Adam Wanner, MD

University of Miami Miller School of Medicine – Miami, FL

Alpha-1 Foundation – Miami, FL

Approximately six percent of the population has COPD, and COPD has become the third leading cause of death in the United States¹. Although the awareness of COPD as a global health problem is growing and although there has been a recent surge in COPD research, spawned by industry and government funding agencies, our understanding of the pathogenetic mechanisms underlying this highly prevalent disease remains fragmentary. As a result, disease-modifying treatment is still elusive. Inhaled smoke, especially cigarette smoke, has been identified as the major cause of COPD, but it is not clear why only a minority of those exposed or exposing themselves to smoke develops clinical COPD. Genetic predisposition comes to mind as a possible explanation for this phenomenon, and the study of COPD genetics therefore is in full swing^2,3.

The lung disease of patients with alpha-1 antitrypsin (AAT) deficiency resembles COPD, including emphysema, chronic bronchitis and bronchiectasis. However, in contrast to COPD, the genetic defect that is responsible for and the biology of the structurally and functionally defective alpha-1 antitrypsin protein that is linked to lung disease have been well characterized in AAT deficiency^4-7. Furthermore, strides are being made to discover additional gene mutations that seem to be needed for the full expression of the AAT deficiency phenotype. In this sense, AAT research has had it easier than generic COPD research, and this is exemplified by the impressive advances that have been made in our understanding of the physiopathological consequences of the misfolded and polymeric alpha-1 antitrypsin protein, the basic defect that is responsible for both the liver and lung disease of AAT deficiency^8,9.

Historically, the concept that an imbalance between proteases and antiproteases is an important contributing factor to the pathogenesis of COPD in general arose from the discovery of AAT deficiency and its association with COPD⁹. Subsequent research showed that exogenous proteolytic enzymes cause lung damage in experimental animals that resembles human emphysema as seen in COPD, that human neutrophil elastase is inhibited by AAT, and that cigarette smoke oxidizes critical residues in AAT thereby compromising its anti-elastase function^10-14. These observations were used to explain why individuals with AAT deficiency are especially susceptible to cigarette smoke induced lung disease. But the interaction between cigarette smoke and AAT also suggested that it contributes to the protease-antiprotease imbalance in smokers without AAT deficiency who develop COPD.

One could argue that the protease-antiprotease principle has had its day in the scientific world and that no other pathogenetic links should be expected between AAT deficiency and COPD. New discoveries tend to refute this argument. For example, it has now been shown that the unfolded AAT protein forms polymers, and that polymeric AAT made by lung cells or reaching the lung through the blood circulation can lead to the local release of chemokines and the recruitment of inflammatory cells to the lung, thereby contributing to neutrophilic inflammation, characteristic of COPD^15-17. Another consequence of the unfolded, polymeric AAT protein is that it accumulates in the endoplasmic reticulum of hepatocytes and to a lesser extent lung epithelial cells and cannot be effectively secreted into plasma; hence the name AAT deficiency. The intracellular aggregation of unfolded AAT initiates a brisk unfolded protein response that involves caspase-3 and apoptosis¹⁸. Apoptosis of lung epithelial cells has been shown to participate not only in the pathogenesis of the lung disease associated with AAT deficiency but also in the pathogenesis of COPD in general¹⁹. Since tobacco smoke can induce the unfolded protein response and disposition of unfolded proteins that are present in low abundance in lung cells of subjects without AAT deficiency, the pro-apoptotic pathways that have been identified in AAT deficiency could shed light on the development of COPD as well²⁰.

Another lesson to be learned from AAT research is the use of AAT as an anti-inflammatory and anti-apoptotic molecule in the treatment of COPD unrelated to AAT deficiency. In other words, exogenous AAT could be thought of as a therapeutic agent, not only as a replacement as in AAT deficiency. There is growing evidence for this paradigm. Human AAT inhibits the inflammatory activity of human monocytes in vitro, and this effect is still seen with modified forms of AAT that lack anti-protease effects²¹. With respect to the anti-apoptotic actions of AAT, it has it been shown in a mouse-model that AAT inhibits apoptosis-dependent lung destruction that is not caused by AAT deficiency¹⁸, and a recent paper has also demonstrated that AAT attenuates cigarette smoke induced apoptosis in vitro²². In addition to these anti-apoptotic actions in vitro and in animal models of lung disease, AAT appears to have broad anti-inflammatory effects in humans. Thus, treatment of patients with cystic fibrosis with aerosol AAT has been reported to reduce sputum neutrophil numbers, IL-8 concentration and unopposed elastase activity²³. As a serin protease inhibitor, it is not surprising that inhaled AAT attenuated free elastase activity in the lung. More significant was the effect of AAT on IL-8, a chemoattractant for neutrophils, and on neutrophil recruitment to the lung. Inasmuch as neutrophils are thought to have a major role in the pathogenesis of COPD, one might consider exploring the clinical benefit of AAT in COPD. Currently, only intravenously administered AAT is available for the purpose of augmentation therapy in AAT deficiency. Intravenous AAT is hardly a therapeutic option in patients with generic COPD. However, with the likelihood that aerosol AAT will soon become available for clinical use, the idea of treating cystic fibrosis and COPD with AAT to prevent disease progression may not be far fetched.

