Jeffrey Teckman

α1-Antitrypsin deficiency

α1-Antitrypsin deficiency (A1ATD) is an inherited disorder caused by mutations in SERPINA1, leading to liver and lung disease. It is not a rare disorder but frequently goes underdiagnosed or misdiagnosed as asthma, chronic obstructive pulmonary disease (COPD) or cryptogenic liver disease. The most frequent disease-associated mutations include the S allele and the Z allele of SERPINA1, which lead to the accumulation of misfolded α1-antitrypsin in hepatocytes, endoplasmic reticulum stress, low circulating levels of α1-antitrypsin and liver disease. Currently, there is no cure for severe liver disease and the only management option is liver transplantation when liver failure is life-threatening. A1ATD-associated lung disease predominately occurs in adults and is caused principally by inadequate protease inhibition. Treatment of A1ATD-associated lung disease includes standard therapies that are also used for the treatment of COPD, in addition to the use of augmentation therapy (that is, infusions of human plasma-derived, purified α1-antitrypsin). New therapies that target the misfolded α1-antitrypsin or attempt to correct the underlying genetic mutation are currently under development.

Lack of Effect of Oral 4-Phenylbutyrate on Serum Alpha-1-Antitrypsin in Patients with α-1-Antitrypsin Deficiency: A Preliminary Study

Objective: In homozygotes with ZZ genotype alpha-1-antitrypsin (α1AT) deficiency, mutant α1ATZ protein (α1ATZ) accumulates in hepatocytes, rather than being secreted into the blood. Homozygous individuals experience emphysema as a result of reduced levels of circulating α1AT in the lung with which to inhibit connective tissue breakdown. Homozygotes may also experience liver disease from the accumulation of α1ATZ within hepatocytes, which causes liver damage. A previous study indicated that the compound 4-phenylbutyrate (4-PBA) mediated a significant increase in release of α1ATZ from cells in tissue culture and in a mouse model of α1AT deficiency. The authors hypothesized that 4-PBA could be used to treat both the liver and lung disease of humans with α1AT deficiency. Methods: In this preliminary, open label study the authors evaluated the effect of 14 days of oral 4-PBA therapy on α1AT blood levels in 10 patients with α1AT deficiency. Results: There was no significant increase in α1AT blood level associated with 4-PBA administration. Symptomatic and metabolic side effects were significant. Conclusion: 4-PBA did not increase α1AT blood levels in humans with α1AT deficiency in this preliminary trial.

Sustained miRNA-mediated knockdown of mutant AAT with simultaneous augmentation of wild-type AAT has minimal effect on global liver miRNA profiles.

α-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AATs antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concomitant Z-AAT knockdown in the liver and increased expression of M-AAT. Transgenic mice expressing the human PiZ allele treated with dual-function rAAV9 vectors showed that serum PiZ was stably and persistently reduced by an average of 80%. Treated animals showed knockdown of Z-AAT in liver and serum with concomitant increased serum M-AAT as determined by allele-specific enzyme-linked immunosorbent assays (ELISAs). In addition, decreased globular accumulation of misfolded Z-AAT in hepatocytes and a reduction in inflammatory infiltrates in the liver was observed. Results from microarray studies demonstrate that endogenous miRNAs were minimally affected by this treatment. These data suggests that miRNA mediated knockdown does not saturate the miRNA pathway as has been seen with viral vector expression of short hairpin RNAs (shRNAs). This safe dual-therapy approach can be applied to other disorders such as amyotrophic lateral sclerosis, Huntington disease, cerebral ataxia, and optic atrophies.

In vivo post-transcriptional gene silencing of alpha-1 antitrypsin by adeno-associated virus vectors expressing siRNA

