depletion reduces cystine accumulation without improvement in proximal tubular function in experimental models for cystinosis.

Cystinosis is an autosomal recessive lysosomal storage disorder, caused by mutations in the gene, resulting in an absent or altered cystinosin (CTNS) protein. Cystinosin exports cystine out of the lysosome, with a malfunction resulting in cystine accumulation and a defect in other cystinosin-mediated pathways. Cystinosis is a systemic disease, but the kidneys are the first and most severely affected organs. In the kidney, the disease initially manifests as a generalized dysfunction in the proximal tubules (also called renal Fanconi syndrome). MFSD12 is a lysosomal cysteine importer that directly affects the cystine levels in melanoma cells, HEK293T cells, and cystinosis patient-derived fibroblasts. In this study, we aimed to evaluate mRNA levels in cystinosis patient-derived proximal tubular epithelial cells (ciPTECs) and to study the effect of knockout on cystine levels. We showed similar mRNA expression in patient-derived ciPTECs in comparison with the control cells. CRISPR knockout in a patient-derived ciPTEC () resulted in significantly reduced cystine levels. Furthermore, we evaluated proximal tubular reabsorption after injection of translation-blocking morpholino (TB MO) in a zebrafish model. This resulted in decreased cystine levels but caused a concentration-dependent increase in embryo dysmorphism. Furthermore, the TB MO injection did not improve proximal tubular reabsorption or megalin expression. In conclusion, mRNA depletion reduced cystine levels in both tested models without improvement of the proximal tubular function in the zebrafish embryo. In addition, the apparent toxicity of higher TB MO concentrations on the zebrafish development warrants further evaluation. In this study, we show that depletion with either CRISPR/Cas9-mediated gene editing or a translation-blocking morpholino significantly reduced cystine levels in cystinosis ciPTECs and zebrafish embryos, respectively. However, we observed no improvement in the proximal tubular reabsorption of dextran in the zebrafish embryos injected with translation-blocking morpholino. Furthermore, a negative effect of the morpholino on the zebrafish development warrants further investigation.