Development of robust antiviral assays using relevant apical-out human airway organoids.

While breakthroughs with organoids have emerged as next-generation in vitro tools, standardization for drug discovery remains a challenge. This work introduces human airway organoids with reversed biopolarity (AORBs), cultured and analyzed in a high-throughput, single-organoid-per-well format, enabling milestones towards standardization. AORBs exhibit a spatio-temporally stable apical-out morphology, facilitating high-yield direct intact-organoid virus infection. Single-cell RNA sequencing and immunohistochemistry confirm the physiologically relevant recapitulation of differentiated human airway epithelia. The cellular tropism of five severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains along with host response differences between Delta, Washington, and Omicron variants, as observed in transcriptomic profiles, also suggest clinical relevance. Dose-response analysis of three well-studied SARS-CoV-2 antiviral compounds (remdesivir, bemnifosbuvir, and nirmatrelvir) demonstrates that AORBs efficiently predict human efficacy, comparable to gold-standard air-liquid interface cultures, but with higher throughput (∼10-fold) and fewer cells (∼100-fold). This combination of throughput and relevance allows AORBs to robustly detect false negative results in efficacy, preventing irretrievable loss of promising lead compounds. While this work leverages the SARS-CoV-2 study as a proof-of-concept application, the standardization capacity of AORB holds broader implications in line with regulatory efforts to push alternatives to animal studies.

Post-zygotic rescue of meiotic errors causes brain mosaicism and focal epilepsy.

Somatic mosaicism is a known cause of neurological disorders, including developmental brain malformations and epilepsy. Brain mosaicism is traditionally attributed to post-zygotic genetic alterations arising in fetal development. Here we describe post-zygotic rescue of meiotic errors as an alternate origin of brain mosaicism in patients with focal epilepsy who have mosaic chromosome 1q copy number gains. Genomic analysis showed evidence of an extra parentally derived chromosome 1q allele in the resected brain tissue from five of six patients. This copy number gain is observed only in patient brain tissue, but not in blood or buccal cells, and is strongly enriched in astrocytes. Astrocytes carrying chromosome 1q gains exhibit distinct gene expression signatures and hyaline inclusions, supporting a novel genetic association for astrocytic inclusions in epilepsy. Further, these data demonstrate an alternate mechanism of brain chromosomal mosaicism, with parentally derived copy number gain isolated to brain, reflecting rescue in other tissues during development.