Recent advances in α-1-antitrypsin deficiency-related lung disease

Abstract

α-1-antitrypsin deficiency (A1ATD) is an under-recognized hereditary disorder associated with the premature onset of chronic obstructive pulmonary disease. There is considerable heterogeneity in the phenotypic expression of lung disease in A1ATD and the pathophysiology is complex, involving the interaction of multiple pathways. Other genetic factors that may contribute to emphysema risk in A1AT-deficient individuals are beginning to be identified. Methods of monitoring disease progression have evolved, including the use of computed tomography densitometry and biomarkers of disease activity. Progress in the development of novel treatment strategies continues, including the hope for a potential cure through the use of gene therapies. In this article, the authors review the recent advances in this field and outline potential future directions of research in A1ATD.

Keywords::

• α-1-antitrypsin
• augmentation
• biomarker
• chronic obstructive pulmonary disease
• emphysema
• gene therapy
• polymerization

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Release date: 4 June 2013; Expiration date: 4 June 2014

Learning objectives

Upon completion of this activity, participants will be able to:

• • Assess the pathophysiology of A1ATD

• • Analyze the clinical presentation of patients with A1ATD

• • Compare methods to predict the progression of emphysema among patients with A1ATD

• • Evaluate treatment options for patients with A1ATD

Financial & competing interests disclosure

EDITOR

Elisa Manzotti

Publisher, Future Science Group, London, UK.

Disclosure: Elisa Manzotti has disclosed no relevant financial relationships.

CME AUTHOR

Charles P Vega, MD

Associate Professor and Residency Director, Department of Family Medicine, University of California, Irvine, CA, USA.

Disclosure: Charles P Vega, MD, has disclosed no relevant financial relationships.

AUTHORS AND CREDENTIALS

Judith A Brebner

The ADAPT Project, Lung Function and Sleep Department, University Hospital Birmingham, Birmingham, UK.

Disclosure: JA Brebner has disclosed the following relevant financial relationships: funded by an educational grant from Grifols.

Robert A Stockley

The ADAPT Project, Lung Function and Sleep Department, University Hospital Birmingham, Birmingham, UK.

Disclosure: RA Stockley has disclosed the following relevant financial relationships: unrestricted research grant from Grifols and acts in an advisory role for Baxter, CSL Behring and Kamada.

Figure 1. a-1-antitrypsin structure.

Reproduced with permission from [153].

Figure 2. Complex interaction of pathogenic mechanisms responsible for the development of emphysema in α-1-antitrypsin deficiency.

Polymerization of Z A1AT in the lung (1) leads to local ER stress (2) and establishment of a postinflammatory cascade (3) including increased neutrophil recruitment (4). Lung polymers are also chemoattractants, recruiting and localizing neutrophils further (4). Polymerization in the liver (5) results in serum and lung deficiency (6), causing unopposed neutrophil elastase activity (7) as a result of the recruited neutrophils (4), which in turn can activate other classes of enzymes (8) in addition to other uninhibited serine proteinases. The net result is proteolytic degradation of lung tissue (9), leading to emphysema. In addition, neutrophil elastase-derived peptide (10) and chemokines (11) can amplify the neutrophilic load and accelerate parenchymal damage through further release of proteinases.

A1AT: α-1-antitrypsin; ER: Endoplasmic reticulum; LTB4: Leukotriene B4;

MMP: Matrix metalloproteinase; PR3: Proteinase 3.

Figure 3. Mechanism of lung disease in α-1-antitrypsin deficiency.

Local deficiency of A1AT leads to excess unopposed neutrophil elastase activity (1). Elastase binds to macrophages stimulating release of leukotriene-B4 (2), a potent neutrophil chemotactic mediator. This promotes neutrophil recruitment (3), causing further release of neutrophil elastase and tissue damage (4). In addition, mutant A1AT polymers colocalize with neutrophils, retaining them in the interstitium and promoting further connective tissue degradation (5).

Figure 4. Declining carbon monoxide transfer coefficient (corrected gas transfer) over time, despite relatively stable forced expiratory volume in 1 s, in a patient with α-1-antitrypsin deficiency monitored in the UK registry.

FEV1: Forced expiratory volume in 1 s; KCO: Carbon monoxide transfer coefficient.

α-1-antitrypsin (A1AT) deficiency (A1ATD) is an under-recognized hereditary disorder first described by Laurell and Eriksson in 1963, after they linked the absence of an α1-globulin band on serum protein electrophoresis to the presence of premature emphysema [1]. A1AT is a 52 kDa, single-chain glycoprotein with a 394 amino acid sequence [2]. It is synthesized predominantly in the liver and functions as a serine proteinase inhibitor or serpin, providing essential protection to lung tissue against the actions of proteolytic enzymes such as neutrophil elastase (NE) and proteinase 3 (PR3).

The A1AT protein is encoded for by the SERPINA1 gene, which is composed of seven exons on the long arm of chromosome 14 (14q31–32.3) [2]. There have now been over 500 single nucleotide polymorphisms reported at this gene locus [201] (although many of these are yet to be validated) and inheritance occurs in an autosomal codominant manner. The traditional proteinase inhibitor (Pi) nomenclature uses alphabetical abbreviations to denote the speed of migration of the different allelic variants on gel electrophoresis [3]. The M allele is the ‘normal’ variant and the commonest deficiency variants are S and Z, which migrate more slowly. The Pi phenotype refers to the type of circulating A1AT identified by serum electrophoresis, whereas genotyping relies on the use of specific probes to identify abnormal allelic sequences. The majority of individuals are homozygous for the normal M allele. Rarely, individuals can also inherit ‘null’ alleles, which do not produce any detectable A1AT protein by routine quantitative methods and hence cannot be phenotyped. The most common deficient phenotype is PiZ, which encompasses both PiZZ and PiZ null genotypes. Deficiency of circulating A1AT is associated with multiple conditions including emphysema, hepatic cirrhosis [4], panniculitis [5], bronchiectasis [6] and vasculitis [7].

In this review, the authors focus on lung disease in A1ATD with respect to its underlying pathophysiology and factors affecting variability in disease severity and clinical phenotypes. The authors also discuss the methods of monitoring disease progression, current treatment strategies and the recent advances in the development of novel therapeutic agents.

