Gene therapy for alpha-1 antitrypsin deficiency: an update

ABSTRACT

Introduction

Altering the human genetic code has been explored since the early 1990s as a definitive answer for the treatment of monogenic and acquired diseases which do not respond to conventional therapies. In Alpha-1 antitrypsin deficiency (AATD) the proper synthesis and secretion of alpha-1 antitrypsin (AAT) protein is impaired, leading to its toxic hepatic accumulation along with its pulmonary insufficiency, which is associated with parenchymal proteolytic destruction. Because AATD is caused by mutations in a single gene whose correction alone would normalize the mutant phenotype, it has become a popular target for both augmentation gene therapy and gene editing. Although gene therapy products are already a reality for the treatment of some pathologies, such as inherited retinal dystrophy and spinal muscular atrophy, AATD-related pulmonary and, especially, liver diseases still lack effective therapeutic options.

Areas covered

Here, we review the course, challenges, and achievements of AATD gene therapy as well as update on new strategies being developed.

Expert opinion

Reaching safe and clinically effective expression of the AAT is currently the greatest challenge for AATD gene therapy. The improvement and emergence of technologies that use gene introduction, silencing and correction hold promise for the treatment of AATD.

KEYWORDS:

• Alpha-1 antitrypsin deficiency
• AAV vectors
• CRISPR-Cas9
• editing guide RNA gene therapy

1. Introduction

1.1. Alpha-1 antitrypsin deficiency

Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder caused by mutations in the SERPINA1 gene, which encodes alpha-1 antitrypsin (AAT), the most abundant protease inhibitor in human serum [1]. AAT is a 52 kD glycoprotein synthesized mainly by hepatocytes and secreted into the circulation, through which it reaches the lungs [2]. The main function of AAT is to inhibit neutrophil elastase (NE), and other proteolytic enzymes and pro-inflammatory products, to protect the lung from excessive proteolytic degradation of interstitial elastin and other connective tissue components [3]. The most common deficient variant of AAT (a single amino acid substitution of lysine for glutamic acid at position 342, E342K), named as the Protease Inhibitor (Pi)*Z phenotype. The Pi*Z and Pi*ZZ are the terms for heterozygous and homozygous mutant genotypes, respectively, and accounts for over 90% of disease-causing alleles, with frequencies ranging from 3% in North America to 23% in the countries of Northern Europe [4]. The wild-type (WT) AAT protein is termed M-type (M-AAT) and the protein produced by the PI*Z allele is termed Z-type (Z-AAT). This missense mutation leads to misfolded and polymerized AAT protein aggregates which accumulate within the endoplasmic reticulum (ER) of the hepatocytes, impairing its secretion into the plasma and potentially resulting in liver disease (e.g. chronic hepatitis, cirrhosis) [5]. The reduction in AAT serum levels [to less than 11 µM (570 g/mL)], results in progressive destruction of alveolar epithelium and chronic airway inflammation, which are associated with an increased risk of lung disease (e.g. chronic obstructive pulmonary disorder, bronchiectasis) [6,7].

1.2. Treatments

At present, serum AAT augmentation therapy, which uses protein purified from pooled healthy human plasma, is still the only specific pharmacological intervention available for the treatment of AATD [1]. The AAT protein replacement strategy sufficiently raises the serum and alveolar epithelial lining fluid (ELF) to the U.S. Food and Drug Administration (FDA) and the European regulatory agencies considered protective levels (over 11 mM and 1.2 mM, respectively), for about 4.5 days [8]. However, this form of treatment only reduces symptom severity and is costly, limited in supply, variable in purity and activity, and can also be a burden for patients due to the need for lifetime weekly intravenous (IV) administration to maintain therapeutic concentrations [9]. Moreover, this therapy addresses only AATD-lung manifestations and does not treat the potential liver disease [10].

To overcome the disadvantages associated with human plasma-derived AAT, the possibility of producing recombinant AAT (rAAT) by alternative sources, such as bacteria, yeast, plants, mammalian cell lines, and animal models has been investigated [11–16]. Despite being successfully expressed in these models, producing rAAT has encountered obstacles, such as incorrect glycosylation, which affects renal clearance, and was not advanced to the FDA approval stage [17].

These concerns stimulated the development of more consistently efficacious treatments which are capable of maintaining a safe, robust, and prolonged stable AAT protein lifespan in an effort to be cost-effective and more convenient for patients [18]. In this regard, gene therapy has emerged as a promising solution for the efficient and sustained expression of therapeutic AAT, aiming to correct at the genetic basis of AATD instead of simply preventing the disease progression [19]. Treating AATD with gene therapy could also theoretically offer the advantage of a single dose administration, relieving the burden and cost of weekly IV-AAT infusions [20].

