little interpatient variability.

Intellia intends to submit a clinical trial application for NTLA-3001 by year-end.

Lightning Fast Wave Life Sciences Demonstrates High ADAR Editing Rates Across Targets

There was a time, not that long ago, when 1-3% ADAR editing rates in tissue culture cells were typically reported in the field.  The hope then was that with further chemical optimization, editing rates could be increased high enough to have a clinically relevant impact in the setting of a gain-of-function approach.  Mathematically speaking, go from nothing to something is an immeasurable relative increase.

In the realm of biology, this is pertinent to diseases like Duchenne Muscular Dystrophy or Spinal Muscular Atrophy where relatively small, 10-20% target engagement by RNA Therapeutics have been demonstrated (splice modulator SPINRAZA) or are expected (exon skippers) to have big disease modifying activity when given to patients essentially genetically null (=not expressing) dystrophin and SMN1, respectively.

In theory, the upper limit of ADAR Editing should be extremely high since near-complete editing of ion channel and neurotransmitter receptor pre-mRNAs are seen in neurobiology.

The perception that ADAR Editing mediated by oligonucleotides could be generally low in clinical applications started to shift when Monian and colleagues at Wave Life Sciences reported in a groundbreaking Nature Biotech paper a year ago robust 50%+ editing rates of the alpha-1 antitrypsin Z allele.  It suggested that this can be achieved by painstaking chemical optimization at a ‘lucky' target site.  This is not much different from how small molecules get chemically matured following an initial low-affinity, low-specificity hit.

For the type of blockbuster market opportunity like alpha-1-antitrypsin this effort is well worth it.  Still, finding potent editing-enabling oligos in an efficient manner would open many doors such as testing scientific hypotheses faster, especially as the ADAR Editing pioneers sift through their list of candidate targets and indications.

Enter Wave Life Sciences and their PRISM platform.  I have never quite understood what exactly is behind this platform for oligonucleotide discovery (RNAi, exon skipping and ADAR Editing).  It does appear, however, to test many combinations of chemical modifications at the base, sugar, and internucleotide linker while considering their chemical neighborhoods and stereochemistry, ultimately coming up with design principles for potent oligonucleotide therapeutics candidates.

Certainly, given that modern lab automation allows for increased throughputs (see biotechtv tour of Aera Therapeutics), such a Big Data, Artificial Intelligence approach to oligonucleotide drug development makes a lot of sense and can give biotech companies crucial competitive advantages in terms of time and better molecules.  

Two chemistry insights regarding ADAR technology that have emerged from PRISM stand out so far.  Firstly, the zwitterionic phosphorylguanidine (PN) backbone linker, preferably in a stereopure format.  The PN chemistry is shown to allow for improved unassisted cellular uptake into various cell types in mice (immune cells, various liver cell types, renal cells etc) while stereopurity brings advantages in terms of ADAR recognition of the duplex substrate.  This sounds similar to the morpholino chemistry that has proven to be quite safe following systemic administration (e.g. eteplirsen by Sarepta), but is very rapidly eliminated from the circulation into urine.  It will therefore be important to show in future studies that the dose demands for oligos with predominantly PN backbone are not as high.

With regard to the orphan base, the base opposite the adenosine to be edited, Wave is honing in on deoxy-N3 uridine as its preferred chemistry.

In addition to alpha-1-antitrypsin (now partnered with GSK), robust 50-90% editing is demonstrated for a number of clinically relevant genes such as UGP2 (à epileptic encephalopathy 83), and Nrf2-Keap signaling (stress regulation in chronic disease).  For the latter two targets, not just one, but several highly potent editing oligos could be identified.  This reflects increased emphasis by Wave in applying RNA Editing to targets that are not for correcting specific mutations, but where the goal is to increase expression of a protein where it could be helpful, but without necessarily changing its inherent function or sequence. 

This allows Wave to scan for potent editing oligos often along the entire target (pre-)mRNA instead of being limited to just one site!  Also, a number of genetic diseases are caused by a various mutations dispersed throughout a gene so that a mutation-correction approach may require the development of multiple editing oligos and some sites will not be amenable to AàI approaches at all.  In the end, this increases the market potential of a given oligonucleotide.

In summary, being able to consistently achieve 50%+ editing rates will be sufficient for most therapeutic editing approaches.  Going from say 2% of something to 50% is a 25-fold increase, but maxing out at 100% for another 2x may not give that much of additive benefit.  Of course, biological pathways can sometimes be complex and the responses may not be as linear.  At 80%+ editing, diseases caused by dominant-negative mutations would also come within the realm of therapeutic possibilities of ADAR editing and this includes liver-related diseases of piZZ alpha-1-antitrypsin, but here ADAR Editing may face inherently more potent knockdown mechanisms such as RNAi.

Disclosure: I am long WVE, as I am impressed by the speed with which they have chemically matured editing oligos and their dystrophin exon skipper is showing intriguing early clinical results (RNA, not protein level).  However, I have not taken a full position yet as I want to see the company first demonstrate robust target engagement in the clinic (including for the dystrophin exon skipper).  So far, all the clinical results have greatly disappointed with claims of 10-20%-type target knockdowns