α-1 Antitrypsin (AAT) deficiency is one of the most common genetic diseases in North America, with a carrier frequency of approximately 4% in the US population. Homozygosity for the most common mutation (Glu342Lys, PI*Z) leads to the synthesis of a mutant protein, which accumulates and polymerizes within hepatocytes rather than being efficiently secreted. This lack of secretion causes severe serum deficiency predisposing to chronic lung disease. Twelve to fifteen percent of patients with PI*ZZ also develop liver disease, which can be severe, even in infancy. This is thought to be due to toxic effects of the accumulated mutant Z-AAT within the hepatocyte. Thus, an approach to reduce AAT-deficient liver disease will likely require some mechanism to decrease the amount of Z-AAT within hepatocytes. In this report, we describe studies of small-interfering RNAs (siRNAs) designed to downregulate endogenous AAT within hepatocytes. Three different siRNA sequences were identified and cloned into a recombinant adeno-associated virus (rAAV) backbone, either singly or as a trifunctional (3X) construct. Each had activity independently, but the levels of AAT expression in cell culture models showed the greatest decrease with the 3X construct, resulting in levels that were five-fold lower than controls. The rAAV-3X-siRNA was then packaged into AAV8 capsids and used in vivo to transduce the livers of human Z-AAT overexpressing transgenic mice. Those studies showed a decrease in total human AAT, a clearing of Z-AAT accumulation by immunohistochemistry, and a decrease in monomer Z-AAT within the liver within 3 weeks after vector injection. The rAAV8-3X-siRNA vector may hold promise as a potential therapy for patients with AAT liver disease.

Diagnosis and Management of Patients With α1-Antitrypsin (A1AT) Deficiency

Alpha(1)-antitrypsin (A1AT) deficiency is an autosomal codominant disease that can cause chronic liver disease, cirrhosis, and hepatocellular carcinoma in children and adults and increases risk for emphysema in adults. The development of symptomatic disease varies; some patients have life-threatening symptoms in childhood, whereas others remain asymptomatic and healthy into old age. As a result of this variability, patients present across multiple disciplines, including pediatrics, adult medicine, hepatology, genetics, and pulmonology. This can give physicians the mistaken impression that the condition is less common than it actually is and can lead to fragmented care that omits critical interventions commonly performed by other specialists. We sought to present a rational approach for hepatologists to manage adult patients with A1AT deficiency.

Sustained Knockdown of a Disease-Causing Gene in Patient-Specific Induced Pluripotent Stem Cells Using Lentiviral Vector-Based Gene Therapy

Patient‐specific induced pluripotent stem cells (iPSCs) hold great promise for studies on disease‐related developmental processes and may serve as an autologous cell source for future treatment of many hereditary diseases. New genetic engineering tools such as zinc finger nucleases and transcription activator‐like effector nuclease allow targeted correction of monogenetic disorders but are very cumbersome to establish. Aiming at studies on the knockdown of a disease‐causing gene, lentiviral vector‐mediated expression of short hairpin RNAs (shRNAs) is a valuable option, but it is limited by silencing of the knockdown construct upon epigenetic remodeling during differentiation. Here, we propose an approach for the expression of a therapeutic shRNA in disease‐specific iPSCs using third‐generation lentiviral vectors. Targeting severe α‐1‐antitrypsin (A1AT) deficiency, we overexpressed a human microRNA 30 (miR30)‐styled shRNA directed against the PiZ variant of A1AT, which is known to cause chronic liver damage in affected patients. This knockdown cassette is traceable from clonal iPSC lines to differentiated hepatic progeny via an enhanced green fluorescence protein reporter expressed from the same RNA‐polymerase II promoter. Importantly, the cytomegalovirus i/e enhancer chicken β actin (CAG) promoter‐driven expression of this construct is sustained without transgene silencing during hepatic differentiation in vitro and in vivo. At low lentiviral copy numbers per genome we confirmed a functional relevant reduction (−66%) of intracellular PiZ protein in hepatic cells after differentiation of patient‐specific iPSCs. In conclusion, we have demonstrated that lentiviral vector‐mediated expression of shRNAs can be efficiently used to knock down and functionally evaluate disease‐related genes in patient‐specific iPSCs.

Oxidative stress contributes to liver damage in a murine model of alpha-1-antitrypsin deficiency

Alpha-1-antitrypsin deficiency is a genetic disorder resulting in the expression of misfolded mutant protein that can polymerize and accumulate in hepatocytes, leading to liver disease in some individuals. Transgenic PiZ mice are a well-characterized model, which express human alpha-1-antitrypsin mutant Z protein (ATZ protein) and faithfully recapitulate the human liver disease. Liver tissue expressing alpha-1-antitrypsin mutant Z protein exhibits inflammation, injury and replacement of damaged cells. Fibrosis and hepatocellular carcinoma (HCC) develop in aging PiZ mice. In this study, microarray analysis was performed comparing young PiZ (ZY) mice to wild-type (WY), and indicated that there were alterations in gene expression levels that could influence a number of pathways leading to liver disease. Redox-regulating genes were up-regulated in ZY tissue, including carbonyl reductase 3 (CBR3), glutathione S-transferase alpha 1 + 2 (GSTA(1 + 2)) and glutathione S-transferase mu 3 (GSTM3). We hypothesized that oxidative stress could develop in Z mouse liver, contributing to tissue damage and disease progression with age. The results of biochemical analysis of PiZ mouse liver revealed that higher levels of reactive oxygen species (ROS) and a more oxidized, cellular redox state occurred in liver tissue from ZY mice than WY. ZY mice showed little evidence of oxidative cellular damage as assessed by protein carbonylation levels, malondialdehyde levels and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8oxodG) staining. Aging liver tissue from PiZ older mice (ZO) had elevated ROS, generally lower levels of antioxidant enzymes than younger mice and evidence of cellular damage. These data indicate that oxidative stress is a contributing factor in the development of liver disease in this model of alpha-1-antitrypsin deficiency.