2. Gene therapy

2.1. AATD gene therapy

Gene therapy is a long-term strategy for the treatment of AATD-lung disease, where the approach is to express the WT allele to drive production of the normal human M-AAT gene into cells that will secrete the protein into the blood, ultimately augmenting the lung levels of AAT and preventing alveolar proteolytic destruction [21]. As a monogenic disorder, AATD appears to be a perfect candidate for gene therapy since the correction of a single gene should be enough for the reversal of the disease phenotype [20]. Another great advantage is that AAT has a short coding sequence, allowing it to be easily packaged within small-sized vectors, such as adeno-associated virus (AAV) [21].

Additionally, because AAT is a secreted protein, the variety of tissues and cell types that can be targeted for gene delivery is wide, while still protecting the lungs from proteolytic injury through the bloodstream [1,21]. Furthermore, there is a broad therapeutic range for serum AAT concentrations, from baseline protective levels of >570 g/mL up to supraphysiological levels, with no adverse clinical effect reported [1,2]. Ideally, gene therapy for AATD would allow for continuous, long-term expression of the gene under its normal regulatory mechanisms to treat all affected individuals to prevent the development of the disease rather than slow the development of its terminal pathology. This would also alleviate the comparatively minor symptoms of AATD such as dermatitis and connective tissue defects.

2.2. Pioneering studies

The earliest successful incorporation of the human AAT (hAAT) gene into cells was in 1986, with the in vitro transduction of a murine fibroblast lineage (NIH3T3) using recombinant retrovirus, demonstrating the feasibility of genetic therapy for the treatment of AATD [22,23]. Subsequently, other in vitro studies demonstrated that hAAT gene transfer resulted in the production of a glycosylated protein capable of inhibiting neutrophil elastase [24].

The first attempt at treating AATD was an ex vivo gene therapy using a retrovirus vector to genetically modify mouse fibroblasts, which were subsequently transplanted into the peritoneal cavities of nude mice. After 4 weeks, hAAT expression was detected in both the sera and the epithelial surface of the animal’s lungs [25].

In 1991, the first in vivo transfer was performed, using an adenoviral vector carrying the hAAT gene delivered by intratracheal (IT) administration to the respiratory epithelium of cotton rats. Expression of hAAT was detected in epithelial lining fluid for at least 1 week after administration [26].

Successive studies using retroviruses and adenoviruses were performed in larger animal models, such as rabbits and dogs, showed transient hAAT expression and/or treatment-related side effects due to high immunogenicity, along with presenting a potential risk of insertional mutagenesis and further cytotoxicity [27–30]. The disadvantages of these delivery systems have discouraged their use in human trials. To overcome these, current studies are focusing mostly on recombinant adeno-associated virus (rAAV) as a vector for gene delivery. As a vector for gene therapy, rAAVs have the advantages of being non-pathogenic to humans, induce low immunogenicity, are able transfect dividing and non-dividing cells, do not integrate into the genome of the target cells, and are able to produce prolonged expression of the transgene (in non-dividing cells), making them promising candidates for safe and efficient AAT gene therapy [31]. Furthermore, AAVs have many capsid variations, which affects their affinities for extracellular receptors on many different cell and tissue types, allowing for a range of therapeutic targets [32,33]. This is advantageous because there is concern that viral vector delivery to the liver could exacerbate the liver disease, which currently only develops in 5–10% of AATD patients and could be triggered or accelerated by environmental factors [19,34].

The pioneer in vivo study using rAAV-mediated hAAT gene delivery demonstrated that a single intramuscular (IM) dose (up to 1.4 × 10¹³ particles) of rAAV2-AAT in mice resulted in sustained and therapeutic levels of AAT, establishing proof-of-concept for IM rAAV2-AAT gene therapy trials in humans [35]. Further, toxicological and biodistribution studies were performed in C57B16 mice and New Zealand white rabbits following IM and IV delivery to establish evidence of possible germline transmission and adverse effects resulting from the therapy. These studies have found that both routes of administration show encouraging safety profiles and that there was less widespread dissemination via the IM route than IV [36].

