Therapeutic splice modulation of COL4A5 reinstates collagen IV assembly in an organoid model of X-linked Alport syndrome

Kidney organoids are an emerging tool for disease modeling, especially genetic diseases. Among them, 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 5 subunit of type IV collagen [5(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 wild-type 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 5(IV) at basement membranes in organoids carrying the splicing variants identified in patients, we demonstrate the efficacy of ASO treatment for 5(IV) restoration. These data contribute to our understanding of the development of GBM and pave the way for a therapeutic screening platform for patients.