Lucía Bañuls

Sara Pastor was not included as an author in the original publication

In the 1970s, the term “rare disease” was coined to describe a category of inherited metabolic diseases with low prevalence and a wide range of symptoms

Background: The study of hematic concentrations of alpha1 antitrypsin (AAT) is currently one step in the diagnosis of AAT deficiency. To try to clarify the relevance of the laboratory techniques, we carried out a systematic review of the literature. Methods: Studies evaluating the quantification of AAT in peripheral blood were searched in PubMed in July 2021. The selection criteria included (1) any type of study design that included a quantification of AAT in peripheral blood; (2) studies written in English or Spanish; (3) studies evaluating human beings; and (4) studies involving adults. Results: Out of 207 studies, the most frequently used techniques were nephelometry (43.9%), followed by ELISA (19.8%) and turbidimetry (13.5%). Altogether, 182 (87.9%) cases expressed their results in units of gram, while 16 (7.7%) articles expressed them in units of mole. Only 2.9% articles referred to the standard used, 43.5% articles indicated the commercial kit used, and 36.2% indicated the analyzer used. Conclusions: The technical aspects of these determinations are not always reported in the literature. Journals should be attentive to these technical requirements and ensure that they are included in the works in which AAT is determined in order to ensure a correct interpretation of the study findings.

Introduction: Alpha-1 antitrypsin (AAT) protein is the most abundant protease inhibitor in serum, but it also presents immunomodulator and anti-inflammatory activity. AAT expression is codified by the SERPINA1 gene. Mutations in this gene lead to AAT deficiency (AATD), a highly underdiagnosed respiratory condition. AAT is mainly produced by hepatocytes and, in smaller quantities, by neutrophils, monocytes, or alpha and beta-pancreatic cells, among others. Natural killer cells (NK) are a type of lymphocyte of the innate immune system. Their main function is to kill infected cells and help regulate immune response by cytokine secretion. The present study aims to characterise NK cells regarding AAT expression, due to the implication of both cells and protein, in the immune system response and regulation. Methods: NK cells were isolated from whole blood by immunomagnetic negative selection from five healthy donors and seven AATD patients. Total RNA and proteins were isolated from NK cells and AAT expression was assayed by RT-qPCR and Western Blotting. To determine AAT secretion, NK cells were cultured for 72 hours and AAT levels were determined from culture supernatants by ELISA. Results:SERPINA1 gene expression was detectable by RT-qPCR, showing similar expression levels to the GAPDH constitutive gene. AAT protein was detected by western blot in cell lysates and by ELISA in culture supernatants. Conclusion: For the first time, we have shown that AAT is expressed and secreted by NK cells derived from healthy individuals and AATD patients. Funded by 2016 Sociedad Valenciana de Neumología grant, SEPAR grant, GVA grant ACIF/2019/231

Several studies have shown that some rare respiratory diseases, such as alpha-1 antitrypsin deficiency (AATD), idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF), and primary ciliary dyskinesia (PCD) present oxidative stress (OS) and endoplasmic reticulum (ER) stress. Their involvement in these pathologies and the use of antioxidants as therapeutic agents to minimize the effects of OS are discussed in this review.

Several studies have shown the importance of oxidative stress (OS) in respiratory disease pathogenesis. It has been reported that the nasal epithelium may act as a surrogate for the bronchial epithelium in several respiratory diseases involving OS. However, the sample yields obtained from nasal biopsies are modest, limiting the number of parameters that can be determined. Flow cytometry has been widely used to evaluate cellular OS profiles. It has the advantage that analyses can be performed using a small amount of sample. Therefore, we aimed to set up a new method based on flow cytometry to assess the oxidative profile of human nasal epithelial cells which could be used in research on respiratory diseases. Levels of total nitric oxide, superoxide anion, peroxynitrite, and intracellular peroxides were measured. Reduced thiol levels, such as antioxidant-reduced glutathione and oxidative damaged lipids and proteins, were also analysed. The intracellular calcium levels, plasma membrane potential, apoptosis, and percentage of live cells were also studied. Finally, a strategy to evaluate the mitochondrial function, including mitochondrial hydrogen peroxide, superoxide anion, mitochondrial mass, and membrane potential, was set up. Using small amounts of sample and a non-invasive sampling technique, the described method enables the measurement of a comprehensive set of OS parameters in nasal epithelial cells, which could be useful in research on respiratory diseases.