2.3. Subsequent studies and clinical trials

In 2000, the first clinical trial ever performed for AAT gene transfer used aerosolized cationic liposomes to carry the WT gene to the nasal epithelium of five subjects with AATD. This trial failed to reach therapeutic levels, but the levels expressed were found to have anti-inflammatory properties, which established proof-of-concept for AATD gene therapy [37]. Following this proof-of-concept and promising results from preclinical studies with rAAV2-AAT, a Phase I trial (NCT00377416) was conducted in AAT-deficient adults. Despite a transient and clinically insufficient level of AAT expression as well as the presence of pre-existing anti-AAV2 capsid antibodies which increased after vector administration, the study proved to be safe and no vector-related adverse events were reported. This has motivated further studies using different serotypes and administration routes for more efficient gene delivery [38,39].

Studies using other serotypes identified AAV1 as a better alternative for gene delivery to skeletal muscle, showing a greater transfer efficiency compared to AAV2 and resulting in 200-fold higher serum AAT levels [21]. The success of the IM rAAV1-hAAT strategy in animal models has encouraged renewed attempts to increase AAT serum expression levels in humans.

Brantly et al. demonstrated a much higher and sustained serum dose-dependent expression of M-AAT, achieving expression for at least 1 year after intramuscular injection of nine Pi*ZZ patients, although this did not reach therapeutic target levels (NCT00430768). Also, a positive T cell response to the AAV1 capsid epitope was detected, but no immune response against the AAT transgene was observed [40].

To promote greater AAT expression and hopefully reach therapeutic AAT concentrations, Flotte et al. moved forward to a dose escalation Phase II trial (NCT01054339). In this study, the rAAV1 vector was produced by a herpes simplex virus-1 helper system to increase transgene expression, and showed a 10-fold increase in vector-derived AAT level (3% of the therapeutic target) in AAT-deficient patients’ blood compared to the Phase I trial, which persisted for at least 90 days. However, for the therapeutic target expression level to be reached, 100 IM injections had to be performed to deliver enough of the AAV vector, since it could not be further concentrated without causing viral aggregation and precipitation. The therapy was well tolerated, and again immune responses were observed to the AAV, but not to the AAT transgene. These results support the use of rAAV1 gene transfer for AATD, although alternative methods of administration are needed to increase transgene expression to therapeutic levels [41].

Mueller et al. published a follow-up on this study and reported persistent hAAT expression 1-year later at 2.5–3.8% of the target therapeutic level after the single IM administration [42]. Aiming to understand the long-term transgene expression, a cytotoxic T cell response directed against the AAV vector capsid was observed and the inflammatory infiltrates in the injected muscle were characterized. A population of AAV1 capsid-specific regulatory T (Treg) cells were identified in muscle tissue. This finding raised the hypothesis that a Treg-induced tolerance response could be allowing the sustained AAT expression despite the presence of tissue inflammation and T cell responses to the capsid [43,44].

In an update on this trial, Mueller and colleagues found sustained AAT expression for 5 years after IM injection, which was accompanied by persistent Treg infiltration. Furthermore, the presence of an exhausted CD8 T cell response to AAV1 capsid was also observed at the vector administration site. These results suggest that IM AAV-based gene transfer could be acting as a chronic viral infection, modulating the immune response. In addition to the immune findings, it was also noted that a low-level consistent AAT expression (still less than 5% of therapeutic target) resulted in partial correction of disease-associated neutrophil defects. Therefore, it was proposed that further dosage increases of rAAV1-AAT by IM delivery would be feasible and could result in persistent therapeutic AAT levels, which could result in clinically relevant inhibition of neutrophil elastase, even at lower serum levels than were previously deemed clinically relevant [45] [Figure 1].

Figure 1. M-AAT serum levels over 5 years after single intramuscular administration of rAAV1-CB-hAAT. AAT serum levels were assayed using a M-AAT-specific ELISA. The doses used were 6.0 × 10¹² vg/kg (high), 1.9 × 10¹² vg/kg (mid), and 6.0 × 10¹¹ vg/kg (low). The dotted line indicates ~2.5% of the target therapeutic serum concentration (572 µg/mL, 11 µM). Reproduced from [45], © 2017 Mueller et al., licensed under CC BY-NC-ND 4.0.

With clinical data on the low sustained expression levels and T cell responses, more work in animal models was needed to improve the AAT gene therapy efficacy. Towards the purpose of further exploring the Treg tolerance response, Arjomandnejad et al. generated chimeric antigen receptor regulatory T (CAR-Treg) cells, which were created from AAV-CAR T cells, to modulate capsid-specific immune responses against AAV (AAV-CAR-Tregs). The CAR Tregs inhibited effector T cell proliferation and cytotoxicity in vitro, produced immunosuppressive cytokines, reduced tissue infiltration, and mediated continued AAT transgene expression up to 26 weeks post-IM injection in mice [46]. This approach paves the way for a possible strategy to induce immunotolerance to AAV therapy to reduce vector clearance and increase transduction efficiency.

