技术资料

A major challenge in developing gene-based therapies for airway diseases such as cystic fibrosis (CF) is sustaining therapeutic levels of transgene expression over time. This is largely due to airway epithelial cell turnover and the host immunogenicity to gene delivery vectors. Modern gene editing tools and delivery vehicles hold great potential for overcoming this challenge. There is currently not much known about how to deliver genes into airway stem cells,of which basal cells are the major type in human airways. In this study,helper-dependent adenoviral (HD-Ad) vectors were delivered to mouse and pig airways via intranasal delivery,and direct bronchoscopic instillation,respectively. Vector transduction was assessed by immunostaining of lung tissue sections,which revealed that airway basal cells of mice and pigs can be targeted in vivo. In addition,efficient transduction of primary human airway basal cells was verified with an HD-Ad vector expressing green fluorescent protein. Furthermore,we successfully delivered the human CFTR gene to airway basal cells from CF patients,and demonstrated restoration of CFTR channel activity following cell differentiation in air-liquid interface culture. Our results provide a strong rationale for utilizing HD-Ad vectors to target airway basal cells for permanent gene correction of genetic airway diseases. View Publication

Extracellular matrix (ECM) composition and stiffness are major driving forces for the development and persistence of fibrotic diseases. Lysyl oxidase (LOX) and LOX-like (LOXL) proteins play crucial roles in ECM remodeling due to their collagen crosslinking and intracellular functions. Here,we systematically investigated LOX/L expression in primary fibroblasts and epithelial cells under fibrotic conditions,Bleomycin (BLM) induced lung fibrosis and in human IPF tissue. Basal expression of all LOX/L family members was detected in epithelial cells and at higher levels in fibroblasts. Various pro-fibrotic stimuli broadly induced LOX/L expression in fibroblasts,whereas specific induction of LOXL2 and partially LOX was observed in epithelial cells. Immunohistochemical analysis of lung tissue from 14 IPF patients and healthy donors revealed strong induction of LOX and LOXL2 in bronchial and alveolar epithelium as well as fibroblastic foci. Using siRNA experiments we observed that LOXL2 and LOXL3 were crucial for fibroblast-to-myofibroblast transition (FMT). As FMT could only be reconstituted with an enzymatically active LOXL2 variant,we conclude that LOXL2 enzymatic function is crucial for fibroblast transdifferentiation. In summary,our study provides a comprehensive analysis of the LOX/L family in fibrotic lung disease and indicates prominent roles for LOXL2/3 in fibroblast activation and LOX/LOXL2 in IPF. View Publication