Biology subjects

Hilkin, B. M.

Publications and source records attributed to Hilkin, B. M..

3 recordsLinked to original sources

bioRxiv2026

In Utero CFTR Modulation Alleviates Disease in G551D Cystic Fibrosis Pigs

Previous studies indicate that pigs with CFTR-null and CFTR-{Delta}F508 mutations develop multiorgan disease similar to that in people with cystic fibrosis (CF). At birth, their airways exhibit host defense defects that predispose to airway infection, inflammation, and mucus accumulation. The CFTR-G551D mutation causes CF by producing CFTR channels that localize correctly but have reduced channel activity. Ivacaftor (VX-770) is a small molecule drug developed to potentiate CFTR activity. To test the phenotype of the CFTR-G551D mutation in pigs and determine whether ivacaftor can rescue CF abnormalities, we developed CFTRG551D/G551D (CF-G551D) pigs through homologous recombination in fetal fibroblasts and somatic cell nuclear transfer. Newborn CF-G551D piglets exhibited phenotypes similar to CF-null piglets, including meconium ileus, exocrine pancreatic destruction, micro-gallbladder, vas deferens destruction, and airway structural abnormalities. Compared to wild-type pigs, CF-G551D pigs had reduced forskolin-stimulated short-circuit current in airway and intestinal tissues. Ivacaftor increased the single-channel open state probability of CFTR-G551D and increased short-circuit current to near wild-type levels. Similar to our other CF pig models, we found that 100% of CF-G551D pigs were born with meconium ileus. To test whether in utero ivacaftor treatment could prevent or alleviate meconium ileus, pregnant sows were treated with ivacaftor beginning at day 35 of gestation and continuing until delivery. This treatment rescued the pancreas, gallbladder, and vas deferens phenotype in the majority of CF-G551D pigs. Animals that were spared from meconium ileus were able to survive without ivacaftor treatment. Airway disease developed similar to other CF pig models. These findings indicate that this model may be useful for studies in which CFTR function can be reversed, for investigating in utero CFTR correction strategies, and for longitudinal studies in CF pigs.

physiology↗

bioRxiv2024

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.

physiology↗

bioRxiv2024

Mucociliary Clearance is Impaired in Small Airways of Cystic Fibrosis Pigs

RationaleCystic fibrosis is a genetic disorder characterized by recurrent airway infections, inflammation, and progressive decline in lung function. Autopsy and spirometry data suggest that cystic fibrosis may start in the small airways which, due to the fractal nature of the airways, account for most of the airway tree surface area. However, they are not easily accessible for testing. ObjectivesHere, we tested the hypothesis that mucociliary clearance is abnormal in the small airways of newborn cystic fibrosis pigs. MethodsCurrent mucociliary clearance assays are limited therefore we developed a dynamic positron emission tomography scan assay with high spatial and temporal resolution. Each study was accompanied by a high-resolution computed tomography scan that helped identify the thin outer region of the lung that contained small airways. Measurements and Main ResultsClearance of aerosolized [68Ga]macro aggregated albumin from distal airways occurred within minutes after delivery and followed a two-phase process. In cystic fibrosis pigs, both early and late clearance rates were slower. Stimulation of the cystic fibrosis airways with the purinergic agonist UTP further impaired late clearance. Only 1 cystic fibrosis pig treated with UTP out of 6 cleared more than 20% of the delivered dose. ConclusionsThese data indicate that mucociliary transport in the small airways is fast and can easily be missed if the acquisition is not fast enough. The data also indicate that mucociliary transport is impaired in small airways of cystic fibrosis pigs. This defect is exacerbated by stimulation of mucus secretions with purinergic agonists.

physiology↗