Genetic epidemiology of A1ATD

The majority of individuals with ‘severe pathophysiological deficiency’, classified by a serum A1AT level below the putative protective threshold (11 µM), have the PiZZ genotype [8]. The highest prevalence of the Z variant is seen in northern Europe and it is thought to have arisen from southern Scandinavia [9]. The S variant distribution differs, as the highest gene frequency is seen in southern Europe in the Iberian Peninsula [10] and generally it leads to a mild deficiency not thought to be central to the pathophysiology of lung disease even when combined with a Z gene (SZ heterozygotes).

Recently, de Serres and Blanco used information from genetic epidemiological studies to develop estimates of the frequency of the two main deficiency alleles (PiS and PiZ) in 97 countries worldwide. More than 190 million individuals are estimated to have a deficient genotype, defined as having at least one of these alleles. In terms of severe deficiency, 0.1% of these individuals have the ZZ genotype with the majority living in Europe and North America [11].

Less is known about the so-called ‘rare’ A1AT variants (non-Z and non-S deficient). It is likely that many of these cases have historically been misclassified as having the PiZ variant due to their absence or low levels and difficulty in their identification using isoelectric focusing phenotyping methods. Genotyping using PCR and DNA sequencing, in combination with measurement of serum A1AT levels, is becoming the new gold standard for diagnosing A1ATD and several defects have been identified, including major gene deletions, point mutations and frame shift mutations leading to the absence or truncated forms of the protein with intracellular polymerization or degradation [12]. Our knowledge of the true prevalence of such rare variants and their associated clinical phenotypes is therefore likely to expand. A recent study of the prevalence of rare alleles in Spain highlighted the importance of standardization of methodology to obtain conclusive prevalence rates among different populations [13]. In 2007, the Alpha One International Registry, incorporating data from 21 countries, reported that 5.3% of its severely deficient patients had rare A1AT alleles [14]. Another more recent study evaluating A1AT serum protein concentrations in different A1ATD phenotypes found 6.7% (111 out of 1664) of subjects with two deficiency alleles had a rare variant [15]. They are known to be more common in certain regions of Italy, such as Sardinia, where there is a high prevalence of ‘Mmalton’. This mutation is characterized by the deletion of the entire codon for residue Phe52, leading to intracellular polymerization and lower serum levels [16]. Results from the Italian registry show a medium to low prevalence of the common PiZZ genotype and Ferrarotti et al. raised the interesting question of whether rare variants are more prevalent in populations with a lower PiZ gene frequency [17].

Screening

In 2003, a joint statement from the American Thoracic and European Respiratory Societies recommended genetic screening for all symptomatic adults with chronic obstructive pulmonary disease (COPD) or adult onset asthma with incompletely reversible airflow obstruction [18]. Indeed, the number of patients being tested is increasing annually in the USA [19]. However, the majority of severely A1AT-deficient patients predicted epidemiologically remain undiagnosed. The concept of population-based screening has been entertained, but at present, only targeted testing of high-risk groups is recommended [18,20]. The benefits of early diagnosis can include smoking cessation advice, avoidance of occupational exposures and opportunities for preconception genetic counseling. However, before undertaking widespread population screening, these benefits must be weighed up with the cost and impact on services of diagnosing a genetic disease in potentially asymptomatic individuals.

The molecular pathophysiology of A1ATD

Serpins such as A1AT are structurally composed of three β-sheets (A–C) and eight or nine α-helices (A–I) with an exposed mobile reactive loop containing residues that act as pseudosubstrates for the targeted proteinase [21,22]. In the case of A1AT, a methionine residue at position 358 in the polypeptide chain (Figure 1) is critical for the interaction with NE. The process of proteinase binding brings about a conformational change within the A1AT protein, whereby the enzyme cleaves the reactive center loop, which moves to the opposite pole of the protein taking the tethered proteinase with it, before being inserted into β-sheet A. The structural deformation of the proteinase that occurs as a result is key to the inhibitory function of the serpin [23]. Serpins have a metastable native state that becomes more stable during proteinase inhibition. This makes them prone to aberrant structural formation and protein misfolding as a result of genetic mutations [24].

Polymerization

The Z mutation occurs owing to the substitution of lysine for glutamic acid at position 342 in the polypeptide chain. The resulting structural change promotes the interaction of the reactive center loop of one molecule and the gap in the β-sheet A of another, causing molecular linkage or so called ‘loop sheet polymerization’ [25].

Yamasaki et al. challenged this polymerization theory when they used protein crystallography to assess the molecular structure of a stable dimer from plasma-derived antithrombin. They reported extensive ‘domain swapping’ of more than 50 residues and reported the intermolecular linkage included insertion of two β-strands (4A and 5A) of one molecule into the β-sheet A of another. They further demonstrated that A1AT polymers also formed via this domain-swapping mechanism. They postulated this as the reason for the hyperstability of serpin polymers [26]. However, in this study, A1AT polymerization was induced by guanidine hydrochloride, and Ekeowa et al. have subsequently demonstrated that 2C1 (a monoclonal antibody that recognizes pathological polymers of A1AT [27]) only recognizes heat-produced polymers not those produced by denaturants, questioning the validity of the data used to support this model of polymerization [28]. In addition, Ekeowa et al. used other methods including the use of synthetic peptides, limited proteolysis and mass spectrometry to help define the mechanism of A1AT polymerization and concluded that the single stranded loop-sheet model remained the best explanation. However, Yamasaki et al. have subsequently crystallized trimeric A1AT polymers (recognized by 2C1) and have now reported that linkage involves domain swapping of carboxy-terminal residues [29]. As the debate regarding the molecular basis of polymerization continues, clarification of the possible variation of linkage among the different mutations and serpins is needed if therapeutic polymerization prevention strategies are to be successful.

Intracellular accumulation of Z variant A1AT polymers within the endoplasmic reticulum (ER) of hepatocytes can lead to neonatal hepatitis, hepatic cirrhosis and hepatocellular carcinoma [30]. The resulting lack of circulating A1AT predisposes individuals to proteolytic attack of their lung tissue, resulting predominantly in emphysema. However, this explanation of the pathogenesis of lung disease in A1ATD is oversimplified as we now recognize that the manifestation relies on a complex combination of both pathogenic mechanisms (Figure 2) and other environmental and genetic factors.

