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

Korro Bio To Become Third Publicly Listed ADAR Editing Company

Today, pure-play ADAR Editing Korro Bio announced that it will reverse merge into biotech shell Frequency Therapeutics (current ticker: FREQ, to be changed to KRRO).  It will thus become the 3^rd publicly traded RNA Editing company following ProQR (pure-play) and Wave Life Sciences, the latter entertaining a broader mix of oligonucleotide therapeutics modalities (ADAR editing, exon skipping, RNAi).

Following two private founding rounds of ~$210M in 2020 and 2022, the transition into the public markets which is being accompanied by another $117M cash injection from mainly existing venture backers led by Surveyor Capital and Cormorant Asset Management, is to prepare the company making the transition to the clinic.  Its lead candidate is to address both the lung and liver manifestations of alpha-1-antitrypsin disease (AATD) caused by the prevalent piZZ genotype.

Today’s development explains their surprising announcement earlier this year to adopt liposomal delivery instead of GalNAc-targeted chemically modified oligonucleotides as is practiced by industry leaders ProQR and Wave Life Sciences.  Without the prospect of a clinical candidate, such an ‘IPO’ would not have been possible. 

As I had noted in an earlier blog entry though, such a development candidate is likely to fail both from a clinical and commercial point of view.  Firstly, for AATD patients at high risk of liver disease or actually manifesting liver disease, a chronically administered LNP seems like a bad idea. Indeed, Korro Bio today revealed significant liver enzyme elevations in animal models at doses (2mg/kg) that are likely required for robust SERPINA1 editing and are substantially higher than what is used for the clinically approved MC3-based LNP formulation Patisiran in ATTR amyloidosis by RNAi Therapeutics company Alnylam.

From a competitive point of view, the LNP approach suffers from the need of frequent, possibly weekly intravenous infusions whereas less frequent (I expect monthly) subcutaneous administration schedules should be feasible with Wave’s first clinical GalNAc editing oligo and possibly less frequently as oligo chemistry advances (similar to RNAi).  As Wave is likely to be a year ahead of Korro in the clinic, this alone makes it a head-scratcher approach for a fast-follower.

As we have learned from the RNAi Therapeutics field, further stabilizing Korro’s oligonucleotide is unlikely to extend dosing frequency as LNPs release most of their cargo into the cytoplasm almost instantaneously whereas the long duration of action by oligo-conjugates is explained by their gradual release from endosomes.

There is one scenario, however, where I can see Korro Bio’s candidate to have staying power, namely in being the only approach among the ADAR Editing and CRISPR genome editing and gene therapy candidates that can successfully treat both the lung and liver manifestations of AATD by achieving >90%-type editing levels.  As we have learned from Arrowhead Pharmaceuticals' liver AATD program (partnered with Takeda), even with near complete removal of toxic alpha-1-antitrypsin expression in the liver, prolonged treatment will likely be necessary to see a robust clinical response (phase 3 involves >3 years of dosing).

It is for this reason that I view the ADAR and genome editing approaches mainly aimed at those suffering from lung disease (which RNAi cannot address), including those with mixed phenotypes.  I will discuss clinical development landscape further in my next blog entry.

What I most like about Korro Bio, a prolific IP filer, is their mix of ADAR Editing programs encompassing genetic correction for AATD and Parkinson’s (LRRK2), anti-protein aggregation (TDP43 in ALS), modulating ion channel (NAV1.7 in pain), disrupting protein-protein interaction in alcoholic hepatitis and activating kinases.  But to extract the full value from applying RNA Editing to these attractive disease areas, Korro Bio needs to catch up on oligonucleotide chemistry and designs.

Roche Impresses with Effective RNA Editing of Polyglutamine Repeat mRNA

Roche has shown interest in RNA Editing through its 2021 partnership with Shape Therapeutics.  The goal of this partnership was to use Shape’s AAV-delivered, DNA-directed RNA editing nucleic acids for neuroscience and rare disease applications.