If the protease-antiprotease principle, which was fueled by the discovery of AAT deficiency, initiated decades of COPD research, why couldn't other scientific observations derived from AAT research continue to fertilize the study of COPD? Perhaps a renewed interest in the study of the AAT deficiency could again inform COPD at large, as it has in the past. Such research could identify novel drug targets for the treatment of COPD associated or not associated with AAT deficiency. From a scientific perspective it may no longer be acceptable to think of AAT deficiency as a rare disease that is of little interest to investigators studying COPD, to COPD research funding agencies, to the pharmaceutical industry, and ultimately to patients who suffer from COPD.

Adam Wanner, MD

Promoting research with the vision of finding better treatments and ultimately a cure for alpha-1 antitrypsin deficiency (AATD) is a core mission of the Alpha-1 Foundation. Investigator-initiated grants have been the driving force of the Foundation's research program from its inception in 1999, based on the time-tested principle in biomedical discovery that innovative ideas typically arise in the research community. Of the 31 million dollars spent on research over the decade between 1999 and 2008, a significant portion has been directed at the peer-reviewed annual "in-cycle" investigator-initiated grants program, with the remainder used to support scientific conferences, and the DNA & Tissue Bank and Research Registry as resources for basic and clinical researchers, respectively.

Foundation-sponsored research has had a significant impact on our understanding of the mechanisms leading to the clinical manifestations of AATD, and most of this information has been obtained through investigations supported by the "in-cycle" grant program, recently complemented by Foundation initiated requests for applications (RFA) to address issues deemed important but somewhat neglected by the research community. This document is meant to summarize the scientific contributions of investigators supported by the "in-cycle" grant program between 1999 and 2008.

Today we know the genetic mutations responsible for the unfolded alpha-1 antitrypsin protein (AAT), generally understand how the unfolded AAT polymerizes and leads to cellular processes that injure the liver and the lung (gain of function), and how the defect in AAT secretion into the blood circulation can expose the lung to the elastolytic actions of neutrophil elastase and other serine proteases with resultant development of COPD (loss of function). From clinically oriented studies that have complemented these discoveries, we have learned more about social aspects and the management of the disease. The areas of research that have contributed to this body of knowledge include basic liver research, basic lung research, clinical and epidemiological research, research to promote the detection of AATD, research into the social and ethical aspects of the condition, and new treatment modalities at basic or clinical levels (Appendix 1). The investigations summarized below are organized accordingly. Of the 99 grants awarded to scientists in and outside the US, 55 address the pathogenesis of AATD, 22 are clinical studies, and 24 deal with novel therapeutic solutions, with overlaps in some of the investigations. The AATD-related publications by Foundation grant recipients' titles of the funded grants are listed in Appendix 2. A few of the grants did not lead to a meaningful result although negative results can often be of scientific importance as well; however those studies they are not included in the summary.

Basic research that addresses the pathogenesis of liver disease

Polymerization of AAT

Intracellular polymerization of unfolded ZZ AAT is believed to have a pivotal role in the pathogenesis of AATD by leading to cytotoxic processes in cells that produce AAT at high rates, notably liver cells. The process of polymerization was investigated by Merszal and by Krishnan. Although those studies suggested that special proteins could promote ZZ AAT polymer formation, their identity has not yet been established, and the precise mechanism of polymerization remains to be clarified. Possibly the polymerization of the mutated ZZ AAT is directly related to the misfolding of the protein and is not a regulated process.

