Transfection of cystinosis patient (CYS) derived proximal tubular epithelial cells (PTECs) and podocytes (PODOs) with CTNS-3HA mRNA results in lysosomal expression of cystinosin-3HA, reduction of cystine levels and reduced inulin leakage at 24 h post-transfection. (a) Cystinosin-3HA expression (green) after transfection of CYS PTECs and PODOs with 500 ng/ml CTNS-3HA mRNA (bottom) as compared with untreated cells (top). Scale bar = 100 µm. (b) Quantification of PTECs and PODOs expressing cystinosin-3HA in randomly acquired microscopical fields (each dot is one field from 3 independent experiments). Images were obtained using a Operetta CLS High Content Screening Microscope. Median and 95% CI are indicated. Data were analysed with Mann–Whitney tests. *p < 0.05. (c–f) Lysosomal localization was demonstrated at 24 h post-transfection by co-staining of cystinosin-3HA (green) with LAMP1 (red) in confocal images in a 2D cell model (c,d) (scale bar = 20 µm) and in PTECs on the hollow fiber membranes (HFM—e,f) (scale bar = 50 µm and 20 µm). (g,h) Reduction of lysosomal cystine accumulation in PTECs (g) and PODOs (h) at 24h post-transfection with increasing concentrations of CTNS-3HA mRNA. Cellular cystine levels were analysed by LC–MS. CTNS-3HA mRNA treated cells were compared with the GFP control using Kruskal Wallis test (PTECs) and Welch’s ANOVA (PODOs) (*). A second comparison between the transfected cells and wildtype control (Kruskal–Wallis—^#) was performed. Median and 95% CI are represented for PTECs and mean with SEM for PODOs (n = 3 independent experiments). *p < 0.05; ***^,###p < 0.001; ****p < 0.0001. (i) At 24 h post-transfection, cells treated with CTNS-3HA mRNA show a reduced perinuclear LAMP1 signal (% of total, Kruskal–Wallis test—*). The (peripheral) distribution in the CTNS-3HA treated PTECs was comparable to the wildtype control (Kruskal–Wallis test—^#) (n = 120 cells). Median with 95% CI is indicated. **p < 0.01; ****^,####p < 0.0001. (i) The fold change leakage of FITC-inulin (relative to untreated PTECs) was evaluated in PTECs cultured on the hollow fiber membranes at 24 h post-transfection (n = 3 biological replicates). Data were evaluated with an one-way ANOVA. Mean and SEM are shown. **p < 0.01; ****p < 0.01.

Transfection of cystinosis patient (CYS) derived proximal tubular epithelial cells (PTECs) and podocytes (PODOs) results in functional cystinosin-3HA expression for up to 4 and 10 days, and reduces cystine levels for up to 10 and 18 days, respectively. (a,b) Time course of cystinosin-3HA expression in PTECs (a) and PODOs (b). Images were obtained using the Operetta CLS High Content Screening Microscope and percentage of cells with detectable cystinosin-3HA expression quantified (each dot represents one observed field from 3 independent experiments). Data were analysed using a Kruskal Wallis test (PTECs) and Welch’s ANOVA test (PODOs). Median and 95% CI are represented for PTECs, and mean and SEM are shown for the PODOs. *p < 0.05; ***p < 0.001; ****p < 0.0001. (c,d) Time course of cystine reduction in CYS PTECs (c) and PODOs (d) that were transfected with CTNS-3HA mRNA. Cystine levels were measured at 4, 10, 14 and 18 days post-transfection and were expressed as percentage (%) of levels measured in cells transfected with GFP mRNA. Each dot represents a replicate, coming from 2 independent transfections. Statistical significance was evaluated using individual tests for each time point, one-way ANOVA (****p < 0.001) and Kruskal Wallis test (^$$p < 0.01; ^$$$p < 0.001; ^$$$$p < 0.0001). Median and 95% CI are indicated for all time points.

