Development and Initial Characterization of Pigs with DNAI1 Mutations and Primary Ciliary Dyskinesia.

Mutations in more than 50 different genes cause primary ciliary dyskinesia (PCD) by disrupting the activity of motile cilia that facilitate mucociliary transport (MCT). Knowledge of PCD has come from studies identifying disease-causing mutations, characterizing structural cilia abnormalities, finding genotype-phenotype relationships, and studying the cell biology of cilia. Despite these important findings, we still lack effective treatments and people with PCD have significant pulmonary impairment. As with many other diseases, a better understanding of pathogenic mechanisms may lead to effective treatments. To pursue disease mechanisms, we used CRISPR-Cas9 to develop a PCD pig with a disrupted DNAI1 gene. PCD pig airway cilia lacked the outer dynein arm and had impaired beating. MCT was impaired under both baseline conditions and after cholinergic stimulation in PCD pigs. Neonatal PCD pigs developed neonatal respiratory distress with evidence of atelectasis, air trapping, and airway mucus obstruction. Despite airway mucus accumulation, lung bacterial counts were similar between neonatal wild-type and PCD pigs. Sinonasal disease was present in all neonatal PCD pigs. Older PCD pigs developed worsening airway mucus obstruction, inflammation, and bacterial infection. This pig model closely mimics the disease phenotype seen in people with PCD and can be used to better understand the pathophysiology of PCD airway disease.

High ionic strength vector formulations enhance gene transfer to airway epithelia.

A fundamental challenge for cystic fibrosis (CF) gene therapy is ensuring sufficient transduction of airway epithelia to achieve therapeutic correction. Hypertonic saline (HTS) is frequently administered to people with CF to enhance mucus clearance. HTS transiently disrupts epithelial cell tight junctions, but its ability to improve gene transfer has not been investigated. Here, we asked if increasing the concentration of NaCl enhances the transduction efficiency of three gene therapy vectors: adenovirus, AAV, and lentiviral vectors. Vectors formulated with 3-7% NaCl exhibited markedly increased transduction for all three platforms, leading to anion channel correction in primary cultures of human CF epithelial cells and enhanced gene transfer in mouse and pig airways in vivo. The mechanism of transduction enhancement involved tonicity but not osmolarity or pH. Formulating vectors with a high ionic strength solution is a simple strategy to greatly enhance efficacy and immediately improve preclinical or clinical applications.

Culturing Immortalized Human Airway Epithelial Cells at an Air-Liquid Interface for Measles Virus Infection.

Measles virus (MeV) infection of airway surface epithelial cells provides a site for final amplification before being released back into the environment via coughing and sneezing. Multiple cell lines have served as models of polarized epithelia for MeV infection, such as Caco2 cells (intestinal derived human epithelia) or MDCK cells (kidney derived canine epithelia). In this chapter, we describe the materials and air-liquid interface (ALI) culture conditions for maintaining four different cell lines derived from human airway epithelial cells: 16HBE14o-, Calu-3, H358, and NuLi-1. We provide methods for confirming transepithelial electrical resistance (TER) and preparing samples for microscopy as well as expected results from apical or basolateral MeV delivery. Polarized human airway derived cells serve as tissue culture models for investigating targeted questions about how MeV exits a human host. In addition, these methods are generalizable to studies of other respiratory viruses or the biology of ALI airway epithelial cells.

High ionic strength vector formulations enhance gene transfer to airway epithelia.

A fundamental challenge for cystic fibrosis (CF) gene therapy is ensuring sufficient transduction of airway epithelia to achieve therapeutic correction. Hypertonic saline (HTS) is frequently administered to people with CF to enhance mucus clearance. HTS transiently disrupts epithelial cell tight junctions, but its ability to improve gene transfer has not been investigated. Here we asked if increasing the concentration of NaCl enhances the transduction efficiency of three gene therapy vectors: adenovirus, AAV, and lentiviral vectors. Vectors formulated with 3-7% NaCl exhibited markedly increased transduction for all three platforms, leading to anion channel correction in primary cultures of human CF epithelial cells and enhanced gene transfer in mouse and pig airways in vivo. The mechanism of transduction enhancement involved tonicity but not osmolarity or pH. Formulating vectors with a high ionic strength solution is a simple strategy to greatly enhance efficacy and immediately improve preclinical or clinical applications.