Mechanisms of lung disease in A1ATD

Proteinase: antiproteinase theory

Our knowledge of the pathophysiology of emphysema in A1ATD started with the ‘proteinase:antiproteinase’ theory. Following the observations made by Laurell and Eriksson, investigation into why the inherited deficiency of A1AT would cause premature emphysema ensued. It was subsequently demonstrated that NE could reproduce changes in animal models suggestive of emphysema [31]. The development of this animal model and the recognition of A1AT as an NE inhibitor formed the basis of the proteinase:antiproteinase hypothesis, whereby imbalance in favor of NE leads to excessive tissue destruction and hence emphysema [32].

There is extensive literature on the role of NE in the pathogenesis of emphysema in A1ATD, but other proteinases may also be relevant. PR3 is also a serine proteinase found in the azurophil granules of neutrophils which also causes emphysema in animal models [33]. PR3 activity has been shown to be greater than that of NE in sputum of A1AT-deficient individuals especially during exacerbations, suggesting a role in the pathophysiology of COPD in these individuals [34]. Matrix metalloproteinases (MMPs; particularly MMP-12) and cysteine proteinases such as cathepsin B have also been implicated as having a direct role in proteolytic alveolar destruction [35,36]. Interestingly, NE has been shown to upregulate MMP-2 and cathepsin B expression in vitro [37] as well as inactivating their relevant inhibitors resulting in a proteinase cascade [38]. A better understanding of the interaction of different proteinases and their inhibitors in the pathogenesis of emphysema is needed as this could have important ramifications with respect to developing novel therapeutic agents.

Chemotactic mediators

High neutrophil counts have been observed in bronchoalveolar lavage (BAL) specimens from A1AT-deficient patients [39]. Neutrophils are key effector cells in airway inflammation and have the potential to accelerate parenchymal damage through release of their proteinases. Multiple factors affecting neutrophil recruitment and activation have been suggested. Leukotriene B4 (LTB4) is a potent neutrophil chemotactic mediator found in increased concentration in the sputum of A1AT-deficient patients [40]. Furthermore, studies of sputum chemotactic activity identified both LTB4 and IL-8 as significant contributors to chemotaxis [41], although only the former correlated with the total migratory potential. in vitro experiments demonstrated that LTB4 is released following the binding of alveolar macrophages and NE, supporting the concept that a proteinase imbalance could promote neutrophilic inflammation through the excess production of this chemokine [42]. A reduction in sputum LTB4 concentrations in A1AT-deficient individuals occurs in response to augmentation therapy, confirming this as a likely mechanism [43].

In a murine emphysema model, elastin degradation products found in BAL fluid have been shown to be chemotactic for monocytes. Using a monoclonal antibody to inhibit these elastin fragments eliminated the chemotaxis in vitro. Administration of the same monoclonal antibody at the time of cigarette exposure was also shown to reduce the accumulation of macrophages in the lung and abrogated the development of emphysema in vivo [44]. Since elastin degradation is likely to be a key process in the development of emphysema in A1ATD, it is possible that elastin fragments also play a role in amplifying neutrophilic inflammation but direct evidence is lacking.

A1AT polymerization & the lung

Z A1AT polymers have been identified in the BAL fluid from A1AT-deficient patients [45]. This extrahepatic polymerization may serve to further exacerbate the deficiency in these individuals, given the lack of functional antiproteinase activity of polymerized A1AT. The Z polymers have also been shown to activate neutrophils, manipulate neutrophil shape, promote adhesion and stimulate myeloperoxidase release in vitro [46]. Mahadeva et al. subsequently demonstrated that polymers of A1AT colocalize with neutrophils in the interstitium of PiZ individuals, which may also add to the connective tissue degradation (Figure 3). Furthermore, Mahadeva et al. also showed that instilling polymers into the lungs of mice resulted in a significant neutrophil influx, which may reflect a direct or indirect chemoattractant process [47].

Although the majority of A1AT is produced in the liver, it is also synthesized by other cells including bronchial and alveolar epithelial cells [48], supported by the presence of Z A1AT polymers in the BAL fluid of a patient 9 years post-liver transplant [49]. The potential proinflammatory properties of polymerized, locally produced Z A1AT in the lung may partly explain the progression of emphysema despite augmentation therapy in some individuals or the delay in efficacy seen in clinical trials [50,51] by perpetuating the inflammation [49]. Z polymerization is concentration dependent [25] and since the A1AT concentration in the blood is higher than in the lung, it is likely that the blood is the site of highest polymer concentration. The addition of MM A1AT would therefore dilute the Z protein and reduce polymer formation, although this would also require clearance of lung tissue polymers before their proinflammatory effect would be lost. Whether this would result in total abrogation of the inflammatory process with time remains unknown.

The importance of recognizing these so-called gain of function effects within the lung, including the concept of ER stress (brought about by the accumulation of misfolded Z A1AT protein in the ER) and the activation of associated inflammatory signaling pathways, has been emphasized in recent years [52]. ER stress can have multiple effects including NF-κB activation, promotion of ER-associated degradation and apoptosis [53]. The contribution of local ER stress to the pathogenesis of emphysema in A1ATD is an important concept as even augmentation therapy would not prevent this local effect and combined therapy with small molecules to prevent intracellular accumulation in lung cells may be required.

Clinical manifestations of A1ATD in the lung

Emphysema

The premature onset of emphysema was the first identified clinical manifestation of A1ATD described in 1963. The classical distribution has a lower zone predominance [54]; however, all zones can be affected. In one study of 102 A1AT-deficient patients, 65 (64%) had predominantly basal changes and 37 (36%) had a greater degree of apical emphysema [55]. It was also noted in this study that basal emphysema had a greater association with forced expiratory volume in 1 s (FEV₁) impairment than apical emphysema, confirming previous observations in usual COPD [56].

Bronchiectasis

Earlier reports of the incidence of bronchiectasis in A1AT-deficient individuals were as high as 43%, although based on small study populations [6,57]. In a larger study of 74 (PiZ) subjects, Parr et al. reported the incidence of ‘clinically relevant’ bronchiectasis to be 27%, which is similar to the reported incidence in usual COPD [58]. Using a different approach, Cuvelier et al. looked at the frequency of deficiency alleles in 202 patients with known bronchiectasis compared with a control group and concluded that there was no association. They did, however, note a higher Z allele frequency in bronchiectasis associated with emphysema [59]. Whether or not A1ATD is an independent risk factor for bronchiectasis or that bronchiectasis simply occurs in association with emphysema remains unresolved and requires a sufficiently powered case–control study with usual COPD [60].