Cell lines and animal models

Basic research in AAT related liver disease requires the availability of cell lines and animal models that mimic the human condition. Zern developed a human liver cell line for cell transplantation and bioartificial assist devices with novel therapeutic approaches for AATD in mind. Theoretically, such a cell line could be engineered to reflect the liver cells of individuals with AATD by ZZ AAT gene transfection. Brantly and colleagues were able to generate a hepatocyte in which native AAT production was knocked down with siRNA technology. While the model did not reflect the naturally occurring Z mutant hepatocyte, as intracellular ZZ AAT accumulation and its hepatotoxic effects were not reproduced, it established the feasibility of silencing the AAT gene in the study of AAT deficiency. Brenner reported the development and functional characterization of a combined homozygous CFTR and heterozygous ZZ knock-in cell line to study interactions between cystic fibrosis and AATD. Steffen went as far as isolating proteasome complexes from hepatocytes and found phosphokinase A and phosphatase 1 to be associated with them. It is not clear what the role of these might be in the disposal of ZZ-AAT in proteasomes. Although several attempts at developing transgenic animal models of AATD failed (e.g. Houghton reported that AAT gene deletion was embryonically lethal in mice), targeted mutagenesis using human ZZ gene transfection was successfully used in mice in some of the studies summarized here.

Trafficking and disposal of AAT

The liver disease of AATD is critically linked to abnormal trafficking and disposal of the unfolded ZZ AAT protein. It therefore is not surprising that the study of these processes has been at the center of AATD liver research. In 2001, Pearlmutter et al. used gene chip analysis to conduct a genomic analysis of proteins involved in AAT degradation in model cell cultures. The researchers found that ZZ AAT retained in the endoplasmic reticulum (ER) upregulates 7 transcripts including AAT and that transfecting normal liver cells with human ZZ AAT upregulates the unfolded protein response and heat shock proteins. Brantly confirmed those findings by demonstrating differential expression of genes involved in protein folding, degradation, apoptosis and post-translational modification in cells expressing MM AAT and ZZ AAT. These observations are important in that they link the up-regulated unfolded protein response to apoptosis, a concept that now has been widely accepted in the hepatotoxicity of AATD. One wonders if these differences in gene expression are a consequence of the upregulated unfolded protein response or reflect modifier genes that may determine the severity of liver disease in AATD. A number of proteins involved in AAT intracellular trafficking and degradation have been identified. These include L-6, an AAT cargo protein that transports AAT to the Golgi apparatus (Conkright), selenoproteins that attenuate the ZZ AAT-induced unfolded protein response (Greene), fibrinogen beta/gamma chains that decrease cellular ZZ AAT levels through a mechanism that remains to be clarified (Glenn), ADD66 that promotes proteasome-associated ZZ AAT degradation (Brodsky), and various ribozymes that can cleave ZZ AAT (Grabowski).

A highly significant contribution to our understanding of ZZ AAT's disposal was made by Seifers and colleagues. Supported by several grants over an 8 year span, they clarified some of the processes involved in the degradation of MM and ZZ AAT in different cell types including liver cells that are highly relevant to AATD. In particular, the investigators found that ER-associated degradation is orchestrated by mannosidase I and II, enzymes that are known to act on sugar residues, but in this context sort misfolded AAT to proteasomes for degradation. The mode of action involves the release of AAT monomers from polymers, another beneficial effect. Furthermore, the investigators reported that ER mannosidase I is upregulated in liver cells in AATD and that mutations in the mannosidase I gene increase hepatotoxicity in AATD. Thus mannosidase I polymorphism could be considered a modifier gene in AATD, possibly explaining the variability in the clinical expression of liver disease. Interestingly, it was also observed that wild-type ER mannosidase I is rapidly downregulated; this raises the possibility of using inhibitors of this downregulation for the treatment of AATD associated liver disease.

Apoptosis and autophagy

Apoptosis and autophagy have an important role in the liver disease of AATD. Teckman studied both processes and found that ZZ AAT activates caspase and induces apoptosis, and that inhibiting a mitochondrial trifunctional protein can attenuate apoptosis; this is a potential target for new drug development. He also reported the presence of autophagy, accelerated cell death and proliferation of liver cells in a mouse model of AATD. This raises the possibility of exploring the therapeutic potential of rapamycin, a drug that inhibits this process.

Inflammation

Brantly and colleagues showed that inflammatory cytokines upregulate AAT production and the unfolded protein response in hepatocytes (Brantly, Lin), suggesting that inflammatory processes could promote the liver disease of AATD. Teckman showed that indomethacin, a non-steroidal anti-inflammatory agent, promotes liver toxicity in his AATD mouse model. Clearly, these basic observations will have to be further investigated with respect to their relevance to human AATD.