Muscle-directed gene therapy delivery has limitations in the concentration of a viral dose that can be administered, and so strategies for a different method of administration were required. Gruntman et al. used the isolated limb infusion (ILI) method to deliver AAV vectors into the limb vasculature of non-human primates. The limb was first isolated from the systemic circulation by a tourniquet, which allows for a greater total viral volume and wide-ranging muscle distribution in the pelvic limb. This achieved sustained therapeutic levels of AAT expression with some increase in expression efficiency in the ILI-dosed animals compared to the IM-dosed animal [47]. The ILI method was also shown to induce a Treg response and to lead to T cell exhaustion in this non-human primate model [48].

IM injection is a common route of administration because of its minimally invasive accessibility and the long-lived non-dividing nature of myocytes, promoting a sustained transgene expression [49]. Furthermore, IM administration has been supported by the European Medicines Agency (EMA) for the first ever approved gene therapy product, Glybera, which is an rAAV1 vector delivered by IM injection. Nevertheless, muscle AAV delivery has so far been proven limiting in the deliverable dose and expression level achieved for AATD. Future additional work will be required to determine if this is a viable route to producing clinically relevant serum (and pulmonary tissue) levels of AAT. Concurrently, alternative delivery routes will need to be investigated.

2.4. Alternate routes to the lung

Aiming to directly deliver AAV vectors to the lungs, and therefore increase AAT levels in the local environment, IT and intrapleural (IP) administration has been used to target the respiratory epithelium and pleura, respectively [50]. Due to the lung’s direct contact with the external environment, it presents both physical and immune barriers to pathogen entry, which can limit the transduction efficiency [51]. Previous studies have shown that AAV2 and AAV5 IT administration presented low transduction rates, thus requiring high viral dosage, which consequently promotes inflammatory reactions [52,53]. Adding polyethylene glycol (PEG), a neutrally charged hydrophilic biocompatible polymer, to the rAAV formulation seemed to prevent the vector from binding to the mucous layer as well as protecting it from neutralizing antibodies [54]. Likewise, the construction of PEG-conjugated nanoparticles (muco-penetrating particles, MPPs) based on polymers has shown efficiency in the treatment of diseases that affect the lung, such as asthma [55,56]. However, these methods all present challenges to persistent AAT expression from the dividing cell populations targeted by airway delivery.

To overcome the lung barriers, pleural administration is an alternative to lung parenchyma transduction, which reaches the systemic circulation and ultimately the liver. This would potentiality achieve sufficient AAT protein threshold levels both in the serum and alveolar lining fluid. De et al. compared IM versus IP administration of AAV2 and AAV5 vectors carrying the hAAT gene in mice. For both routes of administration, AAV5 achieved a 10-fold higher level of hAAT compared to AAV2 in both serum and lung. IP administration of both serotypes achieved a 1.6-fold higher AAT therapeutic concentration than muscle delivery for over 40 weeks [50]. Pleural administration was safe and more efficient than IM delivery in promoting a therapeutic protein level expression in the respiratory system with lower vector dosage [57]. However, it is noteworthy that antibodies against AAV2 and AAV5 are highly prevalent in the human population, limiting their use in gene therapy. AAV9, AAV8, and AAV6 vectors seem to be preferable serotypes for lung transfer, showing safe and improved transduction of the airways, although they do not overcome the problems of pre-existing immunity in some populations or the issues with delivery and persistent expression issues surrounding lung dosing [58–60].

In a subsequent study, De and colleagues screened 25 AAV vectors derived from humans and nonhuman primates for AAT IP delivery to mice. The rhesus AAVrh.10 vector resulted in the highest AAT expression levels, both in the serum and lung, proving to be the most effective known serotype for IP gene transfer, besides escaping neutralization by preexisting AAV-antibodies [61]. Next, a Phase I/II clinical trial was performed in six patients with AAT deficiency to evaluate the safety, dose, and efficiency following IV administration of AAVrh.10-hAAT, with the possibility of delivering follow-up doses via IP administration. An increase in the plasma concentration of M-specific AAT was observed at week 52 following IV administration. However, subjects experienced some treatment-related adverse events along with abnormal changes in clinical laboratory parameters, such as a rise in neutrophil count and alanine transaminase levels (NCT02168686). All six patients were enrolled in a long-term follow-up study (NCT03804021) for a 2-year post-treatment period for continued safety monitoring. No follow-up results for this study have been reported at the time of writing this review, but the study has been discontinued due to low levels of M-AAT being produced [62].