Readers of this blog will know that I have not been a great fan of DNA-directed approaches to ADAR editing, not least because the expressed editing RNAs are unmodified.  This means that they do not benefit from chemistry to optimize efficacy.  In terms of specificity, the simple, but very effective strategy of modifying the base opposite non-target adenosines (e.g. 2’-O-methyl) to abolish off-target editing is not available to DNA-directed RNA editing.  

To compensate the efficacy disadvantage, the concomitant gene therapy-directed overexpression of ADAR enzymes has been attempted.  Unfortunately, this is a no-go since it causes extensive genome-wide off-targeting.  

It therefore comes as no surprise that Roche has also been evaluating synthetic editing oligonucleotides as revealed earlier this month in patent publication WO2023/052317A1.  This patent application addresses CAG/polyglutamine repeat expansion diseases such as Huntington’s disease, but also other neurodegenerative polyGln diseases including a number of the spinal cerebellar ataxias.  Since the number of polyGln repeats critically determines whether a person will manifest the disease and is correlated with protein aggregation, disrupting stretches of CAG-encoded uncharged glutamines with even a few positively charged, CGG-encoded arginines may stop the pathogenic process and is thus a highly attractive therapeutic hypothesis.

Beyond CAG triplett expansion diseases, similar logic may apply to diseases caused by repeat expansions in non-coding regions- as long as the repeat contains an ‘A’ such as in Friedreich’s ataxia (frataxin GAA repeat in intron 1).  Regardless of the specific disease-causing mechanism, disrupting the repeat is likely to be beneficial.  

While attractive in theory, I had been wondering how easy it actually would be to target these repeats by ADAR editing as the target sequence is quite unusual in its repetitiveness which may result in impenetrable higher-order structures.  The use of repetitive oligonucleotides as therapeutic agents is also unusual because of potential structural and manufacturing issues.  Finally, once one of the target adenosines has been converted to an inosine, the target mRNA sequence is altered (=mismatch) and consequently may become a weaker target site.

On the other hand, long repeats may turn out to be excellent targets in that they provide for a high local concentration of target sequence.

Actual data

Unfortunately, conducting casual molecular biology experiments in the basement of private homes is frowned upon in Germany and fraught with legal risks (this has to change), so it’s nice that Roche has actually conducted initial tissue culture experiments to find out about the practicality of the approach. 

Employing ~50-60nt long CUG repeats (the complement of CAG), their editing oligonucleotides were above the typical length of ~30nt as now generally practiced by the leading RNA Editing companies ProQR and Wave Life Sciences.  These were transfected into HeLa cells expressing ATXN3 mRNA with 21-22 repeat CAGs all in the apparent absence of ADAR overexpression.  

The oligonucleotides were modified with 2’-o-methyl only in the 5 nucleotides on the 5’ and 3’ ends each; phosphorothioation of the backbone was also practiced at the wings of the oligos, but extended further into the center than the 2'-o-methyls.  The central part consisted of pure RNA. 

An orphan C was placed towards the 3’ end of the targeting oligo.  This creates a mismatch to the target A as is commonly practiced in the field.  Interestingly, an inosine follows 3’ of the orphan C and this is also practiced by some other companies as e.g. evidenced in last year’s high-profile paper on long-lived and potent ADAR editing in non-human primates by Wave Life Sciences in Nature Biotech.

Remarkably, robust 20-50% AàI conversions were seen for many As in the ATXN3 CAG repeat with more pronounced editing towards the 5’ end of the repeat region consistent with the 3’ placement of the orphan C in the targeting oligonucleotide.  Moreover, less than 2% of the ATXN3 mRNAs was unmodified for each editing oligo.  If you consider that a huntingtin allele with say 33 CAG repeats does not result in Huntington’s disease, but one with 37 repeats typically does, you can imagine the impact that just a single or two successful editing events should have on pathogenicity of the resulting protein.

This experiment thus is an important de-risking step for RNA Editing in repeat expansion diseases and should whet the appetite of Roche which is already heavily invested in oligonucleotide therapeutics for Huntington’s through its collaboration with Ionis Pharmaceuticals (RNaseH mechanism), including research on improving the convenience and efficacy of intrathecal oligo administration.

