Targeting mucus hyperproduction in COPD using siRNA-loaded switchable lipid nanoparticles to silence SPDEF

Abstract

Introduction

Mucus hyperproduction is a key component of chronic obstructive pulmonary disease (COPD), participating to airflow limitation and associated with an increased all-cause mortality. SAM-pointed domain-containing ETS transcription factor (SPDEF) is an intracellular transcription factor required for goblet cell differentiation. Downregulating SPDEF expression using siRNA is a therapeutic option to reduce mucus hyperconcentration and restore mucociliary clearance. However, several biological barriers such as potential immunostimulatory effects, mucus penetration and bronchial epithelial delivery still hamper the efficacy of RNA therapies for lung diseases. Here, using switchable lipid nanoparticles (LNPs) to deliver SPDEF siRNA, we aim at evaluating the potential of targeting SPDEF in relevant human models including an air-liquid interface.

Methods

Primary bronchial epithelial cells derived from pulmonary samples were collected after thoracic surgery of COPD and non-COPD patients. Cells were cultivated at the air-liquid interface to obtain a fully differentiated epithelium with a mucus layer. Lipid nanoparticles were prepared by rapid mixing of lipids in ethanol with siRNA targeted against SPEDF in PBS buffer. After the exposure of epithelial cells to siRNA-LNPs during 4 h, siRNA uptake was evaluated by flow cytometry and confocal imaging. Silencing efficiency was assessed by RT-qPCR and western blot 48 and 72 h after exposure.

Results

The siRNA-LNP were able to efficiently penetrate into differentiated cells in ALI culture. Confocal imaging confirmed that siRNA have crossed the mucus layer and penetrated within the cytoplasm of epithelial cells. SPDEF silencing was achieved 48 hours after siRNA-LNPs exposure at the RNA level, and at 72 h at the protein level. The level of silencing was unchanged in cells derived from control subjects compared with those obtained from patients with COPD.

Conclusion

LNPs are able to overcome the mucus layer and are internalized into differentiated epithelial cells of a translational patient-derived model. This strategy can be used to deliver SPDEF siRNA, that efficiently downregulate SPDEF expression. These results highlight the potential of this switchable lipid nanoparticle formulation to carry out siRNA drugs for COPD treatment.