It is worth mentioning that, to date, all AAT gene therapy studies that have progressed to Phase I/II clinical trials were based on rAAV (AAV2, AAV1, or AAVrh.10) vectors for the delivery of the M-AAT gene to patients’ muscle, systemic circulation, or pleura with no significant adverse events, which is encouraging for continued efforts in this therapeutic approach.

2.5. AAT therapy for liver disease

Correcting AATD through the introduction of the normal AAT gene (M-AAT) is a long-term solution for the pulmonary repercussions of the deficiency. However, many AATD patients suffer from liver disease, which is derived from the accumulation of misfolded AAT protein in the ER of hepatocytes and is not improved with this strategy [10]. As augmentation strategies are continuously implemented more widely and their efficiency improves, the AATD patients slow the development of lung disease, but develop more severe liver disease. Patients who develop liver disease can develop impaired liver function, increased levels of liver enzymes, liver cancer, and cirrhosis, but this presentation is very heterogeneous and there is a lack of histologic or biochemical markers that are specific to AATD [63]. So far, there are no viable treatments available for AATD-associated liver disease until end-stage cirrhosis is reached, when liver transplantation is the only option. Unfortunately, organ transplantation presents many complications, such as chronic rejection and infections due to immunosuppressive treatments, which can lead to death, limited availability of donor tissue, and high cost and expertise required [64]. Therefore, in addition to providing the WT gene, the ideal therapy should also knock-out the pathogenic PI*Z allele to eliminate the expression of the malformed Z-AAT protein to treat the liver disease.

One strategy that is being studied is the suppression of the expression of the mutant gene in an attempt to reduce the production of malformed Z-AAT protein and prevent its toxic hepatic accumulation. The challenge of this approach is being able to simultaneously inhibit the mutant Z-AAT gene expression and induce wild-type M-AAT expression in the same patient. Ozaki et al. attempted this by transducing human hepatoma cells with a retroviral vector expressing a ribozyme-encoding sequence that specifically recognizes abnormal SERPINA1 mRNA along with wild-type SERPINA1 cDNA which had been modified to be ribozyme-resistant. The bi-functional vector was able to reduce mutant AAT protein expression by 50% while expressing functional AAT protein, and this strategy was termed ‘gene replacement’ [65].

Following the same concept, Mueller et al. incorporated microRNA (miRNA) sequences and the M-AAT gene within the same transcript and packaged it into rAAV9. This allowed for silencing of the mutant SERPINA1 mRNA by 80% while promoting increased serum M-AAT expression, which was resistant to the miRNA-induced knockdown in mice [66]. Large animal and human studies still need to be performed to ascertain the viability of this therapeutic approach.

Ribonucleic acid interference (RNAi) has been investigated as another gene therapy tool to accomplish silencing of genes containing pathogenic mutations. Currently, Phase I/II and II/III clinical trials using RNAi to silence expression of the Pi*Z allele in order to prevent synthesis of the misfolded protein are in progress as a treatment for liver disease caused by malformed AAT.

The purpose of the Phase I study was to evaluate the safety profile, tolerance, pharmacokinetics, and pharmacodynamics of a single subcutaneous injection of the drug belcesiran (dose cohorts: 0.1, 1.0, 3.0, 6.0, and 12.0 mg/kg), in participants with no AATD-associated liver disease (NCT04174118). Interim results from the study report that the drug is well tolerated, with no serious adverse events, and there are dose-dependent reductions in serum AAT (maximum reduction of 91%). Follow-up study of the cohort given the highest dose (12.0 mg/kg) is ongoing, but the data are not available for inclusion in this review. The Phase I trial interim results have encouraged the initiation of Phase II clinical trials in 2021 in patients with AAT liver disease.

Phase II/III trials are underway and aim to evaluate the safety, efficacy, and tolerability of multiple subcutaneously doses of the ARO-AAT product Fazirsiran in patients with AATD (NCT03945292 and NCT03946449). The first study (NCT03945292) was started in 2019 and has an estimated completion date of 2023, but no results are posted at the time of this writing [62]. The second (NCT03946449) has reported preliminary data and found that the 16 patients with baseline fibrosis demonstrated a dramatic dose-dependent (25 mg, 100 mg, or 200 mg) reduction in the level of Z-AAT in the serum (74%, 89%, and 94%, respectively) and liver (median reduction of 83%) at 48 weeks post-treatment. The therapy was well tolerated in patients, who showed a decrease in several markers of liver disease, including fibrosis (METAVIR scoring system) and histologic PAS-D globule burden (69% reduction), in liver biopsies, in addition to reduced inflammation markers. The results demonstrate the treatment potential to reverse AATD-induced liver disease and encouraged the initiation of the Phase III trial. The next step is to achieve regression of histologic fibrosis in a larger sample size of 160 patients at 106 weeks post-treatment [67].