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The following is currently known about the pathogenesis of COPD, the principle lung disease associated with AATD: a) the deficiency in AAT leads to a protease-antiprotease imbalance that has a role in the development of COPD, b) AAT has anti-apoptotic actions and its deficiency can facilitate the apoptotic process involved in the pathogenesis of COPD, c) lung epithelial cells and macrophages also produce AAT, and in the case of AATD, polymerized ZZ AAT in the lung is pro-inflammatory and contributes to the development of COPD, and d) cigarette smoke accelerates the development of COPD in AATD. Several research projects supported by the Alpha-1 Foundation have contributed to this body of knowledge, and some of them may lead to novel therapeutic possibilities. The studies were carried out in cell cultures and animal models of COPD.

Protease-antiprotease imbalance

In support of the protease-antiprotease paradigm, Guerassimov showed as early as in 1999 that the pallid mouse, a strain that has intrinsically low levels of circulating AAT, is more susceptible to cigarette smoke-induced emphysema, and that the severity of emphysema is inversely related to the level of AAT. This study not only supported the notion that AAT protects against the development of experimental emphysema, but also linked cigarette smoking to AATD-related emphysema. In keeping with this concept, Anlak reported that modification of the AAT molecule by tyrosine nitration diminishes AAT's anti-elastase activity; inasmuch as cigarette smoke contains NO, this observation shows that cigarette smoke induced emphysema could in part be explained by the smoke's action on AAT. Shifren et al. also found that elastin-mutant mice are more susceptible to cigarette-smoke induced emphysema; since neutrophil elastase has a proven role in the pathogenesis of emphysema, Shifren's finding further validates the protease-antiprotease paradigm.

Other study results fitting this concept include D'Armiento's observation that cigarette smoke induces another protease, MMP-1 in lung epithelial cells, and McElvaney's report suggesting that SLPI mutagenesis reduces its anti-neutrophil elastase activity; SLPI is an antiprotease that is highly expressed in the lung airways. Finally, certain less known proteins including SCCA 1 and SCCA 2 can also inhibit protease-mediated lung epithelial cell injury in a mouse model (Luke).

Apoptosis

Lung epithelial cell apoptosis has now been recognized as an important contributor to the development of emphysema in animal models, and as an extension probably to human COPD as well. This is highly relevant to the pathogenesis of AATD-related lung disease as AAT has anti-apoptotic actions and its deficiency is likely to facilitate the development of COPD through this mechanism. Several animal studies have provided the basis for this premise. Thus, Tucker et al. showed that in a murine model of VEGF receptor blockade-induced emphysema, transfection with the human AAT gene attenuated emphysema formation. This effect was probably related to AAT's inhibition of caspase-3, a protease that is critically involved in the process of apoptosis: caspase-3 was reduced in the lung of the transgenic animals. Petrache further investigated this model and observed that ceramide synthesis mediates apoptosis and emphysema formation. Of interest is the subsequent finding in mice that loss of VEGF gene expression during the post-natal period is associated with abnormal lung development including changes resembling emphysema (Jun). There is at present no evidence for a pathogenetic role of VEGF inhibition in the development of AATD related emphysema, although AAT has been shown to protect against the emphysema induced by VEGF inhibitors in animal models. However, these experiments demonstrated the protective effect of AAT against caspase-3 induced apoptosis, an acknowledged pathway involved in emphysema formation in general. AAT may also protect against chronic bronchitis, a component of COPD that is characterized by increased mucus production in the conducting airways. This has been suggested by a report from Shao showing that AAT modulates cigarette smoke extract induced airway mucin gene expression (MUC5ac); AATD could sensitize the airway to these cigarette smoke effects

Apoptosis

Cigarette smoke-induced emphysema in mice could be considered a more realistic model of human COPD, and lend itself to studying the relation between AATD and COPD. Not all mouse strains are equally susceptible to cigarette smoke induced emphysema, calling for a genome-wide association study (Cosio); this remains to be carried out. In currently known susceptible mouse models, Tuder reported increases in pulmonary stress response genes that could be involved in the generation of emphysema. Another mechanism that has been suggested to contribute to cigarette smoke-induced emphysema in mice is cigarette smoke induced polymerization of AAT, akin to the spontaneous ZZ AAT polymerization in AATD (Mahadeva). This could lead to an upregulated unfolded protein response in the lung with is detrimental consequences including apoptosis as has been shown in liver cells. In support of this possibility, a recent investigation of human lungs by Kelsen et al. revealed an upregulation of several unfolded protein response proteins in cigarette smokers (study not supported by a Foundation grant).

