Investigating the influence of nanoparticle-mediated FOXJ1 messenger RNA delivery on human bronchial epithelial cell differentiation
File(s)
Author(s)
Konstantinidi, Rafaela
Type
Thesis
Abstract
Synthetic messenger RNA (mRNA) presents a promising tool for transient expression of any protein of interest, when efficiently delivered to target cells. Culturing primary human bronchial epithelial cells (HBECs) at air-liquid interface (ALI) results in a pseudostratified differentiated epithelium that closely resembles the cellular architecture found in the human airway. This model provides a powerful tool for studying HBEC biology and manipulating gene expression to modulate differentiation. However, the impact of synthetic mRNA delivery on epithelial differentiation and barrier integrity remains unclear. This thesis first aimed to establish non-viral mRNA transfection of primary HBECs and investigate the impact of reporter mRNA delivery on HBEC differentiation and integrity at ALI. In Chapter 3, formulations of poly (-amino ester) (PBAE), a cationic polymer known for its gene delivery capabilities, were optimised for efficient reporter mRNA delivery to submerged primary HBECs. Transfection of primary HBECs with reporter mRNA prior to air exposure did not hinder their differentiation or barrier integrity at ALI. Building on these findings, Chapter 4 explored the impact of delivering a physiologically relevant mRNA encoding the transcription factor (TF), Forkhead box J1 (FOXJ1), on HBEC integrity and differentiation at ALI. PBAE-mediated FOXJ1 mRNA delivery successfully induced the expression of FOXJ1 downstream genes involved in ciliogenesis. FOXJ1 mRNA transfected cells maintained at ALI for 28 days exhibited differentiation, epithelial integrity, and cilia ultrastructure comparable to non-transfected controls. Finally, Chapter 5 investigated PBAE modifications to enhance mRNA transfection efficiency in HBEC ALI cultures. A novel peptide-modified PBAE, DD90-Gly-Sar, improved mRNA transfection of day 7 and 14 HBEC ALI cultures. Overall, this thesis demonstrates the potential of PBAE-mediated mRNA delivery to primary HBECs and the utility of mRNA-encoded TF as a platform technology for modulating HBEC differentiation in ALI, opening new avenues for investigating gene function implicated in airway diseases.
Version
Open Access
Date Issued
2024-01-13
Date Awarded
01/07/2024
Advisor
Patel, Asha K
Lloyd, Clare
Saglani, Sejal
Yates, Laura
Sponsor
Imperial College London
Publisher Department
National Heart & Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)