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.

Reconsidering RNA Editing for Alpha-1-Antitrypsin Disease

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

Arrowhead Pharmaceuticals SEQUOIA data

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

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

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

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

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

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

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

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

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

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

RNAi and Antisense Targeting the Same Gene: Not a Zero-Sum Game

Transthyretin-mediated amyloidosis has become the single-most important factor for the 3-5 year valuation of certainly Alnylam, and possibly ISIS Pharmaceuticals as well. After TTR, Z-alpha-1-antitrypsin (Z-AAT) is gearing up as the next Alnylam(RNAi)-ISIS(antisense) battleground. Expect to hear competitive language from the two camps why their approach will prove superior over the other.  It is, however, worth keeping in mind that having two candidates race towards approval can also have significant pie-enlarging benefits.

Raising Awareness and Pressuring Regulators

A good competition will attract attention, from patient groups, physicians, regulators, and the investor community.  Patient groups and physicians will be primed that after years of lacking treatment options, the time for change has come.  We have seen this with the trifecta ofweight-loss drug candidates, two of which have recently been virtually willed into approval.  Given the enormous problem of obesity, it became politically untenable to further withhold treatment options.  Eventually, the regulators were forced to give in to the pressure despite their original objections over safety.

Closer to home, similar awareness and pressure is being brought to bear on getting exon-skipping oligonucleotides approved for the treatment of Duchenne Muscular Dystrophy, stoked by the competition between Prosensa/GSK and Sarepta .  If you caution about hurried, aka accelerated approval, then you are readily labeled as a heartless misanthrope.  Similarly, the homozygous familial hypercholesterolemia population has already been educated, targeted, and friended by both patient-access specialist Genzyme and competitor Aegerion and probably strongly expect the new drug approvals.  

Understand that these comments are not meant to be judgments on the respective drug candidates, but just the way I view current dynamics.  In fact, I agree that developing drugs for many of the rare and severe (orphan) diseases requires more risk-taking, usually biomarker-based approaches, and that not too long ago patient needs were not sufficiently considered in a highly risk-averse regulatory climate.

The TTR Amyloidosis Race

Given that Pfizer’s drug candidate tafamidis does not appear about to revolutionize the treatment of TTR amyloidosis, I see similar pressure building for the approvals of gene knockdown approaches ALN-TTR02 and ISIS-TTRRx in 2015-6.  The intense competition between ISIS and Alnylam will provide good fodder for the press (just as it does for this blog), engage the minds and pad the pockets of key opinion leaders and consequently raise the expectations of the public and hopes of those suffering from the disease.

While I have 2015-6 approvals baked into my projections of the RNAi Therapeutics story, I wouldn’t mind a year’s delay if the competitive dynamics would allow for it.  In fact, I expect the FDA to manipulate the process in a way that both drug candidates will come before an AdCom panel together, making the obsession with speed over good science even more so nonsensical.

My problem with ISIS and GSK speeding straight into a phase III study after a single phase I study is that they do not seem to have identified a suitable dosing regimen: 400mg/week good efficacy (-81% knockdown), but probably too high a dose from what we know about the safety of phosphorothioate oligonucleotides; 200mg/week could fare better in terms of safety, but has shown only modest knockdown efficacy (-44% knockdown).  In my opinion, another dose-finding study would be in order under normal circumstances.

[Update 29Nov2012: The Clinicaltrials.gov entry for the upcoming phaseII/III ISIS-TTRRx trial shows that the companies have chosen the stab-in-the-dark 300mg/week dose.  Although the January 2012 phase I results press release made no mention of such a cohort, the June 2012 Annual Shareholder Meeting TTR poster shows data from a 300mg cohort (~70% knockdown).  Unlike all the other cohorts were 90+ day data, the 300mg cohort data was less than 50 days.  It thus looks like a 300mg cohort was included post-hoc. Unfortunately, ISIS has chosen not to list the phase I trial on clinicaltrials.gov.]

This concern over uncertain dosing schedules also applies to Alnylam's ALN-TTR02 (-87% peak knockdown, -67% knockdown after 28 days following single dose) where the current ‘multi-dose’ phase II study only seems to cover two doses.  You would hope that Alnylam presses hard to amend the study to include further doses to gain more experience with the repeat-dosing pharmacology of ALN-TTR02 before betting their house on a single pivotal phase III trial.

Once approvals are obtained, however, the beneficial aspects of competition should disappear quickly especially when small orphan disease populations are involved.  But until then, the race towards approval should free energies allowing TTR amyloidosis to become the first real commercial success story of either RNAi Therapeutics or RNaseH antisense.

Who do you think will win the TTR race? Take part in the survey (upper right hand corner).