3. Next generation therapies

3.1. The CRISPR revolution

Another technology that has been revolutionizing research for the treatment of genetic diseases is CRISPR-Cas9 (clustered regularly interspersed short palindromic repeats (CRISPR) associated nuclease 9) gene editing. Adapted from a naturally occurring bacterial immune system, this technique makes it possible to change an organism’s DNA, through the addition, removal, or site-specific alteration of the genetic material [34]. This tool has been embraced by the gene therapy community, and this has opened the door new possibilities in treating AATD. The intuitive thought in the AATD field was to use CRISPR-Cas9 to knock-out the Pi*Z allele and knock-in the WT allele. Correcting the Pi*Z allele in the liver would alleviate the misfolded protein inducing liver disease and serve as a biofactory to produce M-AAT, which would be secreted into the circulatory system and alleviate the lung disease.

Two groups independently developed a combination of CRISPR/Cas9 gene editing tool and AAV gene delivery vectors for the treatment of AATD. They systemically co-administered an AAV vector expressing CRISPR-Cas9 system to disrupt transcription of the Pi*Z allele with an AAV providing a homology-directed repair (HDR) donor template to correct the Pi*Z allele in mice carrying the mutant allele (PiZ transgenic mice). A reduction of Z-AAT in hepatocytes and the serum as well as a modest level of serum M-AAT mRNA was observed in both studies [16, 68].

Another study of Z-AAT correction used a CRISPR-Cas9 system delivered by an adenovirus vector. The adenoviral CRISPR/Cas9 treatment was able to reverse the pathologic liver phenotype, including decreasing Z-AAT protein aggregation and liver transaminase levels, while improving liver histologic inflammation and morphology as well as reducing liver fibrosis in the PiZ mouse model [15]. These findings are encouraging for potential of using CRISPR-mediated gene editing to correct liver and lung disease in patients with AATD. However, at the time of writing this review, no clinical studies have been performed.

As seen, in the past 30 years several experimental therapeutic strategies have been investigated, including viral (retrovirus, adenovirus, gammaretroviral, lentivirus, and adeno-associated virus) and non-viral (naked plasmids and plasmids complexed with liposomes) gene transfer, although only a few have progressed to clinical trials. Despite the efforts of the scientific community, no genetic therapy-based medicine has been implemented for the treatment of AATD, although much progress has been achieved in this field.

3.2. New strategies being developed

Two groups investigated a new strategic concept for treating AATD. They repopulated the liver of PiZ mice with normal donor hepatocytes considering that WT hepatocytes have a competitive advantage over Z-AAT-burdened hepatocytes. Ding et al. injected M-AAT murine hepatocytes in Pi*Z transgenic mice, resulting in a 20–98% replacement of mutant hepatocytes with WT ones. The repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor, which induces mitotic stimulation [69]. Borel et al. explored two different approaches. They xenotransplanted M-AAT human donor hepatocytes into recipient Pi*Z transgenic mice and corrected the PiZ allele in murine hepatocytes by genome editing. This was accomplished by injecting mice with a AAV8 vector carrying the gene for M-AAT and a synthetic miRNA to express functional AAT and silence the Pi*Z allele. Both strategies resulted WT hepatocytes displaying a proliferation advantage over Z-AAT hepatocytes [70]. The studies provide important information on the competitive advantage of hepatocytes producing M-AAT over Z-AAT, which can further inform the other gene therapy approaches as well as establishes the potential for cell-based therapies using gene corrected or WT hepatocytes in AATD patients.

It has been shown that the liver can also be repopulated with induced pluripotent stem cells (iPSCs) differentiated into hepatocytes [71]. Yusa and colleagues demonstrated that the genetic correction of human iPSCs by a combination of zinc finger nucleases (ZFNs), to specifically cleave the Pi*Z mutation site, and a piggyBac transposon bi-allelic excision allows restoration of AAT protein folding and function in derived hepatic-like cells both in vitro and in vivo. This study suggests that using AATD-hiPSC lines could be a viable strategy in clinical applications [72].

A new AAT-null murine model has been generated and recapitulates the features of the clinical lung disease of AAT-null patients, potentially providing a better understanding of the development and progression of respiratory insufficiency associated with AATD-emphysema. When treated with a single IV injection of M-AAT-rAAV8 the AAT-null mice showed a dose-dependent level of functional human M-AAT and a restoration of lung protease-antiprotease balance. Functional and histological analysis showed decreased lung compliance, increased elastic recoil, and preserved alveolar wall integrity in treated mice, indicating prevention of emphysematous changes [73].