Cigarette smoke

Cigarette smoke-induced emphysema in mice could be considered a more realistic model of human COPD, and lend itself to studying the relation between AATD and COPD. Not all mouse strains are equally susceptible to cigarette smoke induced emphysema, calling for a genome-wide association study (Cosio); this remains to be carried out. In currently known susceptible mouse models, Tuder reported increases in pulmonary stress response genes that could be involved in the generation of emphysema. Another mechanism that has been suggested to contribute to cigarette smoke-induced emphysema in mice is cigarette smoke induced polymerization of AAT, akin to the spontaneous ZZ AAT polymerization in AATD (Mahadeva). This could lead to an upregulated unfolded protein response in the lung with is detrimental consequences including apoptosis as has been shown in liver cells. In support of this possibility, a recent investigation of human lungs by Kelsen et al. revealed an upregulation of several unfolded protein response proteins in cigarette smokers (study not supported by a Foundation grant)

Inflammation

Polymerized ZZ AAT is pro-inflammatory in the lung and its deficiency in monomeric form deprives the lung of an important anti-inflammatory principle as shown above. Thus, the two consequences of AATD potentiate each other's effect on inflammation. These processes have been investigated in cells (Greene, Taggart). Of special interest in the context of AATD were two studies that compared inflammatory cell function between individuals with and without AATD. Spencer reported that alveolar macrophages obtained from individuals with the ZZ phenotype overexpress the inflammatory cytokine IL-1β compared to MM phenotypes, and Lee showed that peripheral monocyte derived macrophages of individuals with the ZZ phenotype have an impaired ability to phagocytize apoptotic neutrophils (abnormal epherocytosis). This defect has since been shown to promote inflammation in the lung.

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In this area of research, the Alpha-1 Foundation supported grants that addressed the epidemiology and natural history of AATD, the role of non-M AAT alleles in heterozygous individuals, and the effect of modifier genes on disease expression. Both the liver and lung manifestations of AATD are included in this group of investigations. The program also supported COPD research unrelated to AATD with the understanding that information derived from common COPD can inform AATD related lung disease.

Epidemiology

DeSerres studied the genetic epidemiology of AATD in North America, Australia and New Zeeland. He estimated the deficiency gene prevalence from case control studies reported from those geographic areas. For the US, he calculated 25 million persons with deficiency alleles in 2001: of these, nearly 17 million were of the MS and about 8 million of the MZ genotype. He also estimated 60,000 persons with the ZZ genotype and a four times higher prevalence of SS and SZ genotypes. Another epidemiological study investigated the relationship between atmospheric pollution and mortality in AATD, using the NHLBI research registry and regional EPA air pollution data (Vedal). No effect was found, but only 120 deaths were analyzed; the small number may preclude a definitive conclusion on the susceptibility of persons with AATD to atmospheric pollution.

Liver disease

With respect to liver disease, the presence of non-M alleles seems to accelerate the progression of chronic liver disease not due to AATD. Among 1412 such patients, Schiff found that on average 8.3% were MS (14% in Hispanics) and 2.5% were MZ (14% in non-Hispanic whites and South Americans). He concluded that the Z allele is associated with an accelerated progression of chronic liver disease. In another study, Bezarra reported preliminary results showing that patients with biliary atresia and non-M alleles have worse outcomes. These observations suggest that different types of chronic liver disease have worse outcomes in carriers of the AATD genes.

Lung disease

Stoller and Campos studied the clinical features of AATD-related lung disease. The study of the latter investigator involving over 1000 AlphaNet patients undergoing a comprehensive management program including augmentation therapy revealed that influenza vaccination had no effect on the rate of exacerbations, that the introduction of tiotropium as a new COPD drug improved outcomes, that the exacerbation frequency in AATD was comparable to what has been reported for common COPD, and that the comprehensive disease management intervention significantly reduced exacerbation frequency and slowed the decline in quality of life. The results of the study conducted by Stoller are pending. Another natural history study addressed common COPD (Buchanan). It compared nursing home residents with and without COPD, using a MDS dataset of 445,000 individuals, 69,00 of whom had a diagnosis of COPD. The results showed that COPD patients were physically fitter but had more cardiovascular disease and depression than their counterparts. Guerra investigated lipopolysaccharide receptor polymorphisms in COPD; the results suggested that some SNPs were associated with a higher FEV1, but without a difference in the slope of FEV1 over time. The clinical significance of this observation therefore remains open for discussion. Kheradman studied responses of T-cells harvested from peripheral blood to elastin epitopes in smokers and ex-smokers and demonstrated the feasibility of this method. This is a promising finding in terms of its potential role as a susceptibility marker in COPD, given the involvement of T-cells in lung inflammation and the importance of elastin degradation in COPD.

