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 6

Evaluation of ASO efficacy in correcting splicing defects in patient-derived fibroblasts with deep-intronic COL4A5 variants.

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Evaluation of ASO efficacy in correcting splicing defects in patient-der...
(A and B) Schematic representation of the COL4A5 variants identified in patients P3 (A) (c.277+560T>G) and P14 (B) (c.609+879A>G), which create an intronic splice enhancer (ISE) and new donor splice site, respectively, leading to intron retention in both cases. Splice Site Finder (SSF) (Alamut Visual Plus software) score for each site is shown. (C and D) RT-PCR analysis of ASO-treated and untreated fibroblasts, where ASO treatments reduced the levels of the mutant transcript and increased the proportion of the WT transcript. In P14, 2 faint bands are observed, which are due to the degradation of mutant mRNA mediated by nonsense-mediated decay, and to a low residual WT production. (E and F) Quantification of the relative expression of WT and mutant transcripts across treatments by fragment analysis (n = 3, 2-way ANOVA), showing the increase of WT/mutant transcript ratio after ASO treatment. (G and H) RT-qPCR quantification of COL4A5 mRNA normalized to HPRT1 shows a significant increase after ASO treatments with P value indicated above each bar plot (n = 3, ANOVA with Tukey’s multiple-comparison test). (I and J) Sashimi plots for untreated and ASO-treated samples, showing changes in splice junction usage before and after treatment.