Small airways disease & bronchodilator reversibility

A1ATD can be associated with varying degrees of airflow obstruction, even varying within individual patients. A study of 1052 subjects with A1ATD from the National Heart, Lung and Blood Institute Registry found that 82% reported symptoms of wheeze without a cold and 70% described ‘attacks of wheezing’ associated with breathlessness. The onset of these attacks was around 31 years of age, therefore the initial diagnosis of asthma given in young A1AT-deficient individuals is unsurprising. The study found that 49% of patients demonstrated significant reversibility at some stage of their follow-up, and the average increase in the FEV₁ was 382 ml (±180). Reversibility (defined as an FEV₁ increase of ≥12% and at least 200 ml postbronchodilator) was even seen in 12.5 % of patients with a normal FEV₁, suggesting bronchial hyper-responsiveness may be an early feature of the disease process [61]. Analysis of data from the UK and USA A1AT registries has shown that bronchodilator reversibility is associated with a more rapid decline in FEV₁ [62,63]. The difficulty in distinguishing asthma and COPD in A1AT-deficient patients is particularly challenging, and recognizing overlap between the two conditions is important to ensure appropriate therapeutic strategies are used to prevent an accelerated decline in lung function [64].

Clinical phenotypes

Emphysema and airflow obstruction often coexist; however, as seen in usual COPD, subsets of patients with either marked emphysema and preserved spirometry or severe airflow obstruction with relatively little parenchymal destruction are identifiable. The lung disease associated with A1ATD exhibits considerable heterogeneity, and the recognition of distinct clinical phenotypes is important as this may lead to more individualized therapy. This pathological disparity was first postulated by Parr et al., partly reflecting the distribution of the emphysema with apical disease having little effect on FEV₁ [55]. Holme and Stockley confirmed this in a prospective study of individuals identified with physiological discordance (normal FEV₁ and low diffusing capacity and vice versa) [65].

An observational study of 745 patients with severe A1ATD compared subjects with emphysema, chronic bronchitis and asthma overlap [66]. This study found chronic bronchitis patients were younger with a lower number of pack years and had better preserved lung function. A greater proportion of asthma overlap patients were women (55.2%) compared with the emphysema group (34.8%). This highlights the importance of patient characterization in the study of A1ATD. The implication for the pathophysiology of the disease and role of A1AT remains currently unknown.

Emphysema risk in A1ATD

A multivariate analysis of 378 severely deficient PiZ patients identified male sex, asthma, chronic bronchitis, pneumonia and cigarette smoking as risk factors for severe airflow obstruction (FEV₁ < 50%) [67]. Another study looking at lung function in a cohort of 101 PiZ subjects found FEV₁ decline to be more rapid in those with moderately severe disease, whereas carbon monoxide transfer coefficient declined quicker in severe disease. Within the moderately severe category, the rate of FEV₁ decline was associated with bronchodilator reversibility, exacerbation frequency, low BMI and male sex [63].

Considerable variability in the age of onset and severity of COPD in patients with A1ATD is well recognized. It is likely that this not only reflects environmental factors but also additional modifying genetic factors [68]. In support of this concept, a study assessing familial aggregation of COPD characteristics in 52 A1AT-deficient individuals and 117 relatives found a trend towards lower FEV₁ in PiMZ relatives of PiZ individuals with significant airflow obstruction compared with those without, suggesting the importance of other familial factors [69]. Indeed in the study by Wood et al., physiological discordance was also noted in PiZZ siblings [70]. Although there was concordance with gas transfer and emphysema on CT scan, the FEV₁ valued showed no concordance, indicating the A1ATD and lung disease link is far from simple. One possible explanation for this familial effect is gene-by-environment interactions, whereby family members inherit differing susceptibility to certain environmental factors such as smoking. A study of airflow obstruction in 378 A1AT-deficient patients found the heritability of FEV₁ to be greater only in analysis of smokers, suggesting the importance of at least gene-by-smoking interaction [71]. The nonlinear relationship between smoking and FEV₁ has recently been demonstrated in four large COPD study cohorts containing individuals with a wide range of airflow obstruction [72]. The authors advocated the use of a piecewise linear approach to model smoking in preference to the traditional pack years method when looking for gene-by-smoking interactions.

Whether or not PiMZ individuals are at increased risk of COPD has remained controversial. Heterozygotes for the Z allele have an A1AT level of approximately 60% of normal, which is believed to be above the protective threshold. A meta-analysis found case–control studies often demonstrated an increased COPD risk to PiMZ individuals, whereas cross-sectional studies generally found no difference [73]. A comparison between PiMM and PiMZ individuals from two large study populations containing a total of 4376 subjects demonstrated a 3.5% lower FEV₁/forced vital capacity ratio in the MZ subjects, suggesting a possible minor susceptibility to airflow obstruction [74]. It is possible that the presence of a small amount of proinflammatory Z polymers and the reduced function of Z A1AT could contribute to COPD risk in such individuals with a PiMZ phenotype [75]. However, many MZ individuals are identified through family studies of A1AT-deficient subjects or those with a family history of COPD, which may bias data towards other gene/environment influences.

In recent years, the search for additional ‘susceptibility genes’ has advanced owing to genome-wide association studies using an international collaborative approach to increase statistical power. Suggested modifier genes in A1AT-deficient individuals so far include NOS3 [76], GSTP1 [77], IL-10 [78], TNF-α [79], IREB2 and CHRNA3 [80]. Knowledge of how genetic and environmental interactions relate to phenotypes and severity of disease will improve our ability to assess risk of COPD in A1AT-deficient patients.

FEV₁, out with the old & in with the new?: biomarkers & alternative methods of monitoring disease progression

Traditionally, FEV₁ has been the gold standard for monitoring COPD progression; however, it has potential drawbacks. First, FEV₁ is effort dependent and can be significantly affected by day-to-day variability. Second, the spirometric normal range is wide, and it is recognized that the emphysema component of COPD can exist with ‘normal’ FEV₁ [81].

The EXACTLE trial demonstrated that computed tomography (CT) densitometry is a more sensitive indicator of emphysema progression than physiological and health status parameters [51]. A further post-hoc analysis of the data from this trial found that the lung density decline in A1ATD was reduced by intravenous augmentation with the greatest effect seen at the lung bases, questioning whether targeted sampling by this method is preferable to assessing the whole lung [82]. The procurement of satisfactory CT data for such complex analytical methods is likely to hinder the involvement of smaller centers in future large-scale augmentation studies.

