AAV-Mediated Base Editing for Correction of RSPH4A Mutations in Primary Ciliary Dyskinesia: A Proof-of-Concept Study.

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous disorder, with abnormal ciliary motility, usually due to an ultrastructural defect, with chronic airway infections. Currently, no curative therapy exists for PCD. Given the prevalence of single nucleotide variants (SNVs) among causative mutations, we evaluated a novel base-editing approach. Specifically, we used a nickase Cas9 fused to adenosine deaminase to correct mutations in the radial spoke head component 4 A (RSPH4A) gene, causing PCD. We selected two PCD patients sharing the same SNV in RSPH4A, one with compound heterozygosity (child, patient 1) and one with homozygosity (adult, patient 2). After designing gRNAs, HEK293T cells with or without a DNA fragment containing the SNV in RSPH4A, were co-transfected with base editor plasmids. Complex formation and editing efficiency were validated by Western blot and digital PCR. We then treated patient cells with AAV containing the base editors and assessed ciliary beat frequency and motion pattern using high-speed video and confocal microscopy to evaluate delivery.Base editor complexes formed efficiently in vitro. AAV-mediated delivery in patient 1 cells led to an approximately 30.4% increase in normal motion pattern, with a corresponding reduction in circular motions (p < 0.001) compared with pre-treatment, and a 20% of editing efficiency detected by dPCR in transduced cells. Our data indicate that this limited editing efficiency is due to reduced AAV penetration in the lower layers of cells.This proof-of-concept study demonstrates the therapeutic potential of base editing for PCD, though current limitations include low editing efficiency and restricted delivery to inner cell layers in our experimental model. Future work should focus on optimizing base editors and testing novel delivery strategies to target progenitor cells, thereby enhancing the prospects for personalized gene therapy in PCD.