Development of a Zebrafish model for primary ciliary dyskinesia to validate gene editing approaches applied ex-vivo

Gene therapy aims to restore the normal structure and function of the respiratory cilia of PCD patients. The cells, obtained by nasal brushing, will be cultured and tested before and after treatment with instrumental, cellular, and molecular systems to evaluate the changes and the restoration of normal ciliary function. To confirm the applicability and feasibility of this approach, we intend to demonstrate that it is possible to develop personalized gene therapy for PCD to correct the genetic defect and restore ciliary motility with a significant clinical improvement. This represents the experimental basis to start a preclinical study on the Zebrafish model. Zebrafish mutants for PCD causing genes can be used as a model that replicates the pathology of PCD regarding etiology, pathophysiology, symptomatology, and response to therapeutic intervention. The Zebrafish olfactory pit cilia resemble multiple motile cilia of the human respiratory tract and the use of zebrafish for whole animal testing is an important stage in gene therapy development, providing proof of principle of true efficacy as well as safety in vivo. Analysis of the structural and localization correction of proteins will be used as an outcome measure to evaluate the impact of gene editing on ciliary structure and function in both human cell cultures and zebrafish (immunofluorescence and high-speed video microscopy analysis). Replacement of mutation with wild-type sequence will be confirmed by nucleic acid sequencing.