Traditionally, FEV1 has been considered the principle outcome variable in clinical studies of COPD with or without AATD. This has been challenged in the recent past, based on the fact that COPD is characterized not only by airflow obstruction as reflected by FEV1 but also by lung tissue destruction that can now be assessed quantitatively with sensitive chest CT methods. Therefore, high resolution chest CT has now emerged as another outcome in characterizing the severity and progression of and the effects of therapeutic interventions in COPD. Two studies supported by the Alpha-1 Foundation have contributed to this evolution. Shaker validated the methodology (that is based on the inverse relationship between radiographic lung density and emphysema) in COPD by using the 15th percentile cut-off point on the density distribution curve and employing lung volume correction, and showed that smoking cessation is followed by a rapid decline in lung density, presumably reflecting a resolution of inflammatory changes in the lung. Subsequent disease progression can then be followed by a progressive decline in lung density. His data also suggested that inhaled glucocorticoids may retard the decline in lung density. In the other study, Mergo used a similar method in patients with AATD. This was a 6 month observational study and too short in duration to detect a slope in lung density over time. However, no correlation was found between lung density and FEV1, supporting the notion that the two outcomes are independent of each other.

The controversy about a possible connection between lung disease and the heterozygous state for AAT deficiency alleles has not been resolved and there appears to be ongoing interest in this question. In 2007, for example, McElvaney embarked upon a new genotype-by-phenotype genetic association study to again address COPD risk in individuals with the MZ phenotype. It is too early to expect results from this investigation.

Modifier genes

The search for modifier genes that could explain the inter-individual differences in the severity of lung or liver disease in AATD was supported by several grants, including grants whose primary purpose was to identify gene mutations that are associated with common COPD. Cox looked for modifier genes in AATD related liver disease in children and Hersh and DeMeo conducted genome-wide association studies in COPD. Results are available from the latter studies. Hersh identified SNPs on chromosome 12p, a region that has been associated with post bronchodilator FEV1. DeMeo investigated sex differences in candidate gene polymorphisms in COPD. No sex differences were found, but SERPIN 2 gene polymorphism was linked to COPD. Whether these or other gene mutations will emerge as modifier genes for AATD related COPD remains to be shown. DeMeo also started to examine epigenetic factors as modifiers in COPD with a focus on HLA associated SNPs and methylation sites. The study just started in 2008 and its results are pending.

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With an estimated prevalence of one per 3000-5000 general population in the US, severe AATD may be present in as many as 100,000 persons in 2009. It is estimated that less than 10% of these individuals have been diagnosed, and many patients with COPD and/or chronic liver disease unknowingly may have the genetic defect. In an attempt to determine the reasons for the low detection rate and to increase it with novel approaches, the Alpha-1 Foundation sponsored five detection grants between 1999 and 2008.

In 1999, Fleming conducted a pilot screening study involving direct or provider-mediated patient contact in high risk groups in Florida. IRB issues were identified as an impediment to testing. Ten percent of the patients refused to be tested; provider-mediated patient contact was more successful than direct contact, but overall the testing rate was disappointingly low. Likewise, Campos reported a very low level of testing in a physician-based COPD-targeted screening project in South Florida. A similar approach in Spain appeared to have a higher yield as reported by Miravitlles from two studies. In the first study involving blood spot screening (genotyping) in 87 patients from 5 centers, he identified 12 (14%) MZs and no ZZs. In the second study, the Spanish Registry/ Screening Program involving 1346 patients with COPD, there were 30 MZs and one ZZ. Of note was the absence of a reliable correlation between genotype and serum AAT levels, indicating that there is a need for phenotyping or genotyping in the diagnosis of AATD.

Testing for AATD in the pulmonary function laboratory is another approach to identifying AATD among COPD patients. In a single pulmonary function laboratory-based targeted testing program of 165 patients, reported by Lougee, 1 ZZ, 2 SZs, 4 MZs and 11 MSs were discovered. Perhaps the pulmonary function laboratory is a better site for AATD screening because of the immediate availability of pulmonary function data suggesting the presence of COPD. This approach currently is undergoing evaluation in a large, Foundation-sponsored multi-center targeted AATD detection study.

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These investigations addressed informed consent issues (Coors), health care economics (Mullins), disease comprehension (Fanos), and a general AATD related survey of the parents and providers of children that have undergone AATD testing (Coors). The results showed that 1) 92% of COPD patients wanted to know the results of their test, 2) 80% of these patients studied the educational materials provided, 3) learning about AATD did not increase the level of anxiety, 4) over 50% understood the concept of inheritance, 5) 90% believed heterozygocity has some adverse health effects, and 6) augmentation therapy constitutes 61% of health care costs in AATD. In order to promote the understanding of AATD in the allied health care community, Chatburn developed and validated a CD-based education and testing program, initially for respiratory therapists.

