Combined cellular and gene therapy to treat primary ciliary dyskinesia

Abstract

Introduction

Primary ciliary dyskinesia (PCD) is a genetic disease caused by mutations that alter cilia beating, including in the respiratory airways, resulting in impaired mucus clearance and severe morbidity as well as increased mortality. We hypothesized that we could restore bronchial cilia beating with genetically corrected iPSC differentiated into bronchial progenitors.

Methods

Our project aims to assess the ability of a corrected iPSC line to functionally repair pathological models in vitro. We generated a PCD patient iPSC line reprogrammed using Sendai viruses, and the corresponding CRISPR/Cas9 corrected cell line, as well a wild-type iPSC line and its CRISPR/Cas9 mutated counterpart. We also generated a GFP-iPSC line expressing the fluorescent GFP protein under the human elongation factor 1 alpha promoter (EF1a), allowing us to study the engraftment ability of GFP bronchial stem cells on a control epithelium model. To assess the efficiency of different bronchial progenitors to engraft a bronchial epithelium, we used our previously published air–liquid interface bronchial epithelium model (iALI).

Results

One main issue is to identify the competent cell type for regeneration of the adult bronchial epithelium. Indeed, there are several cell types constituting the bronchial epithelium, as well as several developmentally bronchial progenitor cells that could be considered. Our iALI differentiation process mimics the embryonic development and thus the iALI model may provide any cell type from the definitive endoderm to the mature bronchial epithelium. Our results suggest that lung progenitors at the ventralized anterior foregut endoderm stage, could be the most efficient cells for engraftment. Besides, their self-renewal ability and their capacity to differentiate into the different cell type spectrum of the bronchial epithelium are promising for the development of a long-term and efficient therapy. The second issue for bronchial epithelium cell replacement would be to determine the best strategy to erode the bronchi prior to cell therapy. Such an erosion is considered necessary to promote cell engraftment because of the barrier function of the intact bronchial epithelium and the lack of selection advantage from the corrected cells. To this end, we compared mechanical, chemical and enzymatic erosion strategies on the iALI model. Our results suggest a better efficiency with enzymatic erosion, showing a homogeneous detachment of the cells and a better engraftment of cells from the GFP-iPSC line.

Conclusion

In conclusion, engraftment of corrected lung progenitors to enzymatically eroded bronchial epithelium seems to be a promising therapeutic strategy to treat PCD. Future experiments will refine the best condition regarding enzymatic solution (concentration, time exposure) and graft cell number to assure functional recovery of the mucociliary clearance.