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.

Journal Club: Therapeutic RNAi Delivered via Vitamin A-Coupled Cationic Liposomes Reverses Liver Cirrhosis in Rats

Illustrating how the basic concept of liposomal RNAi delivery can be modified to target specific cell types at low dosages, a study by Sato and colleagues published last week in the online version of Nature Biotech shows how the addition of vitamin A to cationic liposomes carrying siRNAs against Hsp47 can not only efficiently knock down the gene in hepatic stellate (HS) cells and block disease progression, but that this can actually reverse cirrhosis in a number of rat models for this disease [Sato et al. (2008). Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone. Nature Biotech doi:10.1038/nbt1396].

It is known that by adjusting their stability and therefore pharmacokinetics, liposomes can be adjusted to shift their relative accumulation in tissues such as liver, tumour, or lung. For example, recent improvements in formulation methods now allow for the delivery of around 90% of the injected dose to the liver. While for liver applications, this already minimizes the exposure of non-target organs to the RNAi therapeutic, the ability to knock down genes in specific cell types at low dosages should further improve its therapeutic index.

Adding small conjugates such as sugars (e.g. Mirus Bio’s DynamicPolyConjugates to target RNAi to either hepatocytes or Kupffer cells) to a basic delivery formulation appears to be particularly promising. The authors of the present paper reasoned that since the HS cell which is central to liver cirrhosis efficiently takes up vitamin A, adding this vitamin to liposomally formulated siRNAs may facilitate their uptake in HS cells, in addition to endowing the nanoparticle with favorable pharmacokinetics by binding to retinol binding protein in circulation.

Indeed, while some non-specific uptake was observed into phagocytic cells, the modified A-liposomes efficiently entered HS cells in a vitamin A-dependent manner while largely avoiding other cell types of the liver, including hepatocytes. Actually, delivery was truly targeted since overall drug uptake was greatly enhanced in cirrhotic rats versus non-cirrhotic rats, likely a reflection of the HS cell activation/proliferation state.

Importantly, knockdown of Hsp47, required for collagen production and therefore a potential target for treating cirrhosis, was achieved at the clinically relevant low dosages of 0.1mg/kg to 0.75mg/kg, resulting in the survival of rats concomitant with resolution of the cirrhosis both by histology and normalization of liver enzymes.

The paper also shows that as we are learning more about the molecular biology and pharmacological properties of synthetic RNAs, the scientific standards for publishing papers on therapeutic RNAi have risen. Consequently, the sequence-specific nature of the therapeutic effect was demonstrated by the use of two additional Hsp47-targeted siRNAs. Cytokines and interferon-alpha were also looked at, including IL-12 which has recently gained notoriety
as a TLR3-related pro-inflammatory cytokine. Reassuringly, no IFN-alpha, TNF-alpha, or IL-12 induction was detected at the reasonably early 7 hour time-point with this unmodified Dicer-substrate (Dicer substrates are longer and therefore should be more prone to immune recognition). However, some non-specific elevation in the apoptosis of rat liver HS cells was observed following treatment with VA-liposomes carrying a control siRNA. Albeit small, it remains to be determined whether this was related to sequence-specific cytokine induction, or liposomally or vitamine A-induced cytotoxicity.

It is likely that the systematic screening for improved VA-liposome chemistries and optimized siRNAs should considerably improve the therapeutic index of this interesting variation on the cationic liposome for siRNA delivery. Similarly, vitamin A derivatives that are still taken up by HS cells, but do not cause potential vitamin A-related toxicities may be warranted for actual clinical use.