In recent years, there has been much interest in the development of simpler biomarkers as alternative outcome measures for use in studies of potential disease-modifying therapeutic agents, although validation remains a major challenge. As a significant amount of damage must be done to airways before FEV₁ is altered, biomarkers that correlate with FEV₁ may be deemed reflective rather than predictive. The ideal biomarker should be central to the pathogenic process, predict progression and be responsive to treatments or episodes known to influence this [83]. The aim of the ECLIPSE study was to look for surrogate markers superior to FEV₁ in the longitudinal evaluation of COPD patients; however, severe A1ATD was one of the exclusion criteria for enrolment [84].

The measurement of elastin degradation products, such as desmosine/isodesmosine, has been investigated in both A1AT-deficient and usual COPD patients and does correlate with lung function [85]. Fregonese et al. measured the urinary and plasma desmosine levels in 11 A1AT-deficient individuals over a period of 14 months. Both were significantly higher compared with healthy controls, but they found wide variation in urinary values over time [86]. This was echoed in a study of 390 patients with usual COPD where urinary desmosine levels increased during exacerbations [87]. Plasma levels are more stable for longitudinal studies and identify patients with increased elastin degradation and may facilitate recruitment for targeted therapies [86,87].

Aα-Val360 (a NE-specific cleavage product of fibrinogen, which is generated when elastase is released) has been shown to correlate with severity of COPD as measured by FEV₁ in A1AT-deficient patients and is a sensitive marker of exacerbations and response to A1AT replacement therapy [88]. Whether it predicts progression in A1ATD remains unknown at present. Other suggested biomarkers in A1ATD include MMP-9 [89] and leukotriene B4 [43]. However, further longitudinal studies are needed to establish if any of these proposed biomarkers can satisfy all the criteria needed for the development of novel therapeutic strategies in A1ATD (Table 1) [83]. It is possible that no single biomarker will be able to achieve this but alternative uses may include panels of biomarkers to help define phenotypic subgroups of patients, which is a concept that has been investigated in usual COPD [90].

Treatment of A1AT-related COPD

The current mainstay of treatment of A1AT-related COPD is similar to that of usual COPD [18]. This involves the use of inhaled bronchodilators and steroids, pulmonary rehabilitation, long-term oxygen therapy, antibiotics and/or steroids during exacerbations in addition to smoking cessation advice and preventative influenza and pneumonia vaccinations.

In the search for potential disease-modifying drugs for emphysema, interest in retinoids arose as all-trans-retinoic acid was shown to reverse emphysematous lung damage in rats [91]. A randomized, double-blinded, placebo-controlled trial investigating the efficacy of palovarotene (an oral γ-selective retinoid agonist) in 262 patients with severe A1ATD did not, however, show any significant benefit with respect to changes in lung density or functional parameters after 1 year of therapy [92].

The potential to reduce proteolytic lung damage through the use of synthetic NE inhibitors has also been explored. However, the lack of clinical benefit in usual COPD [93] and other conditions such as cystic fibrosis [94] and acute lung injury [95] have meant that trials in A1ATD have not been undertaken.

The evidence for unilateral and bilateral lung volume reduction surgery (LVRS) is limited to a number of case series. Compared with the outcomes in non-A1ATD-related emphysema, the magnitude of improvement seen post-LVRS appears to be less and is not sustained for the same length of time [96–98]. A recent case series assessing endobronchial valve placement as an alternative to surgical LVRS demonstrated a median FEV₁ improvement of 0.575–0.905 l (p = 0.028) with an associated improvement in BODE index [99]. Studies with larger numbers and longitudinal data will be needed before any firm conclusions can be drawn of long-term benefit but this approach may play a role in bridging younger patients to transplant. However, caution should be exercised as the results of a randomized trial of patients with usual COPD showed modest improvements in lung function following endobronchial valve placement at the expense of an increased risk of complications including hemoptysis, pneumonia and increased exacerbation frequency [100].

Intravenous augmentation therapy: the current understanding

Intravenous administration of A1AT derived from pooled human plasma is only available in some countries as doubts remain over its efficacy and cost–effectiveness. The US FDA have approved the following products in the USA: Prolastin^®, Prolastin-C^® (Grifols, Barcelona, Spain), Aralast^™, Aralast NP^™ (Baxter, IL, USA), Zemaira^® (CSL Behring, PA, USA) and GLASSIA^™ (Baxter). Other products available elsewhere include Trypsone^®/Trypsan^® (Grifols) and Alfalastin^® (LFB, Les Ulis, France).

Augmentation therapy can certainly increase and sustain serum levels above the accepted protective threshold (11 µM, 80 mg/dl or 57 mg/dl by nephelometry) at a dose of 60 mg/kg plasma-derived A1AT once weekly and increase the anti-NE capacity in the epithelial lining fluid of the lungs [101].

Evidence regarding the clinical efficacy of augmentation therapy is less clearly defined. The majority of evidence is based upon observational cohort studies as only two small randomized controlled trials investigating this have been published to date. A reduction in mortality risk was reported in patients receiving augmentation therapy in the large observational study of 1129 participants followed up over a 3.5- to 7-year period. However, overall FEV₁ decline was not altered, although in the subgroup with moderate FEV₁ impairment (FEV₁: 35–49% predicted), a significantly slower decline was seen in those receiving augmentation [62]. However, it should also be recognized that FEV₁ is generally a poor surrogate for emphysema. In addition, FEV₁ decline is not linear and seems to be fastest in the moderately impaired range [63]. This would be the range where evidence of efficacy (using FEV₁ as the outcome) would be easiest to detect. Nevertheless, this range of FEV₁ has been widely adopted as the indicator for augmentation therapy as a result of this observation. Other supportive evidence of efficacy comes from comparing the FEV₁ decline in cohorts from countries with and without therapy [102], sequential studies on FEV₁ decline before and after therapy [103], and meta-analyses of spirometric decline in A1ATD from other published cohorts [104]. However, interpreting observational data can be influenced by healthcare delivery systems and other socioeconomic factors both between and within countries and nonlinearity of FEV₁ decline and FEV₁ remains relatively insensitive to the emphysema disease process. Not only can tissue destruction occur even when the FEV₁ is stable [105] but progressive loss of gas transfer can also be detected (Figure 4) and seems to be greatest when the FEV₁ is at its lowest [63]. These data suggest that concentration of effort on measuring FEV₁ (though simple) only provides a superficial view of the disease process and hence treatment decisions.