Gene therapy is an alternative therapy in many respiratory diseases with genetic origin and currently without curative treatment. After five decades of progress, many different vectors and gene editing tools for genetic engineering are now available. However, we are still a long way from achieving a safe and efficient approach to gene therapy application in clinical practice. Here, we review three of the most common rare respiratory conditions—cystic fibrosis (CF), alpha-1 antitrypsin deficiency (AATD), and primary ciliary dyskinesia (PCD)—alongside attempts to develop genetic treatment for these diseases. Since the 1990s, gene augmentation therapy has been applied in multiple clinical trials targeting CF and AATD, especially using adeno-associated viral vectors, resulting in a good safety profile but with low efficacy in protein expression. Other strategies, such as non-viral vectors and more recently gene editing tools, have also been used to address these diseases in pre-clinical studies. The first gene therapy approach in PCD was in 2009 when a lentiviral transduction was performed to restore gene expression in vitro; since then, transcription activator-like effector nucleases (TALEN) technology has also been applied in primary cell culture. Gene therapy is an encouraging alternative treatment for these respiratory diseases; however, more research is needed to ensure treatment safety and efficacy.

Introduction: The emergence some years ago of the CRISPR/Cas9 system allowed gene therapy to be specific, versatile, cheap and approachable to almost every laboratory. Due to these features, many different genetic diseases such as cystic fibrosis or β-thalassemia have been addressed in cellular models using the CRISPR/Cas9 genetic editing tool. Alpha-1 antytripsin deficiency (AATD) is a rare genetic condition that can provoke respiratory and hepatic symptoms. The Z allele of SERPINA1 gene is a well-characterised point mutation that can trigger the whole pathology. Henceforth, Z mutation is a suitable target for genetic edition using CRISPR/Cas9 in order to develop a gene therapy to treat AATD. Aims and Objectives: The aim of this work is to set-up a CRISPR/Cas9 system to revert the Z mutation on in vitro models. Methods: Five different RNA guides were designed. Hek293 cells were transfected with the RNA guides and GFP using Lipofectamine 3000. After a 48 hour-incubation, transfection efficiency was estimated by fluorescence microscopy. Genomic DNA was isolated and the Z mutation locus was amplified by PCR. Gene editing efficiency was verified by the T7 endonuclease assay. Results: Transfection efficiency was estimated to be approximately 10-20%. After performing T7 endonuclease assay, the digested samples showed two smaller DNA fragments in the agarose gel, indicating that CRISPR/Cas9 system has produced a break in the genome at the Z mutation. Conclusion: SERPINA1 Z mutation can be targeted and edited by the CRISPR/Cas9 system in Hek293 cells with the RNA guides designed. This is the first step towards Z mutation correction in others AATD cellular models. Funding: SVN 2016 and 112/2016 SEPAR grants.

Alpha-1 antitrypsin deficiency (AATD) is a rare hereditary condition that leads to decreased circulating alpha-1 antitrypsin (AAT) levels, significantly increasing the risk of serious lung and/or liver disease in children and adults, in which some aspects remain unresolved. In this review, we summarise and update current knowledge on alpha-1 antitrypsin deficiency in order to identify and discuss areas of controversy and formulate questions that need further research. 1) AATD is a highly underdiagnosed condition. Over 120,000 European individuals are estimated to have severe AATD and more than 90% of them are underdiagnosed. 2) Several clinical and etiological aspects of the disease are yet to be resolved. New strategies for early detection and biomarkers for patient outcome prediction are needed to reduce morbidity and mortality in these patients; 3) Augmentation therapy is the only specific approved therapy that has shown clinical efficacy in delaying the progression of emphysema. Regrettably, some countries reject registration and reimbursement for this treatment because of the lack of larger randomised, placebo-controlled trials. 4) Alternative strategies are currently being investigated, including the use of gene therapy or induced pluripotent stem cells, and non-augmentation strategies to prevent AAT polymerisation inside hepatocytes.