Cystic fibrosis (CF) is caused by genetic mutations of the CF transmembrane conductance regulator (CFTR), leading to disrupted transport of Cl
and bicarbonate and CF lung disease featuring bacterial ...colonization and chronic infection in conducting airways. CF pigs engineered by mutating CFTR develop lung disease that mimics human CF, and are well-suited for investigating CF lung disease therapeutics. Clinical data suggest small airways play a key role in the early pathogenesis of CF lung disease, but few preclinical studies have focused on small airways. Efficient targeted delivery of CFTR cDNA to small airway epithelium may correct the CFTR defect and prevent lung infections. Adeno-associated virus 4 (AAV4) is a natural AAV serotype and a safe vector with lower immunogenicity than other gene therapy vectors such as adenovirus. Our analysis of AAV natural serotypes using cultured primary pig airway epithelia showed that AAV4 has high tropism for airway epithelia and higher transduction efficiency for small airways compared with large airways. AAV4 mediated the delivery of CFTR, and corrected Cl
transport in cultured primary small airway epithelia from CF pigs. Moreover, AAV4 was superior to all other natural AAV serotypes in transducing ITGα6β4
pig distal lung progenitor cells. In addition, AAV4 encoding eGFP can infect pig distal lung epithelia in vivo. This study demonstrates AAV4 tropism in small airway progenitor cells, which it efficiently transduces. AAV4 offers a novel tool for mechanistical study of the role of small airway in CF lung pathogenesis in a preclinical large animal model.
In a newborn pig cystic fibrosis (CF) model, the ability of gland-containing airways to fight infection was affected by at least two major host-defense defects: impaired mucociliary transport and a ...lower airway surface liquid (ASL) pH. In the gland-containing airways, the ASL pH is balanced by CFTR (CF transmembrane conductance regulator) and ATP12A, which, respectively, control HCO
transport and proton secretion. We found that, although porcine small airway tissue expressed lower amounts of ATP12A, the ASL of epithelial cultures from CF distal small airways (diameter < 200 μm) were nevertheless more acidic (compared with non-CF airways). Therefore, we hypothesized that gland-containing airways and small airways control acidification using distinct mechanisms. Our microarray data suggested that small airway epithelia mediate proton secretion via ATP6V0D2, an isoform of the V0 d subunit of the H
-translocating plasma membrane V-type ATPase. Immunofluorescence of small airways verified the expression of the V0 d2 subunit isoform at the apical surface of Muc5B
secretory cells, but not ciliated cells. Inhibiting the V-type ATPase with bafilomycin A1 elevated the ASL pH of small airway cultures, in the presence or absence of HCO
, and decreased ASL viscosity. These data suggest that, unlike large airways, which are acidified by ATP12A activity, small airways are acidified by V-type ATPase, thus identifying V-type ATPase as a novel therapeutic target for small airway diseases.
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