CCDC40 mRNA Nanoparticles Improve Ciliary Function in Patient-Derived Airway Cells and Zebrafish
Testing Ciliary Recovery by Replacing the Missing Protein
Delivery of messenger RNA (mRNA) encoding CCDC40 in lipidoid nanoparticles (LNPs) improved ciliary function in patient-derived airway cells and CCDC40-deficient zebrafish. Protein localization was restored in some cells, and animal results differed by administration route. The study assessed functional changes in cultured cells and animals; it was not a clinical trial evaluating patients’ respiratory function.
Primary ciliary dyskinesia (PCD) is a genetic disorder in which impaired ciliary movement reduces the airways’ ability to clear mucus and pathogens. Recurrent airway infections can lead to bronchiectasis and chronic respiratory failure, and pathogenic CCDC40 variants are associated with more severe lung-function decline than several other genotypes. The paper describes current treatments as focused on symptom management, including infection control and secretion clearance, and states that no treatment yet corrects the underlying defect.
CCDC40 helps associated proteins localize to the axoneme, the internal scaffold of cilia. The researchers tested an approach that supplies mRNA so cells can produce the needed protein.
Improved Beating and Local Particle Transport in Patient-Derived Cells
The team cultured nasal respiratory epithelial cells from five patients with pathogenic variants in both CCDC40 alleles at an air–liquid interface. With future inhaled administration in mind, they applied LNP-CCDC40-mRNA to the apical surface of the cultured epithelium once weekly for six weeks.
After four treatments, ciliary beat frequency in patient-derived cells was 3.25 ± 0.08 Hz in the control-mRNA group and 9.48 ± 0.22 Hz in the CCDC40-mRNA group. Cells from six healthy donors measured 8.98 ± 0.08 Hz; there was no statistically significant difference between the CCDC40-mRNA group and the healthy controls. Improved beating was also observed after six treatments, along with improved fluorescent-particle transport. However, particle movement represented local transport within the observed field and does not mean that mucociliary clearance was restored across the entire culture surface.
In cultured cells from four donors for which immunofluorescence analysis was possible, CCDC40 and associated proteins CCDC39, GAS8/DRC4, and DNALI1 were observed to relocalize to the axoneme. CCDC40 localization within the axoneme was restored in 10–74% of multiciliated cells, with a mean of 31.5%, showing substantial donor-to-donor variation. The denominator was the multiciliated cells analyzed; this percentage does not represent delivery efficiency across all airway cells or the treatment success rate in patients. Post-treatment ultrastructure was not assessed by electron microscopy, so full restoration of internal structure was not established.
Route-Dependent Results in Animals and the Need for Clinical Validation
In CCDC40-deficient zebrafish, the researchers tested injection into single-cell-stage embryos and exposure of larvae to drug-containing culture medium beginning 30 hours after fertilization. Medium exposure was not limited to the olfactory pits, and the injection was not delivered directly into them. At five days post-fertilization, olfactory-pit ciliary beat frequency and particle-transport speed in the embryo-injection group approached wild-type levels, and flow directionality improved.
By contrast, the medium-exposure groups at both 10 ng/μL and 160 ng/μL showed no significant difference in beat frequency compared with the untreated deficient group. At the higher concentration of 160 ng/μL, the area showing ciliary beating and particle-transport speed improved, but recovery of flow directionality was partial. Because results differed by administration method, the benefits of embryo injection cannot readily be generalized to inhaled treatment in humans.
This study is a preclinical proof of concept that replacing a deficient protein may improve ciliary function. It used cells from a small number of donors, and the extent of recovery varied. The researchers stated in the paper that they plan a phase 1 trial in people carrying pathogenic CCDC40 variants. The study did not report that a clinical trial had begun or present results establishing safety or efficacy in humans.
Source: Full text of the American Journal of Respiratory Cell and Molecular Biology paper, DOI: 10.1093/ajrcmb/aanag034.
Primary Ciliary Dyskinesia (PCD) is a genetically heterogeneous disorder leading to destructive airway disease with severe bronchiectasis and chronic lung failure in adulthood. Pathogenic variants in CCDC40 are associated with more severe reduction of lung function compared to most other PCD types. Currently, no therapies correcting the underlying disease mechanism are available. Here we investigate the efficacy of lipidoid nanoparticle-formulated mRNA encoding human CCDC40 (LNP-CCDC40-mRNA) as a corrective measure for structural and functional defects in vitro (human cells) and in vivo (zebrafish). Human nasal respiratory epithelial cells cultured at air-liquid-interface from five CCDC40-deficient individuals and a newly generated vertebrate animal model (ccdc40-/- zebrafish) were treated with LNP-CCDC40-mRNA. CCDC40-deficient cells were analyzed by high-speed video microscopy and immunofluorescence microscopy. ccdc40-/- zebrafish olfactory pit cilia were analyzed by high-speed video microscopy and fluid flow assays. Topical application of exogenous LNP-CCDC40-mRNA to CCDC40-deficient cells results in endogenous CCDC40 expression (10-74% of ciliated cells), enabling axonemal integration of CCDC40-associated proteins (CCDC39, GAS8/DRC4, DNALI1). Consistently, ciliary beat frequencies were significantly increased in treated CCDC40-deficient cells and comparable to healthy control cells. Further, we showed improved ciliary transport of fluorescent particles. Injection or topical application of human LNP-CCDC40-mRNA to ccdc40-/- zebrafish significantly increased ciliary motility and established directional flow in olfactory pits. We provide structural and functional evidence in vitro and in vivo for the biological efficacy of LNP-CCDC40-mRNA in CCDC40-deficient respiratory cells and zebrafish. Based on our results, an in vivo human study (Phase 1 trial) is planned in individuals with pathogenic variants in CCDC40.
The study provides a rationale for developing an inhaled treatment candidate that replaces the missing protein in primary ciliary dyskinesia associated with CCDC40 variants. One concrete application is to apply a candidate formulation to patient-derived airway cells and assess axonemal protein localization, ciliary beating, and particle transport together. Measuring beat frequency separately from particle transport can clarify the scope of functional improvement. Before this could become a treatment, studies must determine whether it reaches human airways in sufficient amounts, whether repeated dosing is safe, how long the effect lasts, and whether it translates into improved lung function. Expansion to other PCD genotypes would also require separate validation of gene-specific formulations and delivery performance.