Madeline Tomaske

Abstract Derangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimer’s disease. The Norrin/FZD 4 /TSPAN12 pathway activates WNT/β-catenin signaling, which is essential for BBB and BRB function. However, systemic pharmacologic FZD 4 stimulation is hindered by obligate palmitoylation and insolubility of native WNTs and suboptimal properties of the FZD 4 -selective ligand Norrin. Here, we develop L6-F4-2, a non-lipidated, FZD 4 -specific surrogate which significantly improves subpicomolar affinity versus native Norrin. In Norrin knockout ( Ndp KO ) mice, L6-F4-2 not only potently reverses neonatal retinal angiogenesis deficits, but also restores BRB and BBB function. In adult C57Bl/6J mice, post-stroke systemic delivery of L6-F4-2 strongly reduces BBB permeability, infarction, and edema, while improving neurologic score and capillary pericyte coverage. Our findings reveal systemic efficacy of a bioengineered FZD 4 -selective WNT surrogate during ischemic BBB dysfunction, with potential applicability to adult CNS disorders characterized by an aberrant blood-brain barrier.

Understanding how genes influence drug responses is critical for advancing personalized cancer treatments. However, identifying these gene-drug interactions in a physiologically relevant human system remains a challenge, as it requires a model that reflects the complexity and heterogeneity among individuals. Here we show that large-scale CRISPR-based genetic screens, including knockout, interference (CRISPRi), activation (CRISPRa), and single-cell approaches, can be applied in primary human 3D gastric organoids to systematically identify genes that affect sensitivity to cisplatin. Our screens uncover genes that modulate cisplatin response. By combining CRISPR perturbations with single-cell transcriptomics, we resolve how genetic alterations interact with cisplatin at the level of individual cells and uncover an unexpected link between fucosylation and cisplatin sensitivity. We identify TAF6L as a regulator of cell recovery from cisplatin-induced cytotoxicity. These results highlight the utility of human organoid models for dissecting gene-drug interactions and offer insights into therapeutic vulnerabilities in gastric cancer. CRISPR-Cas9-based screens have allowed the study of gene-drug interactions. Here, the authors develop CRISPR-Cas9 knock-out, activation and repression screens in human gastric 3D organoids, also integrating single-cell CRISPR screens, to identify genes involved in the response to cisplatin in gastric cancer.