Injection of human CTNS-mCherry mRNA in fertilized eggs of ctns^−/− zebrafish results in embryonic protein expression and does not cause toxicity. (a,b) The percentage (%) of dead (a) and dysmorphic embryos (b) was quantified at 5 days post-injection of human CTNS-mCherry (30–150 embryos per experiment, n = 3). Statistical analysis was done by means of a Kruskal Wallis test. Median and 95% CI are indicated. (c,d) CTNS-mCherry mRNA levels were quantified in the untreated (lane 1) and sham injected (lane 2) fish at 24 h (lane 3), 72 h (lane 4) and 120 h (lane 5) post-injection by quantitative RT-PCR (d) and agarose gel electrophoresis (arrow—c). Expression of bactin1 mRNA was used for normalization. Per condition, ΔC(t) is shown for each biological replicate (n = 5) and analysis was done with an one way ANOVA. Untreated and sham group C(t)s were artificially put at 40 cycles for quantification and mean and SEM are shown. ****p < 0.0001. (e,f) Protein expression was quantified at 24 h post-injection by live in vivo microscopy. Mean mCherry-fluorescence per embryo was quantified in a total of 15 embryos and the yolk background subtracted. Images were obtained from the larvae head region using the Olympus IX71 widefield fluorescence microscope. Statistical analysis was performed with a Mann–Whitney test and median and 95% CI are indicated. ***p < 0.001. (g) Expression of cystinosin-mCherry protein at 24 h was confirmed in CTNS-mCherry injected fish (lane 2) in comparison with the sham injected control (lane 1) by a western blot (top) with beta-actin as the loading control (bottom). Original blots and gel are presented in Supplementary Figs. S4 and S5. L=ladder.

Injection of human CTNS-mCherry mRNA in fertilized eggs of ctns^−/− zebrafish decreases cystine accumulation, restores tubular reabsorption and alleviates glomerular proteinuria. (a) Injected ctns^−/− zebrafish larvae were collected in groups of 10–12 fish. Cystine levels in treated larvae were measured at 72 h and 120 h post-injection (n = 5 groups). Statistical analysis was performed using a Student’s t-test. Mean and SEM are indicated. **p < 0.01. (b,c) mRNA treated ctns^−/−[Tg(l-fabp:DBP:eGFP)] larvae were injected with 10 kDa dextran-AF647 for evaluation of low molecular weight proteinuria (LMWP) at 72 h post-injection and fixed for cryosection after 16 h. Sections were stained with Hoechst 33342 (blue) and wheat germ agglutinin (WGA, white), with the DBP-GFP visible in the liver (green). Tubular intensity of dextran-AF647 fluorescence (magenta) was measured and used to quantify low molecular weight protein absorption. Data were analysed with a Student’s t-test and mean and SEM are indicated (n = 9 fish). Scale bar = 100 µm. *p < 0.05. (d) Glomerular proteinuria was evaluated in the ctns^−/−[Tg(l-fabp:DBP:eGFP)] larvae at 120 h post-injection of CTNS-mCherry mRNA. Images were generated with the Acquifer imaging machine. For quantification, an automatized FIJI script was used within a mask determined by segmentation of blood vessels upon traversing blood cells. Data were analysed with a Welch’s t-test. Mean and SEM are indicated (n = 26 fish). ***p < 0.001. (e,f) Sections of injected ctns^−/− fish were prepared at 72 h post-injection and stained for the megalin multi-ligand receptor on the proximal tubular brush border (green), with a nuclear counterstain (blue). Confocal images were obtained and Alexa-488 intensity was measured in each tubule (n = 8 fish) and showed the restauration of proximal tubular megalin expression in treated larvae. Statistical significance was tested using an Student’s t-test and mean and SEM are indicated. *p < 0.05. Scale bar = 20 µm. (g) Paraffin sections of embryos at 120 h post-injection were stained for cleaved caspase-3 and the proximal tubulus identified (red circle). High signal spots are accentuated by an arrow. Scale bar = 20 µm.