As an investor in ProQR I was, of course, pleased to see that when discussing the prior art of ADAR editing in general, all 5 patent applications cited by Roche referred to ones controlled by ProQR. 

Looking forward to the next chapter in this story.

RNAi Also Conquers the Central Nervous System

In the span of a day, RNAi Therapeutics have gone from a mechanism widely viewed as being constrained to the liver only, to a major therapeutic modality for many targets and indications in a variety of tissues.  Due to the demonstrations in the liver, lung (yesterday), and today the central nervous system to potently and specifically knock down genes with infrequent dosing, RNAi will play a prominent role in today’s precision medicine-oriented drug development.

Employing C16 lipid-conjugated, chemically stabilized RNAi triggers, Alnylam and their partner in CNS drug development Regeneron achieved 84-90% maximal target gene knockdown with knockdown persisting at >70% for at least 3 months after a single dose.

Since chemical stability has been key to the successes in the lung, CNS, and also liver, it seems very likely that similar breakthroughs will be achievable for muscle, kidney, adipose tissues, and (in the words of Alnylam's President) 'even tumors' that Alnylam and Arrowhead are working on.

The initial target in the phase I study of ALN-APP was amyloid beta precursor protein (APP). Unlike the armada of antibodies that have targeted every known aggregation form of abeta for the treatment of Alzheimer’s, ALN-APP reduces them all and before they are even made thereby offering a unique angle to this important target.  An even more exciting near-term application of ALN-APP in my opinion is for cerebral amyloid angiopathy (CAA) where abeta accumulation near blood vessels can lead to intracerebral hemorrhage.  Studies with antibodies in Alzheimer’s have actually led to fatal damage to those very intracerebral blood vessels by causing local inflammation, and thereby make them a bad choice for CAA.

Beyond abeta and tau for Alzheimer’s, the CNS in particular abounds with otherwise difficult-to-drug important targets for diseases like Parkinson’s, Huntington’s, ALS, spinocerebellar ataxias for which RNAi is ideally suited.

The prolonged and robust knockdown observed is significantly better than what has been observed for previous RNaseH antisense candidates such as against SOD1 and tau (~50% target gene lowerings).  Safety also appears to be superior to the broadly phosphorothioated antisense molecules with no changes in neuronal markers of damage and inflammation seen with ALN-APP compared to placebo.

The US FDA though slapped a clinical hold on the multi-dose part of the trial based on findings in standard preclinical animal tox studies at doses well above what will be needed in the clinic.  The single dose exploration study, however, has been allowed to continue, and Canada has already allowed the multi-dose part to go ahead.  It therefore seems highly unlikely that the findings could derail ALN-APP or even this technology approach at this point.

With the recent news, the pharmaceutical landscape has changed and Big Pharmaceutical companies will have to think hard whether not having a stake in RNAi as a platform is viable.  The achievement is also one of delivery and stabilization chemistry which can be more broadly applied to other oligonucleotide therapeutics modalities in the CNS.

Oligonucleotides Break Through to the Lung

It is days like today that I live for.  Days when new platform technology data is revealed that will change the practice of medicine and benefit patients for a number of diseases of high unmet need. In this case asthma, IPF, COPD etc. 

Almost a decade after GalNAc started to revolutionize oligonucleotide therapeutics delivery to the liver (hepatocytes) and turned oligonucleotides into the important therapeutic modality it has become today, Arrowhead Pharmaceuticals just reported the equivalent for the lung (lung epithelial cells to be precise).

Employing inhaled delivery of αvβ6 integrin-targeted stabilized RNAi triggers in healthy volunteers, the company found robust, -80% mean maximum target gene (RAGE) knockdown after 2 doses spaced a month apart. 

Since the knockdown reading was based on RAGE protein in serum (sRAGE), the true knockdown in the desired lung epithelium is likely higher.  This is also supported by the observation that more direct bronchoalveolar lavage measurements revealed -75% knockdown after just a single 92mg dose when the corresponding reading in the serum indicated -56%.  Further dose escalation to 184mg is ongoing and there are first indications that the long-lived pharmacodynamic response observed in animals will hold up in the clinic.

