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 3

Immunofluorescence staining of kidney organoids highlighting the localization of various junctional and ECM-associated proteins relevant to podocyte maturation.

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Immunofluorescence staining of kidney organoids highlighting the localiz...
(A) Spatial distribution of podocalyxin (in green) and type IV collagen (in red) in organoid (day 38 of culture). Podocalyxin is mainly localized at the apical side of the podocytes, while type IV collagen (captures all chains α1–α6) is present at the basal side, forming the GBM. Scale bars: 50 μm. (B) Colocalization of tight junction protein (ZO-1) (in orange), nephrin (in red), and collagen α5(IV) (in green), at the basal side. Scale bars: 50 μm. (C) Type IV collagen network localization and abundance at days 22 and 38 of culture. This panel confirms the increased production of collagen α3(IV) and α4(IV) chains at day 38 and their deposition in the GBM (white arrows point to α3α4α5 and arrowheads point to α5α6α5 heterotrimers in the PEC-BM and TBM). Scale bars: 100 μm.