Combined cellular and gene therapy to treat primary ciliary dyskinesia

Abstract

Primary Ciliary Dyskinesia (PCD) is a genetic disease caused by mutations that alter cilia beating, including in the respiratory airways. This results in poor mucus clearance, severe morbidity, and increased mortality. We hypothesized that bronchial cilia beating can be restored using genetically corrected iPSC differentiated into air - liquid interface bronchial epithelium model (iALI) for autologous cell therapy. We have previously demonstrated that corrected cells derived from a PCD patient iPS line can be differentiated into iALI with functional ciliary beating. The current aim of the project is to evaluate the engraftment potential of these corrected cells and their ability to repair the pathological model post-engraftment. Several key issues need to be addressed identifying competent cells for bronchial engraftment, exploring various strategies to pre-treat the bronchial epithelium, and assessing the recovery of the ciliary beating recovery to assure bronchial repair.

Our team has established the differentiation of iPSCs into iALI. We have generated a PCD patient iPSC line using Sendai viruses, a 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 that expresses the fluorescent GFP protein under the human elongation factor 1 alpha promoter (EF1a), which allows us to track the engraftment of GFP-labeled bronchial stem cells in both control and mutated iALI models. Our results suggest that lung progenitors at the ventralized anterior foregut endoderm stage could be the most efficient cells for engraftment. Their self-renewal capability and ability to differentiate into various cell types of the bronchial epithelium are promising for developing a long-term and effective therapy. Regarding bronchial erosion, we found that promoting cell engraftment is crucial due to the barrier function of the intact bronchial epithelium and the lack of selective advantage of corrected cells. Various chemical and enzymatic strategies have shown potential, but their safety for in-vivo use needs further assessment. Furthermore, GFP-expressing engrafted cells displaying cilia suggest successful differentiation into ciliated cells, though functional recovery still requires confirmation.

In conclusion, the engraftment of corrected lung progenitors into eroded bronchial epithelium appears to be a promising therapeutic strategy to PCD. Next step of the project involves developing this therapy for in-vivo application, assessing its safety and efficacy in an immunodeficient mini-pig model.