Another gene editing strategy, called ‘prime editing,’ has been tested in the PiZ mouse model. Prime editing utilizes RNA-directed targeting with a Cas9 derivative fused to a reverse transcriptase (RT) domain to enable precise ‘rewriting’ of mutant sequences. In a recent publication, Liu et al. designed a prime editing guide RNA (pegRNA) to target the PiZ mutation site and rewrite it with a corrected sequence [74]. The feasibility of this precision technique in vivo in the PiZ mice bodes well for future clinical development. However, there are issues of efficient delivery and evasion of immune responses to the AAV capsid, Cas9 molecule, and RT protein can be overcome.

In parallel with the development of new technologies that have allowed novel strategies to be developed, improvements have also been made in the initial augmentation strategies to deliver recombinant proteins to patients. A Phase I clinical trial is underway to evaluate the safety of delivering a recombinant, oxidation-resistant form of the AAT protein (INBRX-101) to patients (NCT03815396).

4. Conclusion

There is great potential in the use of gene therapy for the treatment of diseases associated with AATD, with diverse strategies and enthusiastic scientists. The use of dual-function vectors that concomitantly inhibit the expression of the Z-AAT gene and promote the biological activity of M-AAT is a viable approach for the absolute correction of pulmonary and hepatic manifestations related to AATD. Co-administration of AAT-AAV vectors and capsid-specific Treg cells is a promising way to avoid immune responses, which eliminate the transducing agents, improving their efficiency and reducing adverse events resulting from their administration. Genetic editing strategies, including the use of silencing RNAs and CRISPR-Cas9, have revolutionized many fields of medicine and hold great promise for the correction of site-specific mutations in the SERPINA1 gene. iPSCs are used in a vast number of applications and hold the possibility to recreate entire organs for transplantation or repopulation. Differentiation of these cells into hepatocytes expressing corrected AAT is a potential solution for both AATD liver and lung diseases. In summary, there are many approaches to treat, and even cure, diseases related to AATD, and the success of each of the approaches will determine the direction of the field in the coming years. However, the development of more reliable experimental animal models and the optimization of these technologies is essential to be able to test these therapeutic approaches and facilitate their subsequent clinical translation.

5. Expert opinion

AATD has been simultaneously very attractive and very challenging as a target for gene therapy. The secreted nature of the transgene product makes it straightforward to track the serum or plasma AAT level as the critical biomarker to evaluate the success of gene replacement. This has led to dozens of studies of various gene therapy vehicles expressing the AAT gene by numerous routes of delivery in various animal models. However, challenges of this condition are that the current threshold of expression required for the prevention of lung disease is extremely high compared with other secreted gene therapy targets. Adding to this challenge is the fact that clinical efficacy, in terms of functional clinical improvement, is very difficult to document in such a slowly progressive disorder. The lack of clear, prospective evidence of the lung function benefits of protein replacement (augmentation) therapy more than 30 years after its approval is evidence of the magnitude of this challenge. Further, the liver disease both requires a separate strategy (i.e. silencing or knocking out of the Pi*Z allele) and limits the delivery strategy (i.e. minimizing liver toxicity). There is also less known about the liver disease compared to the lung disease in AATD patients.

However, it is encouraging that many of the most advanced and sophisticated molecular approaches in gene therapy show relatively high efficiency in hepatocytes, which are the target cells for optimal expression of the gene encoding M-AAT. In particular, the promise of ‘next-generation’ CRISPR technologies, such as prime editing, provides significant hope for future effective therapies. The advent of CRISPR technology has also enabled the generation of new animal models of AATD lung disease, such as the ferret model [75], which may prove to be crucial to address the challenges of developing better endpoints for evaluating therapeutic gene correction.

Thus, we are hopeful for the prospects of using gene therapy to treat AATD in the coming years. However, the measure of that success can only be documented through the further development of robust endpoints that can ‘connect the dots’ between biological activity of AAT and clinical efficacy. These newer endpoints may be considered in several specific categories: (1) those reflecting the rate of progression of degradation of the lung parenchyma and extracellular matrix (ECM); (2) those reflecting the inflammatory state of the lung; (3) those reflecting the functional status of patients.