RAGE is a key player in pro-inflammatory signaling in the lung and thought to play a central role in related pulmonary disorders such as asthma.

In addition to clearing the efficacy hurdle, safety seemed excellent, or in the words of the company ‘no patterns of adverse changes in any clinical safety parameters’.

As some may remember, an earlier RNAi candidate targeting the lung (ENaC for Cystic Fibrosis) was shelved by Arrowhead due to preclinical findings in chronic tox studies in the rat.  The reason is thought to be that the sheer amount of material delivered to rat lungs overwhelmed and inflamed the macrophage-based particle clearance system.

What is different this time is that ARO-RAGE utilizes improved stabilization chemistries and therefore only a fraction of the overall tissue exposure is required to achieve the same knockdown. 

This is reminiscent of the early days in GalNAc conjugate-based delivery to the liver when a first-generation GalNAc-TTR RNAi trigger had to be discontinued by Alnylam due to adverse safety in the clinic.  Improved GalNAc RNAi drugs of increased metabolic stability (and reduced 3'-fluoro content) are now well established medicines.

Beyond RNAi Therapeutics, today’s results have important implications for oligonucleotide therapeutics applications in the lung in general, including RNA Editing. 

Most importantly, they establish αvβ6 integrin as a valid target receptor for oligo conjugates.  Moreover, some of the chemistries should be directly translatable for stabilization purposes and together with ARO-ENAC Arrowhead should now have good insights into the chemistry-safety relationship. 

ProQR and Partner Eli Lilly Demonstrate Oligonucleotide-induced RNA Editing in the CNS: A Major De-risking Event for the Industry

With every new oligonucleotide therapeutics modality that feeds into an endogenous cellular mechanism comes uncertainty as to whether the mechanism is sufficiently robust to be of therapeutic utility.  

This is especially true for RNA Editing as in its early days targeted AàI editing was only shown with the concomitant DNA-directed overexpression of ADAR along with an targeting RNA or the introduction of recombinant ADAR-antisense conjugates of little direct therapeutic use.  Similarly, simply introducing into a cell a chemically synthesized antisense oligonucleotide hybridizing to the area surrounding the target adenosine in an mRNA will only give you minute editing efficiencies in cell culture without further structural and chemical optimization.

The liver and CNS, due to their gene target richness and the demonstrated clinical feasibility of delivering oligonucleotides to these organs, are of particular importance to the RNA Editing industry.  The demonstration byscientists from Wave Life Sciences of oligonucleotide-directed RNA Editing in non-human primates was therefore an enormous de-risking event in that it showed that RNA Editing is sufficiently robust in living primate livers.

Of similar importance was the revelation by ProQR and their partners from Eli Lilly last week that this also holds true for the primate nervous system following the intrathecal administration of an editing oligonucleotide.  10-30% editing were seen in the brain depending on the anatomical location investigated.  In both the mice (intracerebroventricular delivery) and cynomolgous monkeys, editing was highest in the cortex.  Even higher editing levels, up to 50%, were observed in the spinal cord of non-human primates.

The spinal cord (motor neurons) also happens to be the location of the most successful oligonucleotide therapeutic currently on the market: SPINRAZA (nusinersen) for spinal muscular atrophy.  Since RNA Editing is quite new and many do not fully appreciate what 10-50% editing efficiencies mean, SPINRAZA can serve as a good example for how impactful such target engagements can be particular for gain-of-function approaches.

SPINRAZA is a splice modulator and works through gain-of-function by obscuring an intronic splice silencer element in the SMN2 pre-mRNA.  Typically, only 10-20% of SMN2 mRNA is ‘correctly’ spliced to yield a functional full-length protein.  With 12mg of SPINRAZA in infants (same dose used for the RNA editing studies in cynomolgous monkeys), this increases 2-3x.  This means that an approximately 10-40% successful target engagement can save babies from certain death and, if given early enough, may allow children with the type I SMA mutations to grow up almost normally.

In the case of the (undisclosed) target gene that Eli Lilly is looking at, these types of target engagements with RNA editing resulted in 5-25x increases in protein function.  Because of the above and because gain-of-function is a particular competitive strength of RNA editing, this application should be prioritized in target selection of industry pipelines.