Reciprocal mutations of lung-tropic AAV capsids lead to improved transduction properties.

Considerable effort has been devoted to developing adeno-associated virus (AAV)-based vectors for gene therapy in cystic fibrosis (CF). As a result of directed evolution and capsid shuffling technology, AAV capsids are available with widespread tropism for airway epithelial cells. For example, AAV2.5T and AAV6.2 are two evolved capsids with improved airway epithelial cell transduction properties over their parental serotypes. However, limited research has been focused on identifying their specific cellular tropism. Restoring cystic fibrosis transmembrane conductance regulator (CFTR) expression in surface columnar epithelial cells is necessary for the correction of the CF airway phenotype. Basal cells are a progenitor population of the conducting airways responsible for replenishing surface epithelial cells (including secretory cells and ionocytes), making correction of this cell population vital for a long-lived gene therapy strategy. In this study, we investigate the tropism of AAV capsids for three cell types in primary cultures of well-differentiated human airway epithelial (HAE) cells and primary human airway basal cells. We observed that AAV2.5T transduced surface epithelial cells better than AAV6.2, while AAV6.2 transduced airway basal cells better than AAV2.5T. We also investigated a recently developed capsid, AAV6.2FF, which has two surface tyrosines converted to phenylalanines. Next, we incorporated reciprocal mutations to create AAV capsids with further improved surface and basal cell transduction characteristics. Lastly, we successfully employed a split-intein approach using AAV to deliver an adenine base editor (ABE) to repair the CFTR R553X mutation. Our results suggest that rational incorporation of AAV capsid mutations improves AAV transduction of the airway surface and progenitor cells and may ultimately lead to improved pulmonary function in people with CF.

Interferon-independent processes constrain measles virus cell-to-cell spread in primary human airway epithelial cells.

Amplification of measles virus (MeV) in human airway epithelia may contribute to its extremely high contagious nature. We use well-differentiated primary cultures of human airway epithelial cells (HAE) to model ex vivo how MeV spreads in human airways. In HAE, MeV spreads cell-to-cell for 3-5 days, but then, infectious center growth is arrested. What stops MeV spread in HAE is not understood, but interferon (IFN) is known to slow MeV spread in other in vitro and in vivo models. Here, we assessed the role of type I and type III IFN in arresting MeV spread in HAE. The addition of IFN-ß or IFN-λ1 to the medium of infected HAE slowed MeV infectious center growth, but when IFN receptor signaling was blocked, infectious center size was not affected. In contrast, blocking type-I IFN receptor signaling enhanced respiratory syncytial virus spread. HAE were also infected with MeV mutants defective for the V protein. The V protein has been demonstrated to interact with both MDA5 and STAT2 to inhibit activation of innate immunity; however, innate immune reactions were unexpectedly muted against the V-defective MeV in HAE. Minimal innate immunity activation was confirmed by deep sequencing, quantitative RT-PCR, and single-cell RNA-seq analyses of the transcription of IFN and IFN-stimulated genes. We conclude that in HAE, IFN-signaling can contribute to slowing infectious center growth; however, IFN-independent processes are most important for limiting cell-to-cell spread. IMPORTANCE Fundamental biological questions remain about the highly contagious measles virus (MeV). MeV amplifies within airway epithelial cells before spreading to the next host. This final step likely contributes to the ability of MeV to spread host-to-host. Over the course of 3-5 days post-infection of airway epithelial cells, MeV spreads directly cell-to-cell and forms infectious centers. Infectious center formation is unique to MeV. In this study, we show that interferon (IFN) signaling does not explain why MeV cell-to-cell spread is ultimately impeded within the cell layer. The ability of MeV to spread cell-to-cell in airway cells without appreciable IFN induction may contribute to its highly contagious nature. This study contributes to the understanding of a significant global health concern by demonstrating that infectious center formation occurs independent of the simplest explanation for limiting viral transmission within a host.

Representative measles virus infection requires appropriate airway epithelia culture conditions.

