Splice modulation of COL4A5 reinstates collagen IV assembly in an organoid model of Alport syndrome
Hassan Saei, Bruno Estebe, Nicolas Goudin, Mahsa Esmailpour, Julie Haure, Olivier Gribouval, Christelle Arrondel, Vincent Moriniere, Pinyuan Tian, Rachel Lennon, Corinne Antignac, Geraldine Mollet, Guillaume Dorval
Hassan Saei, Bruno Estebe, Nicolas Goudin, Mahsa Esmailpour, Julie Haure, Olivier Gribouval, Christelle Arrondel, Vincent Moriniere, Pinyuan Tian, Rachel Lennon, Corinne Antignac, Geraldine Mollet, Guillaume Dorval
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Research Article Genetics Nephrology

Splice modulation of COL4A5 reinstates collagen IV assembly in an organoid model of Alport syndrome

Abstract

Kidney organoids are an emerging tool for disease modeling, especially genetic diseases. Among these diseases, X-linked Alport syndrome (XLAS) is a hematuric nephropathy affecting the glomerular basement membrane (GBM) secondary to pathogenic variations in the COL4A5 gene encoding the α5 subunit of type IV collagen [α5(IV)]. In patients carrying pathogenic variations affecting splicing, the use of antisense oligonucleotides (ASOs) offers immense therapeutic hope. In this study, we develop a framework combining the use of patient-derived cells and kidney organoids to provide evidence of the therapeutic efficacy of ASOs in XLAS patients. Using multiomics analysis, we describe the development of GBM in WT and mutated human kidney organoids. We show that GBM maturation is a dynamic process, which requires long organoid culture. Then, using semi-automated quantification of α5(IV) at basement membranes in organoids carrying the splicing variants identified in patients, we demonstrate the efficacy of ASO treatment for α5(IV) restoration. These data contribute to our understanding of the development of GBM in kidney organoids and pave the way for a therapeutic screening platform for patients.

Authors

Hassan Saei, Bruno Estebe, Nicolas Goudin, Mahsa Esmailpour, Julie Haure, Olivier Gribouval, Christelle Arrondel, Vincent Moriniere, Pinyuan Tian, Rachel Lennon, Corinne Antignac, Geraldine Mollet, Guillaume Dorval

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Figure 1

Multiomics characterization of kidney organoids in prolonged culture.

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Multiomics characterization of kidney organoids in prolonged culture. (A...
(A) Kidney organoid differentiation protocol transitioning from 2D to 3D cultures, with focus on prolonged culture. (B) Immunofluorescence and hematoxylin and eosin (H&E) staining of kidney organoids at day 22 and day 38, demonstrating their structural integrity. (C) Transmission electron microscopy image of an organoid (day 38), highlighting glomerular structures with podocytes (P), primary and secondary-like foot processes (PFP and SFP), glomerular basement membrane (GBM), and parietal epithelial-like cells (PEC) (original magnifications: left image, ×1,250; top image, ×4,300; bottom image, ×8,500). (D) Multidimensional scaling plot for transcriptomic and proteome data, showing distinct clustering of samples from each time point (early, mid, and late). (E) Cross-matching of RNA and protein data (left: mid versus early; right: late versus mid) identified 830 shared differentially regulated biomolecules (genes and proteins) comparing late versus mid groups. (F) Gene Ontology enrichment analysis (GO:CC, cellular components; GO:BP, biological processes; GO:MF, molecular function) of 830 shared differentially regulated genes (fold change, 1.2) and proteins (FDR, 0.05) obtained from late versus mid culture comparison. (G) Heatmap visualization of differentially expressed ECM structural genes and proteins over time comparing early and late. (H) Heatmap of type IV collagen and laminin gene expression from bulk RNA-seq. (I) RT-qPCR quantification of different chains of type IV collagen of kidney organoids harvested at early, mid, and late culture, confirming GBM maturation and dynamics in prolonged culture (2-way ANOVA with Tukey’s multiple-comparison test).