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Many grants supported by the Foundation have directly or indirectly investigated novel therapeutic approaches in AATD. Both basic and clinical experiments have been carried out and some of their results are promising and suggest that exciting new treatment strategies will become available in the foreseeable future.

The types of therapies addressed in these investigations include pharmacologic agents, gene therapy, stem cell approaches, and cell or organ transplantation. Of the 20 funded grants, 13 targeted the liver disease and 7 the lung disease of AATD. In some cases, this separation may not be appropriate as both organs may benefit from the intervention. For example, transfecting liver cells with MM AAT in an individual with ZZ AATD could result in the secretion of the MM protein into the circulation thereby ameliorating AATD and the attendant lung disease; however, this presumably would not change the accumulation of polymerized ZZ AAT in hepatocytes and therefore have no effect on the liver disease.

As in the above reviewed basic and clinical studies that have examined the pathogenesis of liver and lung disease in AATD, the therapy related grants have focused either on preventing the cytotoxic effect of polymerized ZZ AAT or on reversing AATD related disease processes by increasing the concentration of MM AAT in blood and lung or on inhibiting neutrophil elastase and other proteases involved in the pathogenesis of lung disease.

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Zhou observed in a preliminary study that a tripeptide can prevent the polymerization of ZZ AAT in liver cells. In a recent investigation involving a liver cell culture and a ZZ mouse model, Liu found that selected small molecules modify the structure and function of ZZ AAT. He and his coworkers identified 8 molecules that increased ZZ AAT secretion and that the ZZ AAT had anti-elastase activity. Two molecules increased the ZZ AAT level in the blood. In a pilot study, Brantly and colleagues sought to bring this therapeutic concept to the clinical arena by studying 4-phenyl-butyrate mediated AAT secretion rescue. Dose ranging was completed in 12 subjects. While this compound may not have the expected clinical efficacy, the study set the stage for the screening of other molecules. Taken together, these observations support the feasibility of using small molecules for the treatment of AATD, an approach that has gained momentum in the recent past, both in academic research laboratories and the biotech industry.

The effects of new and established drugs on lung inflammation and destruction were evaluated by Cantor in a mouse model, and will be evaluated in a recently initiated clinical study of patients with AATD by Olson. In the former investigation, tobacco smoke induced lung destruction was slightly attenuated by inhaled hyaluronic acid although the treatment increased lung inflammation. It has now been shown that this adverse effect is only seen with low molecular weight hyaluronic acid, and the therapeutic potential of inhaled high molecular weight hyaluronic acid is still being pursued. In the other study, the intent is to determine the effect of AAT augmentation therapy on the expression of several pro-inflammatory cytokines in patients with AATD. The results are pending. Such endpoints may prove useful in the future in the evaluation of new AAT formulations including aerosol AAT, especially in dose ranging studies.

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The ultimate goal of gene therapy in AATD is to silence the mutated gene (typically ZZ) and to transfect cells with the wild type AAT gene (MM) in the liver, lung and other tissues. This approach would target both the cytotoxic effects of the polymerized ZZ protein and increase the level of MM AAT in blood and tissues. This is a challenging biological problem. Although considerable methodological progress has been made in cell systems and animal models, meeting both goals in the same animal has not been achieved to date, and proving the feasibility and effectiveness of gene therapy in humans has been a slow process. The Foundation has supported some of this research.

Basic research in liver-directed gene therapy has included a study by Anthony who showed that by using a CMV vector he was able to transfect murine liver cells and a human hepatoma cell line with the human MM AAT gene; unfortunately, an increase in MM AAT gene expression and in AAT secretion was not seen. Zern tested the efficiency of a SV40 vector in transfecting human hepatoma cells and the murine liver with the human MM AAT. The method was successful. Coincidentally he also found that transfecting the liver of mice expressing the human ZZ AAT with ribozyme decreases ZZ protein expression. MM AAT expression was observed for several months after transfection. Also in the human ZZ AAT expressing mouse liver, Steer subsequently reported that using an RNAi vector he was able to decrease the expression of both the ZZ and MM protein. In long-term experiments, the liver showed repopulation with both ZZ and MM protein expressing cells. Li showed that with liver-specific SiRNA vectors, he was able to knock down wild-type AAT in human ZZ AAT transfected mice. So far, a similar observation for the ZZ protein has not been reported by him. Finally, Flotte used an AAV vector to test the possibility of a post-translational modification of the ZZ protein (trans-splicing gene repair) in the liver of human ZZ AAT transgenic mice. This reduced the ZZ protein accumulation in the liver and increased its level in blood. Collectively, these observations are promising and have made a significant contribution to the field because they have targeted the liver, a prime site of cytotoxicity and the primary site of AAT production and secretion.