The first randomized control trial by Dirksen et al. involved four weekly infusions of either A1AT or albumin. The outcome measures included lung function and quantification of emphysema using CT densitometry. No significant effect on pulmonary function was seen, but CT data showed a trend towards protection against loss of lung tissue in the augmentation group [50]. A similar trend was seen in the second randomized trial, which used a more conventional augmentation regimen involving weekly infusions of 60 mg/kg of Prolastin. The primary analysis of this study also failed to achieve conventional levels of statistical significance, although one of the analysis methods did. In addition, although there was no effect on exacerbation frequency, augmentation did reduce hospital admissions [51]. Subsequent analysis of the CT data did indicate a significant benefit on lung density decline if only the lower zone (where the emphysema is classically localized) was assessed [82]. In addition, despite the differences in methodology, an integrated analysis of the results of these two trials was performed and demonstrated a significant reduction in the decline of lung density in subjects treated with A1AT replacement (p = 0.006) [106], suggesting that both individual studies were underpowered.

The acceptance of such data as evidence of efficacy, although not ideal, may be necessary given the challenges to performing a suitably powered double-blind randomized trial. These include the relative rarity of the disease, ethical justification for intravenous placebo arms, high cost and need for a long duration of follow-up [106,107] and, importantly, experienced centers to ensure consistency of acquisition of densitometry data. A double-blinded, randomized trial of Zemaira (CSL Behring) versus placebo with a 2-year follow-up period has recently been closed to recruitment but the outcome has yet to be analyzed [202].

Some studies have focused on the short-term effects of augmentation therapy by measuring markers of neutrophil recruitment, inflammation and proteinase activity. One such study demonstrated a significant reduction in the sputum concentrations of LTB4 (a neutrophil chemoattractant) in response to augmentation therapy [43]. Whereas this is supportive of an anti-inflammatory effect, it cannot support clinical efficacy.

The variability of lung function and damage even in smokers with A1ATD indicates that management should be individualized. Smoking cessation slows or stops progression and physiology can decline even when the FEV₁ remains stable. Thus, assuming augmentation therapy is effective, it will remain critical to assess progression accurately through a variety of methods and only instigate therapy when clear evidence of decline, above age-related changes, is shown to occur.

Exacerbations

Exacerbations in A1ATD are associated with greater inflammation and elastase activity than in usual COPD [108]. These episodes relate to physiological decline even in patients on augmentation therapy [109]. This suggests that such episodes should be treated perhaps with bolus intravenous augmentation or even inhaled A1AT to boost anti-elastase screen in the airways affected by the exacerbation. Although logical, the practicalities of designing and delivering such a study may prove insurmountable.

Novel therapeutic approaches

Inhaled replacement therapy

Intravenous augmentation via weekly infusions is both expensive and inconvenient for patients. This has promoted interest in finding alternative routes of administration such as inhalation. Superficially, apart from the convenience aspect, there may also be perceived benefits of delivery of A1AT directly to the target organ as only 2–3% of A1AT given intravenously reaches the lung [110]. Preliminary studies demonstrated that the aerosol approach can successfully augment the anti-NE capacity of the respiratory epithelium and inhaled A1AT can pass into the lung interstitium as trace amounts can be detected in the systemic circulation [110,111]. However, protecting against the interstitial damage leading to emphysema poses significant logistical problems. First, the A1AT has to reach the alveolar region in sufficient amounts and this is highly unlikely using conventional nebulizers particularly in areas already affected [112,113]. It could be argued that it is more crucial to protect areas with the most preserved function, although particle size and dose remain challenges to overcome. Even if sufficient amounts of the drug can be deposited onto the alveolar surface, the epithelial integrity will largely prevent access to the interstitium [114]. Nevertheless, it is possible that reducing the activity of elastase in the alveolar space could decrease LTB4 production by macrophages (see earlier). This in its own right would reduce polymorphonuclear neutrophil traffic and hence indirectly protect the interstitial connective tissue. Alternatively, nebulizer drug may be more beneficial for airways disease in those patients with little emphysema but acute or recurrent exacerbations. Indeed, a multicenter, double-blinded randomized controlled trial evaluating the safety and efficacy of inhaled human A1AT is currently underway [203]. Interim reports indicate good safety and tolerability, although definitive results will not be available for approximately 12 months.

Recombinant/transgenic augmentation

The cost and finite supply of human plasma-derived A1AT products, and potential infection risks have prompted research into alternative recombinant and transgenic sources. The A1AT gene has been expressed in a large variety of hosts [115]. Transgenic production of human A1AT has been achieved in mice, rats, rabbits, sheep and goats [116–120]. Spencer et al. raised concern regarding antibody responses seen in some recipients due to impurities in the aerosolized human A1AT produced from the milk of transgenic sheep [121]. The potential for immune-mediated responses to transgenic proteins has currently halted the clinical development of these products. Another issue that has been highlighted is the stability of these recombinant proteins [115]. Problems with absent or aberrant glycosylation can lead to conformational abnormalities, protein misfolding and aggregation with subsequent functional impairment and exposure of immunogenic epitopes normally hidden. Lack of glycosylation also leads to the rapid clearance of recombinant glycoprotein from the circulation [122]. The degree of sialylation of the terminal glycans of the A1AT glycoprotein is essential for its plasma survival [123]. In order to achieve the necessary degree of sialylation in recombinant A1AT derived from an human embryonic cell line (PER.C6), Ross et al. have cotransfected the human A1AT cDNA with a single recombinant human sialyltransferase gene. They reported that the recombinant A1AT produced by selected cell subclones is pharmacokinetically similar to plasma-derived A1AT and has equivalent functional properties in vitro [124]. This emerging technology may yet result in an effective transgenic form of A1AT, although caution and long-term safety studies will be essential.

Polymerization prevention strategies & chemical chaperones

The loop-sheet polymerization mechanism of the Z mutation promotes interaction between the reactive center loop of one A1AT molecule and the gap in the β-sheet A of another. The resulting polymers lead to aggregation in hepatocytes and subsequent plasma deficiency [25]. A strategy to try and prevent polymerization has been the use of small peptides to compete with reactive loops and bind to β-sheet A [125–129]. Although in vitro tests are promising, challenges to this approach being translated to humans have included how to deliver the peptides to the ER of hepatocytes and the fact that they inactivate Z A1AT [127], therefore potentially worsening plasma deficiency, emphysema risk and progression. Nevertheless, such an approach would certainly be protective of the ER stress caused by polymerization and thought to predispose liver damage and failure.