Wave Life Sciences to Focus RNA Editing on Gene Upregulation

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

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

 

Mutations often scattered across genes

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

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

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

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

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

 

mRNA technology

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

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

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

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

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

 

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

 

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

Silicon Valley Bank Failure is Warning Against More RNA Editing Start-Ups

As the collapse of Silicon Valley Bank (SVB) is making the rounds, let's take a step back and ponder what it means for the RNA Editing space.

SVB has been a prominent banking partner for start-ups in tech and biotech, willing to do business where traditional banks did not feel comfortable with the unique risk profiles and needs of such businesses.  Short-term, the failure means that some jobs are at risk as small companies for which SVB was the only banking partner may not be able to make this month’s payroll in time, and 10-20% of uninsured deposits above the $250k FDIC limit may be lost forever. 

SVB’s failure is a crack in the system resulting from rampant inflation and the dramatic rise of interest rates in response.  It’s quite possible that other cracks, also among the more traditional banking sector, emerge soon due to an imbalance of short-term cash demands of bank customers and banking treasuries overweighted in bonds with long maturities that can now only be sold at a loss.   

Too many biotechs!

That SVB, along with a smaller crypto-catering bank (Silvergate), is among the first victims is largely due to biotechnology’s voracious appetite for capital, but an inability to raise more of it in the current environment.  Having too many companies developing the same platforms and targeting the same diseases in parallel, especially in the gene therapy, genome editing, and immune oncology spaces, has only exacerbated the interest rate problem. 

SVB is thus symbolic for the (bio)tech excesses in recent years, culminating with the Covid19 crisis where every little idea was transformed into a start-up with $100M of funding from the get-go housed in glitzy labs and offices in the most expensive hubs, run by entitled executives more focused on ESG issues than bringing their technologies to fruition.   Contrast this to 15 years ago when little biotechs like LNP pioneer Tekmira (Protiva then) did better science, developed platforms more rapidly while fighting off larger rivals, yet spending just $3-4M a quarter.

Take CRISPR genome editing.  It seems like every new Cas enzyme, every new enzyme tethered to Cas9 doing something, anything with DNA or the epigenetics around it needs a new cash-burning start-up.  Instead of for example licensing prime editing to Beam Therapeutics, founded on more advanced base editing technology from the same academic laboratory, the movers and shakers in the VC scene sought to exploit David Liu’s star scientist status to found yet another immature biotech company many years away from a potential product and with even more inexperienced management.  To add insult to injury, this has been taken to the extreme of selling Liu’s* genius to suggest that he has solved nucleic acid delivery where thousands of humble scientists have worked over decades on similar concepts.  Enter Aera Therapeutics with- hold your breath- $193M in start-up funding.  

* correction: the scientific founder behind Aera Therapeutics is another CRISPR researcher from MIT, Feng Zhang, not David Liu.  The message, however, is the same.

Every paper a new biotech it seems. Sorry, and with all due respect to those involved: this type of behavior is unacceptable and comes across as greed and hubris.

 

Not too late for RNA Editing

One of the reasons I like ADAR RNA Editing also as an area of investment is that it is a differentiated technology platform allowing for unique therapeutic approaches and, equally important, where there has not been this hype leading to an overproliferation of companies and inefficient use of capital.

It is also not surprising that the two most advanced companies in the space, ProQR and Wave Life Sciences, are not pure-play startups.  By contrast, the refinement of editing oligonucleotides here happens within companies with a decade of experience in oligonucleotide drug development, and at least in the case of Leiden-based ProQR, at a fraction of the cost of its start-up rivals KorroBio and ADARx based in the Boston and San Diego hubs, respectively.

I am highlighting KorroBio and ADARx because they are the two most prominent start-ups around synthetic oligo-based ADAR RNA Editing that have significantly benefited from the Covid19 boom in biotech financing, but where I fail to understand what they are bringing to the table and thus the point of their existence.  For example, I wrote about my surprise at Korro Bio going with LNP delivery for liver-targeted AATD.  This most likely comes down to their inexperience in oligonucleotide chemistry.