IMPORTANCE: For many years, measles virus (MeV) was assumed to first enter the host via the apical surface of airway epithelial cells and subsequently spread systemically. We and others reported that MeV has an overwhelming preference for entry at the basolateral surface of airway epithelial cells, which led to a fundamental new understanding of how MeV enters a human host. This unexpected observation using well-differentiated primary cultures of airway epithelia from human donors contradicted previous studies using immortalized cultured cells. Here, we show that appropriate differentiation and cell morphology of primary human airway epithelial cells are critical to recapitulate MeV infection patterns and pathogenesis of the in vivo airways. By simply culturing primary cells in media containing serum or passaging primary cultures, erroneous results quickly emerge. These results have broad implications for data interpretation related to respiratory virus infection, spread, and release from human airway epithelial cells.

CFTR-rich ionocytes mediate chloride absorption across airway epithelia.

The volume and composition of a thin layer of liquid covering the airway surface defend the lung from inhaled pathogens and debris. Airway epithelia secrete Cl- into the airway surface liquid through cystic fibrosis transmembrane conductance regulator (CFTR) channels, thereby increasing the volume of airway surface liquid. The discovery that pulmonary ionocytes contain high levels of CFTR led us to predict that ionocytes drive secretion. However, we found the opposite. Elevating ionocyte abundance increased liquid absorption, whereas reducing ionocyte abundance increased secretion. In contrast to other airway epithelial cells, ionocytes contained barttin/Cl- channels in their basolateral membrane. Disrupting barttin/Cl- channel function impaired liquid absorption, and overexpressing barttin/Cl- channels increased absorption. Together, apical CFTR and basolateral barttin/Cl- channels provide an electrically conductive pathway for Cl- flow through ionocytes, and the transepithelial voltage generated by apical Na+ channels drives absorption. These findings indicate that ionocytes mediate liquid absorption, and secretory cells mediate liquid secretion. Segregating these counteracting activities to distinct cell types enables epithelia to precisely control the airway surface. Moreover, the divergent role of CFTR in ionocytes and secretory cells suggests that cystic fibrosis disrupts both liquid secretion and absorption.

WNK Inhibition Increases Surface Liquid pH and Host Defense in Cystic Fibrosis Airway Epithelia.

In cystic fibrosis (CF), reduced HCO3- secretion acidifies the airway surface liquid (ASL), and the acidic pH disrupts host defenses. Thus, understanding the control of ASL pH (pHASL) in CF may help identify novel targets and facilitate therapeutic development. In diverse epithelia, the WNK (with-no-lysine [K]) kinases coordinate HCO3- and Cl- transport, but their functions in airway epithelia are poorly understood. Here, we tested the hypothesis that WNK kinases regulate CF pHASL. In primary cultures of differentiated human airway epithelia, inhibiting WNK kinases acutely increased both CF and non-CF pHASL. This response was HCO3- dependent and involved downstream SPAK/OSR1 (Ste20/SPS1-related proline-alanine-rich protein kinase/oxidative stress responsive 1 kinase). Importantly, WNK inhibition enhanced key host defenses otherwise impaired in CF. Human airway epithelia expressed two WNK isoforms in secretory cells and ionocytes, and knockdown of either WNK1 or WNK2 increased CF pHASL. WNK inhibition decreased Cl- secretion and the response to bumetanide, an NKCC1 (sodium-potassium-chloride cotransporter 1) inhibitor. Surprisingly, bumetanide alone or basolateral Cl- substitution also alkalinized CF pHASL. These data suggest that WNK kinases influence the balance between transepithelial Cl- versus HCO3- secretion. Moreover, reducing basolateral Cl- entry may increase HCO3- secretion and raise pHASL, thereby improving CF host defenses.

Functional correction of CFTR mutations in human airway epithelial cells using adenine base editors.

Mutations in the CFTR gene that lead to premature stop codons or splicing defects cause cystic fibrosis (CF) and are not amenable to treatment by small-molecule modulators. Here, we investigate the use of adenine base editor (ABE) ribonucleoproteins (RNPs) that convert A•T to G•C base pairs as a therapeutic strategy for three CF-causing mutations. Using ABE RNPs, we corrected in human airway epithelial cells premature stop codon mutations (R553X and W1282X) and a splice-site mutation (3849 + 10 kb C > T). Following ABE delivery, DNA sequencing revealed correction of these pathogenic mutations at efficiencies that reached 38-82% with minimal bystander edits or indels. This range of editing was sufficient to attain functional correction of CFTR-dependent anion channel activity in primary epithelial cells from CF patients and in a CF patient-derived cell line. These results demonstrate the utility of base editor RNPs to repair CFTR mutations that are not currently treatable with approved therapeutics.