The discovery of a distinct hydrophobic cavity in the crystal structure of A1AT that is only patent in its nonpolymerized form has provided a further target for drugs preventing polymerization [130,131]. Mallya et al. have performed virtual ligand screening on 1.2 million small molecules to try and identify compounds that could target this cavity and prevent polymer formation in vitro. The leading identified compound named CG was shown to reduce the intracellular accumulation of Z A1AT by 70% in murine hepatoma cells [132], although effects on A1AT function have not been reported.

An alternative approach is the use of synthetic chaperones with the aim of stabilizing the intermediates on the Z A1AT folding pathway and 4-phenylbutyric acid is the most studied chaperone to date. Animal studies were initially encouraging [133] but unfortunately human trials have been disappointing so far. In a preliminary open-label study, ten patients with severe A1ATD were administered oral 4-phenylbutyric acid for 14 days, which caused considerable side effects and did not significantly alter their circulating A1AT level [134].

Gene therapies

Interest in targeted gene therapies to treat A1ATD is gaining momentum. The classical approach of inserting normal genes into cells of patients with the genetic mutation is being investigated in the hope of achieving therapeutic A1AT levels and hence preserving lung function. The first clinical trial in humans involved delivery of the normal A1AT gene in a plasmid–cationic liposome complex into the nasal mucosa [135]. Transient increases were seen in the A1AT levels in the nasal lavage specimens but they returned to baseline by day 14. The results using recombinant adeno-associated virus (rAAV) vectors have been more promising. A nonrandomized, open label, Phase II clinical trial evaluating the safety and efficacy of an rAAV vector is currently ongoing. The aim is to produce sustained serum A1AT levels above the protective threshold (>11 µM), which has not been achieved so far. No gene therapies are currently licensed for use in A1ATD. Details of all the clinical trials to date can be seen in Table 2 [135–139]. However, importantly, gene therapies utilizing this approach will not prevent the production of the defective A1AT Z protein and, therefore, will have no impact on A1AT-related liver disease caused by defective protein accumulating in hepatocytes.

Genetic approaches to preventing Z A1AT liver disease include strategies to inhibit the translation of the mutant gene. Initial attempts to do this have utilized ribozymes to cleave PiZ mRNA [140,141]. In one animal study, a vector system based on simian virus 40 was used to deliver ribozyme directly to the liver of transgenic mice carrying the mutant human A1AT PiZ allele via portal vein catheters. This resulted in marked decreases of human A1AT mRNA and protein in the liver [142]. More recently, studies have looked at the ability of siRNA to downregulate endogenous A1AT production. siRNAs are double-stranded RNA molecules that play a role in post-transcriptional gene silencing. Cruz et al. used adeno-associated virus vectors expressing siRNA packaged in AAV8 capsids to transduce the livers of transgenic mice. A significant reduction in circulating human Z A1AT levels and clearance of Z A1AT protein from hepatocytes was seen within 3 weeks of vector administration [143]. in vivo toxicity has been seen in mice treated with high doses of short hairpin RNA vectors, highlighting the importance of preclinical studies in determining potential risks associated with gene therapies [144]. Again, this approach, although protecting the liver, will result in a further reduction in plasma A1AT, reducing the potential protection of the lung.

For these reasons, the ideal gene therapy would not only knock down the PiZ protein but also increase M-A1AT protein production as a potential treatment for both liver and lung disease. Following reports that RNAi-associated toxicity can be avoided by using miRNAs as opposed to short hairpin RNAs, Mueller et al. created miRNAs that target the human A1AT gene. Using the same rAAV vector, they incorporated miRNA sequences that target the PiZ A1AT gene, while also driving the expression of a wild-type PiM gene (which is resistant to the miRNA-mediated knockdown). This had the desired effect in transgenic mice of reducing circulating Z A1AT and increasing secretion of M A1AT [145]. Such an approach would therefore both protect the liver by switching off the Z protein translation and subsequent polymerization while secreting normal M protein to protect the lung. The complexities and safety of human therapy have yet to be addressed.

A final possibility involves the technique of gene correction. As the Z mutation is known, the addition of the correct M sequence as small DNA fragments lead to recombination and sequence correction. This approach has been used in several single-gene diseases and has been shown to be effective in cells from A1AT-deficient patients with the Z defect [146]. Again, the practicalities and safety of human delivery and efficacy have yet to be addressed.

Stem cell

For a gene therapy to be successful, sustained gene expression is essential. Given their ability to self-renew and capacity for differentiation, the concept of using stem cells as a delivery platform is therefore an attractive prospect. In 2008, Wilson et al. described the approach of A1AT gene transfer by transplanting lentivirally transduced hemopoietic stem cells into mice, demonstrating that this can achieve sustained human A1AT expression in vivo [147].

Other stem cell strategies have focused on utilizing mesenchymal stem cells. Ghaedi et al. differentiated mesenchymal stem cells to produce hepatocyte-like cells, which were subsequently genetically modified to provide a potential in vitro source of A1AT gene containing hepatocytes for autologous transplantation [148]. Other studies have used lentiviral and rAAV vectors to transduce bone marrow and adipose-derived stem cells before transplanting them into mouse models with promising results [149,150].

Induced pluripotent stem cells (IPSCs) are differentiated or ‘adult’ cells that are genetically reprogrammed to an embryonic-like state [151]. The potential to generate unlimited cells for autologous cell-based treatments for a variety of diseases is an exciting prospect in this rapidly advancing field of research. Yusa et al. have successfully used a combination of zinc finger nucleases and piggyBac technology to correct the point mutation in human IPSCs derived from individuals with A1ATD [152]. Furthermore, they demonstrated that these corrected IPSCs can differentiate into hepatocyte-like cells in vitro which can function in vivo when transplanted into the liver of a mouse model. This approach could potentially provide sustained A1AT gene expression. However, the risk of other mutations arising during prolonged culture of the IPSCs was highlighted and careful screening would be essential for their safe clinical use long term.