So here is my plea to the ADAR RNA Editing industry: we do not need to repeat the mistakes made in other areas of biotechnology.  Competition, such as the rivalry between Alnylam and Sirna Therapeutics in RNAi, is a good thing as it focuses the mind, but 2 or 3 strong ADAR Editing-based pure-plays are really enough, plus some platform adoptions by larger oligonucleotide therapeutics companies like Ionis, Arrowhead, or Alnylam and disease-specific licensing activities of Big Pharma; and if there are important advances in academia relevant for the space, tech licensing the old style is appropriate.

Korro Bio Opts for LNP in Liver-Directed Lead Program

Korro Bio yesterday announced that it would collaborate with Genevant to develop liposomally formulated oligonucleotides for the ADAR editing of alpha-1-antitrypsin in the liver.

This is a big surprise for the field since based on the successes in the oligonucleotide therapeutics industry in general and data from competitors Wave Life Sciences and ProQR in particular, it would have seemed obvious to employ GalNAc-conjugated editing oligonucleotides for alpha-1 antitrypsin-related liver disease.

Korro Bio is a privately held pure-play ADAR editing company that has raised more than $200M since 2020 and is developing oligonucleotides, as opposed to DNA-directed small editing RNAs, for mediating AàI conversion.  Given this substantial funding and what appears to be the ready availability of GalNAc, it is a big mystery to me why Korro has chosen intravenously administered LNPs and in the process is giving up substantial ownership in this program through the collaboration.

Just last month, Korro Bio and scientific founder Joshua Rosenthal published a selection strategy for efficient editing oligonucleotides.  The paper (Quiroz et al, 2023) finished off with experiments illustrating the need for extensive oligonucleotide modification, reminiscent of what ProQR and Wave Life Sciences have practiced, for effective ADAR editing.

 

Learnings from RNAi

One explanation for why Korro may have favored an intravenously over a subcutanously administered technology may be potency.  In yesterday’s press release and a recent Nature Biotechnology RNA editing industry article, the company is boasting that it wants to return serum alpha-1-antitrypsin levels to within the normal range.

A lofty goal and perhaps most readily achieved without having to balance the demands of chemical modification for stabilization purposes and inherent ADAR activation potency. 

In the earlier days of RNAi, Alnylam’s LNP-formulated Patisiran actually won out over an internal GalNAc competitor that didn’t quite have the potency and was also associated with toxicity.  Patisiran has also won the commercial race against a subcutaneously administered antisense oligonucleotide by Ionis due to superior clinical data.

Clearly, depending on the stage of chemical modification know-how with regard to a specific oligonucleotide modality, LNPs may be preferable even for the targeting of genes in hepatocytes.

Maybe Korro Bio does believe it still has a potency edge over the competition, and combining its oligonucleotides with LNPs may also get them faster into the clinic.

 

The Vivek Factor  

There is no reason to believe that in AATD an LDL receptor-targeted delivery strategy may be beneficial over an ASGPR-targeted one because of changes in receptor expression levels.

I would, however, not rule out that Korro Bio succumbed to the magic of the bewilderingly fast-talking executives from the Roivant universe (Genevant is a Roivant subsidiary).  I still cannot get over the fact that Tekmira handed over half the company plus LNPs to (now US Presidential candidate) Vivek Ramaswamy for some toxic small molecules scribbled on the back of an envelope.

When you hear Vivek on the campaign trail these days and his sharp fast talk full with twisted arguments that make your head spin, then I understand why people that for whatever reason like this energetic person may throw out reason and just want to trust this guy.  But beware: while making billions for himself and his family, he has lost many more of shareholders’ money for projects like the Alzheimer’s drug that he dug out from a dumpster and IPO’d at a valuation of over a billion USD.  I digress…

 

Until we see non-human primate data from Korro Bio and Genevant, I will count this candidate out of the race in AATD.  Whether ProQR will fill the void and throw down the gauntlet to Wave Life Science will be seen by its pipeline reveal at the end of this month.