Woodall, M. N. J. | Search bioRxiv

bioRxiv2023

Lung viral infection modelling in a bioengineered whole-organ

Lung infections are one of the leading causes of death worldwide, and this situation has been exacerbated by the emergence of COVID-19. Pre-clinical modelling of viral infections has relied on cell cultures that lack 3D structure and the context of lung extracellular matrices. Here, we propose a bioreactor-based, whole-organ lung model of viral infection. The bioreactor takes advantage of an automated system to achieve efficient decellularization of a whole rat lung, and recellularization of the scaffold using primary human bronchial cells. Automatization allowed for the dynamic culture of airway epithelial cells in a breathing-mimicking setup that led to an even distribution of lung epithelial cells throughout the distal regions. In the sealed bioreactor system, we demonstrate proof-of-concept for viral infection with the engineered lung by infecting primary human airway epithelial cells. Moreover, to assess the possibility of drug screening in this model, we demonstrate the efficacy of the broad-spectrum antiviral Remdesivir. This whole-organ scale lung infection model represents a step towards modelling viral infection of human cells in a 3D context, providing a powerful tool to investigate the mechanisms of the early stages of pathogenic infections and the development of effective treatment strategies for respiratory diseases.

bioengineering↗

bioRxiv2022

K978C CFTR restores essential epithelial function with greater efficiency than wildtype CFTR when expressed in CF airway cells.

Class Ia/b CFTR variants cause severe cystic fibrosis (CF) lung disease in ~10% of CF patients and are untreatable with small molecule pharmaceuticals. Genetic replacement strategies offer a potential cure for all patients but so far, have displayed limited efficiency in vivo. We hypothesised that increasing protein abundance and/or activity of introduced CFTRs would more effectively restore function to CF bronchial epithelial cells (CFBE) in the presence of CF sputum (CFS) than wildtype (WT)-CFTR. We investigated codon optimised CFTR (hCAI), increased open probability CFTR (K978C) and codon optimised plus K978C (h^K978C) as candidates for gene therapy. Transfection of HEK293T with hCAI and h^K978C produced ~10-fold more CFTR protein than WT or K978C CFTRs. hCAI and h^K978C also displayed ~4-fold greater anion transport than WT in a halide-sensitive YFP quenching assay. However, functionality of modified CFTR cDNAs expressed in CFBE were profoundly different. 10% transduction of CFBE with K978C, compared to 22% transduction with WT, restored Cl- transport to similar levels as that recorded from non-CF cells. K978C increased ASL height and pH more effectively than WT-CFTR, while hCAI and h^K978C had limited impact. Further investigation indicated that codon optimised CFTRs mis-localised in CFBE and compromised vectoral Cl- transport. These data provide further evidence that codon optimised CFTR cDNAs may be unsuitable for gene therapy practices that employ high activity promoters. However, increased activity CFTR cDNAs such as K978C, that potentially mimic the effect of potentiators, may provide more potent recovery of function than WT-CFTR cDNA in CF airways. Significance StatementCystic fibrosis (CF) disease is associated with genetic malfunction of the Cl- channel CFTR, leading to dehydration and decreased pH in the fluid lining the airways. Replacement of CFTR by gene therapy/gene editing offers potential therapeutic benefit but efficiency is poor. We show that gain of activity K978C CFTR under the control of a high activity promoter fully restored Cl- transport, hydration and pH to CF bronchial epithelial cells (CFBE) in the presence of CF sputum and more efficiently than wild type CFTR. Codon optimised forms of CFTR were much less effective and proteins were mis-localised/mis-processed in CFBE. Thus, K978C could offer improved therapeutic potential.

molecular biology↗