The progression of destruction of the lung tissue is currently being evaluated using both imaging and biochemical biomarkers. The measurement of computed tomography (CT) lung density has been particularly informative in studies of augmentation therapy, including the RAPID trial and the RAPID extension [76, 77]. In those studies, the rate of loss of lung density was reduced by nearly half among the group receiving an early start on augmentation therapy. The preservation of CT lung density corresponds with a preservation of pulmonary function by spirometry as well, but with less variability in the measurement. In a similar fashion, biochemical biomarkers of ECM loss have proven to be valuable outcome measures in evaluating the effects of therapies in this patient population. In particular, the measurement of desmosine and isodesmosine have correlated well with improvements in a wide range of pro-inflammatory cytokines, serum markers of protease/antiprotease imbalance and other indices of AAT biological activity [78].

Strategies to evaluate the inflammatory state of the lung have previously relied heavily on the use of bronchoalveolar lavage (BAL) fluid. While the evaluation of the cellular composition of BAL and of a wide range of pro-inflammatory cytokines in that fluid provide a direct insight into the lung compartment, the scalability of such studies is limited because of the nature of fiberoptic bronchoscopy. Thus, BAL studies may be most appropriate for early phase studies of gene therapy rather than larger scale or long-term studies, which may rely more on measures of inflammatory cells, mediators in the peripheral blood, exhaled gas, and/or exhaled breath condensate. Finally, while the measurement of pulmonary function by spirometry remains a mainstay of AAT clinical research, patient-reported outcomes and functional activity studies derived from mobile technology and wearable devices are making a huge impact in chronic disease management, and could eventually become the ultimate test of ‘real world’ efficacy.

The clinical implementation of AATD genetic correction will be a significant step not only for the treatment of those with the deficiency but also for the advancement of gene therapy itself. The more the technology consolidates, the more affordable and accessible it will be, which will benefit a greater number of people in more areas. In addition, these advancements will contribute to the development of therapies for other unresolved diseases. Therefore, efforts to effectively establish the clinical use of AATD gene therapy remain of utmost importance to the scientific community, and the reality of providing effective and long-lasting treatment for patients suffering from the disease is very promising.

Abbreviations

Alpha-1 antitrypsin (AAT)

Alpha-1 antitrypsin deficiency (AATD)

Alpha-1 antitrypsin deficiency-associated liver disease (AATLD)

Adeno-associated virus (AAV)

Bronchoalveolar lavage (BAL)

Chimeric antigen receptor regulatory T (CAR-T) cells

Chronic obstructive pulmonary disease (COPD)

Clustered regularly interspersed short palindromic repeats CRISPR associated nuclease 9 (CRISPR-Cas9)

Computer tomography (CT)

Extracellular matrix (ECM)

Endoplasmic reticulum (ER)

Epithelial lining fluid (ELF)

Human AAT (hAAT)

Induced pluripotent stem cells (iPSCs)

Interference ribonucleic acid (RNAi)

Intramuscular (IM)

Intraperitoneal (IP)

Intrapleural (IP)

Intratracheal (IT)

Intravenous (IV)

Isolated limb infusion (ILI)

Micro ribonucleic acid (miRNA)

Neutrophil elastase (NE)

Polyethylene glycol (PEG)

Protease inhibitor (Pi)

Recombinant AAT (rAAT)

Recombinant AAV (rAAV)

Regulatory T (Treg) cells

Ribonucleic acid (RNA)

Article highlights

• Alpha-1 antitrypsin deficiency is characterized by progressive lung and liver disease.

• AATD has historically been treated by augmentation therapy, but this is inadequate for patients.

• Preclinical and clinical studies have investigated several approaches, and gene therapy is the most promising so far.

• Clinical studies to date have not achieved long-lasting expression of the M-AAT protein at endogenous levels, but have markedly reduced levels of the pathogenic Z-AAT protein.

• Current strategies are employing CRISPR technology to simultaneously express the M-AAT allele and silence the Z-AAT allele.

• As technology advances in the gene therapy field and across all areas of biomedicine, there is great reason for optimism in achieving a curative treatment for AATD.

This box summarizes key points contained in the article.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

A reviewer on this manuscript has disclosed that they have consulted for Grifols, CSL, Takeda and Inhibrx and have run trials for Grifols, Arrowhead, Mereo BioPharma, Shire/Baxter/Takeda and Adverum. A reviewer on this manuscript has disclosed that they were an investigator for Adverum in a previous gene therapy study for AATD that ended in 2022. They participate and consult for the companies involved in the gene silencing studies for AAT liver disease. A reviewer on this manuscript has disclosed receipt of consulting fees from Takeda. Peer reviewers on this manuscript have no other relevant financial relationships or otherwise to disclose.

Additional information

Funding

This paper was not funded.