Alpha-1-antitrypsin deficiency (A1ATD) is a monogenic inherited liver disease with 180,000 individuals diagnosed worldwide ^[1]. The mutation responsible for the disease results in a misfolding of the Alpha-1 Antitrypsin (A1AT) protein. This is a protein predominantly synthesized and secreted by hepatocyte cells in the liver, subsequently translocating to the lungs where A1AT plays an important role against infections from the effect of neutrophile elastase. When the A1AT protein is misfolded it leads to a systematic disease, affecting both its site of functionality, the lungs and the primary site of production, the liver. The gene which encodes for A1AT, SERPINA1, can undergo various mutations which results in the misfolding of the A1AT protein. However, the most common disease causing mutation in the SERPINA1 gene is the ZZ-mutation (E342K) ^[2]. Since A1ATD is an inherited condition, it currently cannot be pre-emptively avoided, and it can impact individuals of any racial or ethnic background ^[3]. While the condition is generally classified as rare, approximations indicate that 80,000 to 100,000 people in the United States have an A1AT deficiency, implying that the disease often goes unnoticed or is insufficiently diagnosed ^[4]. This is partly due to certain mutations resulting in a milder phenotype characterized by A1AT misfolds without a complete loss of functionality.

Severe misfolds of A1AT, including the most common ZZ mutation result in a complete loss of primary function located in the lungs. The normal function of A1AT is as a protease inhibitor, acting as an “off switch” for a proteolytic enzyme called neutrophil elastase. Neutrophil elastase plays a role in protecting the lungs from infections, however, when it becomes overly active it can result in the damage of healthy lung tissue. After neutrophil elastase has assisted in the fight against an infection, A1AT acts as a "switch" to deactivate neutrophil elastase, thereby reducing the risk of proteolytic damage to healthy lung tissue. When A1AT is dysfunctional, it heightens the vulnerability to Chronic Obstructive Pulmonary Disease (COPD) ^[5]. The misfolded A1AT not only impacts the lungs but also the liver as the misfolded A1AT gives rise to a gain-of-toxic function phenotype as the misfolded insoluble globular protein (known as ATZ) accumulates in the endoplasmic reticulum (ER) of hepatocyte cells. The accumulation of the ATZ protein can give rise to liver fibrosis and, in some cases, increase the risk of developing hepatocellular carcinoma (HCC), a potentially fatal cancer ^[6].

Presently, the available treatment choices for managing lung complications in individuals with A1ATD are quite restricted. One strategy to reduce the risk of COPD involves a lifelong treatment called augmentation therapy. In this therapeutic approach, A1AT protein is derived from the blood of a healthy donor, pooled together, and subsequently administered to patients to elevate their A1AT levels in the lungs, ultimately contributing to the slowing down of lung damage progression ^[7].

Conversely, when it comes to addressing liver-related issues of A1ATD caused by the accumulation of ATZ, a significant shortage of efficacious treatments exists, making liver transplantation the only definitive curative recourse. The demand for liver transplants far surpasses the available supply. This creates a need to find novel therapeutics to address the effects A1ATD has on the liver.

By leveraging DefiniGEN's differentiation platform, it becomes possible to generate disease model hepatocytes manifesting the disease derived from induced Pluripotent Stem Cells (iPSCs). The iPSC’s expressing the A1ATD can be derived through two pathways depending on the client’s preferences. One option utilizes the gene editing tool CRIPSR/Cas9 to introduce the ZZ mutation in the SERPINA1 gene into otherwise genetically healthy iPSCs. Once differentiated out the now Hepatocyte-Like Cells (HLCs) hold the ZZ mutation with an otherwise wild-type form of the genome. The alternative approach involves the reprogramming of somatic cells from patients with A1ATD back to an iPSC state using four specific transcription factors referred to as the “Yamanaka factors”. This is when the two pathways converge as the A1AT deficient iPSCs are differentiated out into HLCs using our OptiDIFF platform. Dr. Carlos Gil a Team Leader scientist at DefiniGEN added that “clinical manifestations in the liver of patients with A1ATD are heterogeneous and can range from asymptomatic to chronic hepatitis and cirrhosis. There are still many questions around co-existing disease modifiers, therefore having both CRISPR-derived and patient-derived cells to study the disease can help cover part of that heterogeneity present in the human population.”

At DefiniGEN, our terminally differentiated A1ATD HLCs derived using our OptiDIFF technology showcase expression levels hepatocyte maturity markers closely resembling those detected in primary human hepatocytes (PHHs). These markers include Albumin (ALB), A1AT and Hepatocyte Nuclear Factor 4 Alpha (HNF4A). This is whilst confirming they display the A1ATD through phenotypic validation utilizing immunostaining with polymer specific A1AT antibody to visualize and confirm the rise in intracellular polymeric A1AT associated with A1ATD.

We have available, multiple A1ATD patient lines derived from patients with varying ages and genders. The differentiation process for each patient cell line has been optimized, utilizing one of the OptiDIFF approaches to ensure the greatest hepatic maturation for each line. Using an immunostaining method with the polymer-specific A1AT antibody showed that each A1ATD patient cell line used at DefiniGEN consistently and reproducibly display A1AT polymer accumulation but more importantly the accumulation pattern of the A1AT polymer varies between patient cell lines. During the conversation with Dr. Gil, he elucidated that the creation of patient cell lines with varying levels of A1AT is significant as “liver biopsies of patients with A1ATD showcase a great degree of variability in polymer burden and other disease markers so it’s important to be able to capture that variability when modelling the disease in vitro.” Our A1ATD cell lines have also shown that the phenotype can be modulated with treatment to the reference drug carbamazepine (CBZ) in a dose-dependent manner and they have also been shown to recover to respond to Nucleotide based therapeutics. Combining all these methods has allowed us to create an accurate in vitro pre-clinical model which aims to shorten the time taken in the drug discovery pipeline.

Explore our case study "Generating Human Preclinical Data for Candidate Therapeutics Against Alpha-1-Antitrypsin Deficiency." This study provides comprehensive insights into the technologies and methodologies utilized by DefiniGEN, as discussed in this blog, offering essential data and information to enhance your comprehension.

1. de Serres, F.J., Alpha-1 antitrypsin deficiency is not a rare disease but a disease that is rarely diagnosed. Environ Health Perspect, 2003. 111(16): p. 1851-4.
2. Kass, I., et al., Conformational properties of the disease-causing Z variant of α1-antitrypsin revealed by theory and experiment. Biophys J, 2012. 102(12): p. 2856-65.
3. de Serres, F.J., Worldwide racial and ethnic distribution of alpha1-antitrypsin deficiency: summary of an analysis of published genetic epidemiologic surveys. Chest, 2002. 122(5): p. 1818-29.
4. McCarthy, C., et al., Epidemiology of Rare Lung Diseases: The Challenges and Opportunities to Improve Research and Knowledge. Adv Exp Med Biol, 2017. 1031: p. 419-442.
5. Wells, A.D., et al., Alpha-1 Antitrypsin Replacement in Patients With COPD. P t, 2019. 44(7): p. 412-415.
6. Mitchell, E.L. and Z. Khan, Liver Disease in Alpha-1 Antitrypsin Deficiency: Current Approaches and Future Directions. Curr Pathobiol Rep, 2017. 5(3): p. 243-252.
7. Miravitlles, M., et al., European Respiratory Society statement: diagnosis and treatment of pulmonary disease in α(1)-antitrypsin deficiency. Eur Respir J, 2017. 50(5).