Characteristics of hepatocellular carcinoma in a murine model of alpha-1-antitrypsin deficiency

Aim:  Individuals with homozygous (ZZ) alpha‐1‐antitrypsin (α1AT) deficiency are at an increased risk for liver damage, cirrhosis and hepatocellular carcinoma (HCC). The transgenic PiZ mouse, expressing the human α1AT mutant Z gene, is a valuable model for this disease. We studied PiZ mice in order to identify and characterize mechanisms involved in the development of HCC.

Hepatic progenitor cell proliferation and liver injury in α-1-antitrypsin deficiency.

Objectives: Homozygous ZZ α-1-antitrypsin (a1AT) deficiency is a common genetic liver disease that causes liver injury and hepatocellular carcinoma (HCC). The a1AT mutant Z gene encodes a mutant protein that accumulates within hepatocytes leading to hepatocellular death and a hepatic regenerative response. However, the mechanisms linking hepatocellular injury to these responses are poorly understood. In this study, we examined liver injury and response in human liver and in transgenic mice for involvement of hepatic progenitor cells. Methods: Liver biopsy specimens of low-grade, early-stage human ZZ liver exhibiting minimal inflammation and minimal fibrosis (grade 1 and stage 1) were examined for hepatic progenitor cell (HPC) proliferation using immunoreactivity for cytokeratin-7 (CK-7). Transgenic mouse model liver and other selected human biopsies were also examined. Results: Increased CK-7-positive HPC proliferation was seen in human ZZ liver compared to normal liver, but was 5-fold less HPC proliferation than in grade- and stage-matched disease control hepatitis C–infected liver. Livers from PiZ mice, a model transgenic for the human a1AT mutant Z gene, which recapitulates the human injury, also showed HPC proliferation. Human ZZ liver and PiZ mice develop dysplasia in the liver and HCC. HCC in PiZ mice was also characterized by HPC proliferation. Progressive hepatic fibrosis with age in the PiZ mice is demonstrated for the first time in the present study. Conclusions: Chronic injury in both ZZ human and PiZ mouse liver is associated with hepatic fibrosis and a unique magnitude of HPC proliferation within the hepatic proliferative response.

Appropriateness of Newborn Screening for α1-Antitrypsin Deficiency

Objective: The Alpha-1 Foundation convened a workshop to consider the appropriateness of newborn screening for &agr;-1-antitrypsin (AAT) deficiency. Methods: A review of natural history and technical data was conducted. Results: Homozygous ZZ AAT deficiency is a common genetic disease occurring in 1 in 2000 to 3500 births; however, it is underrecognized and most patients are undiagnosed. AAT deficiency can cause chronic liver disease, cirrhosis, and liver failure in children and adults, and lung disease in adults. The clinical course is highly variable. Some neonates present with cholestatic hepatitis and some children require liver transplantation, but many patients remain well into adulthood. Some adults develop emphysema. There is no treatment for AAT liver disease, other than supportive care and liver transplant. There are no data on the effect of early diagnosis on liver disease. Avoidance of smoking is of proven benefit to reduce future lung disease, as is protein replacement therapy. Justifying newborn screening with the aim of reducing smoking and reducing adult lung disease-years in the future would be a significant paradigm shift for the screening field. Recent passage of the Genetic Information Nondiscrimination Act (GINA) and the Affordable Care Act may have a major effect on reducing the psychosocial and financial risks of newborn screening because many asymptomatic children would be identified. Data on the risk–benefit ratio of screening in the new legal climate are lacking. Conclusions: Workshop participants recommended a series of pilot studies focused on generating new data on the risks and benefits of newborn screening.