L1077P CFTR pathogenic variant function rescue by Elexacaftor-Tezacaftor-Ivacaftor in cystic fibrosis patient-derived air-liquid interface (ALI) cultures and organoids: in vitro guided personalized therapy of non-F508del patients.

Cystic fibrosis (CF) is caused by defects of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR-modulating drugs may overcome specific defects, such as the case of Trikafta, which is a clinically approved triple combination of Elexacaftor, Tezacaftor and Ivacaftor (ETI) that exhibited a strong ability to rescue the function of the most frequent F508del pathogenic variant even in genotypes with the mutated allele in single copy. Nevertheless, most rare genotypes lacking the F508del allele are still not eligible for targeted therapies. Via the innovative approach of using nasal conditionally reprogrammed cell (CRC) cell-based models that mimic patient disease in vitro, which are obtainable from each patient due to the 100% efficiency of the cell culture establishment, we theratyped orphan CFTR mutation L1077P. Protein studies, Forskolin-induced organoid swelling, and Ussing chamber assays congruently proved the L1077P variant function rescue by ETI. Notably, this rescue takes place even in the context of a single-copy L1077P allele, which appears to enhance its expression. Thus, the possibility of single-allele treatment also arises for rare genotypes, with an allele-specific modulation as part of the mechanism. Of note, besides providing indication of drug efficacy with respect to specific CFTR pathogenic variants or genotypes, this approach allows the evaluation of the response of single-patient cells within their genetic background. In this view, our studies support in vitro guided personalized CF therapies also for rare patients who are nearly excluded from clinical trials.

Drug Repurposing for Cystic Fibrosis: Identification of Drugs That Induce CFTR-Independent Fluid Secretion in Nasal Organoids.

Individuals with cystic fibrosis (CF) suffer from severe respiratory disease due to a genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which impairs airway epithelial ion and fluid secretion. New CFTR modulators that restore mutant CFTR function have been recently approved for a large group of people with CF (pwCF), but ~19% of pwCF cannot benefit from CFTR modulators Restoration of epithelial fluid secretion through non-CFTR pathways might be an effective treatment for all pwCF. Here, we developed a medium-throughput 384-well screening assay using nasal CF airway epithelial organoids, with the aim to repurpose FDA-approved drugs as modulators of non-CFTR-dependent epithelial fluid secretion. From a ~1400 FDA-approved drug library, we identified and validated 12 FDA-approved drugs that induced CFTR-independent fluid secretion. Among the hits were several cAMP-mediating drugs, including ß2-adrenergic agonists. The hits displayed no effects on chloride conductance measured in the Ussing chamber, and fluid secretion was not affected by TMEM16A, as demonstrated by knockout (KO) experiments in primary nasal epithelial cells. Altogether, our results demonstrate the use of primary nasal airway cells for medium-scale drug screening, target validation with a highly efficient protocol for generating CRISPR-Cas9 KO cells and identification of compounds which induce fluid secretion in a CFTR- and TMEM16A-indepent manner.

Infant-derived human nasal organoids exhibit relatively increased susceptibility, epithelial responses, and cytotoxicity during RSV infection.

BACKGROUND: Respiratory syncytial virus (RSV) causes significant morbidity and mortality, especially in young children. Why RSV infection in children is more severe compared to healthy adults is not fully understood. METHODS: We used ex-vivo human nasal organoid platforms from infants and adults to investigate the underlying mechanism of this disease disparity at the initial site of RSV replication, the nasal epithelium. RESULTS: Infant-derived human nasal organoid-air liquid interface (HNO-ALIs) lines were more susceptible to early RSV replication. Moreover, infant-derived HNO-ALIs elicited a statistically significant greater overall cytokine response, enhanced mucous production, and greater cellular damage compared to their adult counterparts. Furthermore, the adult cytokine response was associated with a superior regulatory cytokine response, which could explain less cellular damage than in infant lines. CONCLUSIONS: Our data highlights substantial differences in how infant and adult upper respiratory tract epithelium responds to RSV infection at the cellular level. These differences in epithelial cellular response can lead to impaired mucociliary clearance, a more dysregulated innate immune response predisposing infants to more severe RSV infection compared to adults.

Nasal microbionts differentially colonize and elicit cytokines in human nasal epithelial organoids.

Nasal colonization by Staphylococcus aureus or Streptococcus pneumoniae is associated with an increased risk of infection by these pathobionts, whereas nasal colonization by Dolosigranulum species is associated with health. Human nasal epithelial organoids (HNOs) physiologically recapitulate human nasal respiratory epithelium with a robust mucociliary blanket. We reproducibly monocolonized HNOs with these three bacteria for up to 48 hours with varying kinetics across species. HNOs tolerated bacterial monocolonization with localization of bacteria to the mucus layer and minimal cytotoxicity compared to uncolonized HNOs. Human nasal epithelium exhibited both species-specific and general cytokine responses, without induction of type I interferons, consistent with colonization rather than infection. Only live S. aureus colonization induced IL-1 family cytokines, suggestive of inflammasome signaling. D. pigrum and live S. aureus decreased CXCL10, whereas S. pneumoniae increased CXCL11, chemokines involved in antimicrobial responses. HNOs are a compelling model system to reveal host-microbe dynamics at the human nasal mucosa.

Human Nasal Organoids Model SARS-CoV-2 Upper Respiratory Infection and Recapitulate the Differential Infectivity of Emerging Variants.

The human upper respiratory tract, specifically the nasopharyngeal epithelium, is the entry portal and primary infection site of respiratory viruses. Productive infection of SARS-CoV-2 in the nasal epithelium constitutes the cellular basis of viral pathogenesis and transmissibility. Yet a robust and well-characterized in vitro model of the nasal epithelium remained elusive. Here we report an organoid culture system of the nasal epithelium. We derived nasal organoids from easily accessible nasal epithelial cells with a perfect establishment rate. The derived nasal organoids were consecutively passaged for over 6 months. We then established differentiation protocols to generate 3-dimensional differentiated nasal organoids and organoid monolayers of 2-dimensional format that faithfully simulate the nasal epithelium. Moreover, when differentiated under a slightly acidic pH, the nasal organoid monolayers represented the optimal correlate of the native nasal epithelium for modeling the high infectivity of SARS-CoV-2, superior to all existing organoid models. Notably, the differentiated nasal organoid monolayers accurately recapitulated higher infectivity and replicative fitness of the Omicron variant than the prior variants. SARS-CoV-2, especially the more transmissible Delta and Omicron variants, destroyed ciliated cells and disassembled tight junctions, thereby facilitating virus spread and transmission. In conclusion, we establish a robust organoid culture system of the human nasal epithelium for modeling upper respiratory infections and provide a physiologically-relevant model for assessing the infectivity of SARS-CoV-2 emerging variants. IMPORTANCE An in vitro model of the nasal epithelium is imperative for understanding cell biology and virus-host interaction in the human upper respiratory tract. Here we report an organoid culture system of the nasal epithelium. Nasal organoids were derived from readily accessible nasal epithelial cells with perfect efficiency and stably expanded for more than 6 months. The long-term expandable nasal organoids were induced maturation into differentiated nasal organoids that morphologically and functionally simulate the nasal epithelium. The differentiated nasal organoids adequately recapitulated the higher infectivity and replicative fitness of SARS-CoV-2 emerging variants than the ancestral strain and revealed viral pathogenesis such as ciliary damage and tight junction disruption. Overall, we established a human nasal organoid culture system that enables a highly efficient reconstruction and stable expansion of the human nasal epithelium in culture plates, thus providing a facile and robust tool in the toolbox of microbiologists.