mRNA transcript therapy of primary respiratory epithelial cells derived from PCD individuals with bi-allelic CCDC40 mutations

Introduction Primary Ciliary Dyskinesia (PCD) is characterized by impaired mucociliary clearance and progressive airway damage due to dysfunctional motile cilia in the airways and more than 50 genes are associated with PCD. Clinical care is symptomatic and no causal treatment is available. We studied the feasibility of mRNA therapy to correct the molecular defect in human respiratory epithelial cells (hRECs) from PCD individuals with bi-allelic disease-causing variants in Coiled-Coil Domain Containing 40 (CCDC40). In CCDC40 deficient cells, ciliary beating is hampered due to microtubular disorganization, and loss of CCDC40 binding partners Coiled-Coil Domain Containing 39 (CCDC39), Growth Arrest Specific 8 (GAS8) and Dynein Axonemal Light Intermediate Chain 1 (DNALI1). Here, we provide proof-of-concept (PoC) that Stabilized Non-Immunogenic mRNA (SNIM RNA) rescues ciliary defects.

Methods hRECs were obtained by nasal brush biopsy from healthy donors and CCDC40-individuals, cultured at air-liquid interface and treated once per week over 6 weeks either with LF92-tdTomato-SNIM RNA as control or LF92-CCDC40-SNIM RNA. Video microscopy analysis of the ciliary beat frequency (CBF) and immunofluorescence analysis (IF) was performed to assess correction of ciliary [WK1] defects and motility. LF92-SNIM RNA related cytotoxicity was measured throughout treatment period.

Results After six weeks of LF92-tdTomato-SNIM ​RNA treatment, hRECs of healthy donors beat at a frequency of 8.921±0,096 Hz, whereas hRECs of CCDC40 individuals show a reduced CBF of 2.524±0.160 Hz. After six weeks of LF92-CCDC40-SNIM ​RNA treatment, hRECs of the same CCDC40 individuals show a CBF of 10.28±0.196 Hz, comparable healthy control hRECs. IF demonstrated proper ciliary localization of CCDC40 dependent proteins CCDC39, GAS8 and DNALI1 in healthy control hRECs and in LF92-CCDC40-SNIM RNA treated CCDC40-deficient hRECs, but absence of those proteins in LF92-tdTomato-SNIM ​ RNA treated CCDC40-deficient hRECs. LDH release as indication of cytotoxicity was stable over the experimental period, with values correlating to spontaneous LDH activity from untreated cells.

Discussion Here, we provide PoC that a novel therapeutic approach, based on SNIM RNA, corrects the basic structural defects in ciliary axonemes of CCDC40-PCD individuals. Together with low immunogenic potential, this approach has the potential to become a gene specific treatment option in PCD.