Another approach in gene therapy is to increase MM AAT in the circulation by transfecting cells outside the liver. In one such experiment, Yu developed a vascular stent graft containing genetically engineered vascular smooth muscle cells to deliver AAT. Human MM AAT transfected stents implanted into the aorta of two dogs resulted in peak serum human MM AAT levels of 4-8 μg/ml after a few days; the levels decreased rapidly thereafter. These were modest concentrations; the currently accepted protective threshold serum concentration of AAT is 500 - 800 μg/ml depending on the measurement method used. A more promising approach has been gene therapy involving skeletal muscle. As early as in 1999, Song demonstrated in a primate model that the intramuscular administration of the AAT gene using an AAV vector was safe without inducing a significant immune response and without germ line transmission. A series of subsequent experiments by Flotte and colleagues led to the development of a human-grade AAV2-hMM AAT vector for clinical testing. Such experiments are currently underway.

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Two concepts in cell-based therapy have formed the basis of the grants supported by the Foundation in this category. One of them uses stem cells to regenerate normal organ-specific cells; so far the hepatocyte has been the focus of this approach although theoretically the lung could also be targeted. In the other approach, stem cells transfected with the MM AAT gene are directed to the target organ to increase normal AAT production and secretion into the circulation. So far only preliminary observations have been reported and questions about the efficiency and duration of these interventions in patients remain to be answered by future research.

Zern developed a culture medium that promoted the transformation of human embryonic stem cells into a hepatocyte-like appearance with the expression of hepatocyte-specific genes. He also was able to transfect the cells with additional genes using a liver-specific lentivirus vector, raising the possibility of transgenic human hepatocyte-like stem cells expressing the MM AAT gene. Along the same lines, Miki was able to transform human amnion stem cells into hepatocytes in vitro. Sharma engineered myelomonocytes to fuse with and induce gene expression in hepatocytes with the intent to use hepatocyte-targeted cell fusion to correct AATD. Experiments directed at the MM AAT gene currently are underway. These early in vitro investigations using cell lines are necessary to move the field forward, but major hurdles must still be overcome before their clinical applicability can be evaluated in humans.

Welsh transfected mesenchymal stem cells with the human MM AAT gene using a lentivirus gene. The transfection efficiency was sub-optimal, but no cytotoxicity was seen. The experiments also showed that the mesenchymal stem cells fused with native lung cells in elastase treated lungs. Verfaille successfully transformed marrow-associated progenitor cells to hepatocytes, and Berclaz reported that human AAT transfected hematopoetic progenitor cells expressed the human AAT protein and showed a high transfection rate and long-term engraftment and proliferation in the lung of pallid mice. These marrow stem cell-based strategies are initial steps in finding cell-based therapeutic solutions in patients with AATD-related lung disease.

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Two studies addressed this mode of therapy for AATD. Miki explored the possibility of inducing AAT expression in human fetal hepatocytes by culturing them in a medium containing oncogene M. Under these conditions, AAT gene expression was increased; interestingly other hepatocyte genes were not induced. The ultimate goal of such experiments is liver cell transplantation, a strategy employed by several other investigators in the field of hepatology.

Gildea investigated lung function and other outcomes after double lung transplantation in patients with AATD. In comparison to common COPD, the incidence of rejection was similar while the post-transplant decline in FEV1 over a 3-year period was greater in AATD. The latter observation may have an impact on future criteria governing the organ allocation system for lung transplantation.

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An encouraging conclusion that can be drawn from the review of this research activity is that it has identified new targets for therapeutic interventions with the potential of controlling or curing the liver and lung disease of AATD. It is likely that future therapeutic research will take two directions. One line of investigations will be directed at finding novel drugs, and gene-and cell-based interventions that target the basic defect in AATD; academia and biotech companies are best suited for this and the Foundation has recently established The Alpha-1 Project, a venture philanthropy program to promote this kind of research. The other area of therapeutic research is the development of aerosol formulations of AAT for augmentation therapy and the search for new sources for AAT using recombinant or transgenic technology. The plasma protein industry likely will have a major role in this. While we may be on the verge of finding new treatments for AATD that cannot be developed by academic investigators and require the help of the pharmaceutical industry, the Foundation is committed to continuing its investigator-initiated research grant program as new discoveries can be expected from it that will feed new drug development.

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