Expert commentary

As awareness of A1ATD increases, a growing number of people are being diagnosed with this hereditary condition and its associated complications. Since the original discovery of A1ATD and the classical association with basal panacinar emphysema, much has been learnt about the condition. We now have a better understanding of the complex interactions underlying the pathophysiology of lung disease in these individuals. We also recognize that presentation with lung or liver disease is variable and requires further epigenetic studies to identify relevant features. The variability of lung pathology (airways versus alveolar disease), distribution of emphysema and methods of determining progression will provide further insight into who, when and how to treat the lung disease. Currently, the mainstay of treatment for A1ATD-related lung disease is similar to that of usual COPD with the addition of intravenous augmentation (in some countries) but as we have described, new treatment paradigms are evolving.

Five-year view

In the next 5 years, new ways of generating and delivering A1AT need to be clarified for both efficacy and safety.

Alternative potential ways to treat the deficiency need exploration to determine methods of drug or gene-modifying treatment deliveries. Of importance will be genetic and biomarker studies that will predict individuals at risk for developing the clinical manifestations and progression in A1ATD. This will lead to more personalized treatment strategies.

Table 1. Current status of validation of selected biomarkers in α-1-antitrypsin deficiency.

Biomarker validation criteria	Biomarker
	Desmosine	Aα-Val360	MMP-9	LTB4
Central to the pathophysiological process	✓	✓	Evidence limited	✓
Relates to disease severity	✓	✓	✓	✓
Is stable and only varies with events known to relate to disease progression	✓	✓
Predicts progression	Increasing levels associated with DLCO decline in small study (11 patients)		✓
Is sensitive to intervention factors that are known to be effective		✓

DLCO: Diffusing capacity of the lungs for carbon monoxide.

Table 2. Clinical trials of gene therapy in α-1-antitrypsin deficiency utilizing different vectors of administration.

Study (year)	Phase	Vector	Administration route	Patients (n)	Main results/conclusions	Ref.
Brigham et al. (2000)	I	Plasmid–cationic liposome complex	Intranasal	5	In the A1AT-transfected nostril nasal lavage fluid:	[135]
					A1AT protein concentration increased, peaking at day 5 (at a third of normal) and returning to baseline by day 14
					IL-8 concentration decreased
Brantly et al. (2006)	I	rAAV2-A1AT	im.	12	One subject (who had not received intravenous augmentation) had detectable subtherapeutic expression of wild-type M-A1AT in the serum (82 nM) 30 days after receiving a dose of 2.1 × 10^13 vg	[136]
					Anti-AAV2 capsid antibodies rose after vector injection (residual M-AAT levels from prior augmentation obscured the results for some patients)
Brantly et al. (2009)	I	rAAV1-A1AT	im.	9	In highest dose cohort receiving 6.0 × 10^13 vg, expression of wild-type M-A1AT seen in two subjects at subtherapeutic levels (0.1% of normal) at 1 year	[137]
					Anti-AAV capsid antibodies developed and IFN-γ ELISpot to AAV1 capsid was positive in all patients by day 14
Flotte et al. (2011)	II	rAAV1-A1AT (herpes simplex virus production method used [138])	im.	9	Interim results:	[139]
					Dose-dependent increase in M-A1AT levels seen
					In highest dose cohort receiving 6 × 10^12 vg/kg serum M-A1AT levels peaked on day 30 with a mean value of 572 nM but this is still subtherapeutic
					Transient creatinine kinase rises seen in higher dose cohorts

ELISpot: Enzyme-linked immunosorbent spot; im.: Intramuscular; rAAV: Recombinant adeno-associated virus; vg: Vector genome.

Key issues

• • α-1-antitrypsin (A1AT) is a serine proteinase inhibitor that protects the lung from damage by proteolytic enzymes such as neutrophil elastase.

• • A1AT deficiency (A1ATD) is an under-recognized hereditary disorder associated with the premature onset of chronic obstructive pulmonary disease. Genotyping is becoming the gold standard diagnostic test.

• • The pathophysiology of lung damage in A1ATD is complex, involving the interaction of multiple inflammatory pathways.

• • We are beginning to identify other genetic factors that may contribute to emphysema risk in A1ATD.

• • Computed tomography densitometry is the most sensitive measure of emphysema and its progression.

• • There is growing interest in the development of biomarkers in A1ATD. The most promising candidates include desmosine, Aα-Val360, matrix metalloproteinase 9 and leukotriene B4, but further investigation and validation is still needed.

• • There remains a lack of suitably powered double-blind randomized trials proving efficacy of intravenous augmentation. Supportive evidence is largely based on observational cohort studies.

• • New paradigms of treatment are continuing to evolve including inhaled A1AT replacement, the use of recombinant/transgenic sources of A1AT for augmentation, polymerization prevention strategies and gene and stem cell therapies.

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Recent advances in α-1-antitrypsin deficiency-related lung disease

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1. You are seeing a 30-year-old woman who was recently diagnosed with alpha-1-antitrypsin deficiency (A1ATD). What should you consider regarding the pathophysiology of this disease?

• □ A Most patients with severe deficiency of A1AT have the PiZZ genotype

• □ B Bronchoalveolar lavage usually yields normal counts of inflammatory cells among patients with A1ATD

• □ C Leukotriene B4 (LTB4) levels are reduced in the sputum of patients with A1ATD

• □ D Elastin degradation products function as the principal trigger for the inflammatory response in A1ATD

2. What should you expect regarding clinical manifestations of A1ATD in this patient?

• □ A A1ATD primarily affects the apices of the lungs

• □ B Apical emphysema is predictive of a lower forced expiratory volume in 1 second (FEV1) vs basilar emphysema

• □ C Only 5% of patients demonstrate bronchodilator reversibility at any point in their disease

• □ D Bronchodilator reversibility is associated with a more rapid decline in FEV1

3. Which of the following options is the best means to predict the progression of emphysema in this patient?

• □ A Computed tomography (CT) densitometry

• □ B Her age

• □ C Her current treatment regimen

• □ D Pulmonary function testing

4. Which of the following statements regarding the treatment of this patient is most accurate?

• □ A Intravenous augmentation therapy with A1AT has stronger evidence of physiological vs clinical benefits

• □ B Retinoids are a promising line of therapy

• □ C Lung volume reduction surgery appears particularly effective for patients with A1ATD

• □ D Currently available commercial gene therapy can improve both pulmonary and liver outcomes among patients with A1ATD