Fig. 4: Expression and therapeutic potential of different lysosomal enzymes.

a AAV6 donor used for KI experiments. All enzymes were tagged with hemagglutinin tag (3xHA). b Transcript upregulation of different enzymes in targeted HUDEP-2 upon differentiation (qPCR, n = 2, mean). Fold increase is indicated. c Representative western blot detecting different enzymes (HA-tag and anti-β tubulin) of targeted HUDEP-2 upon differentiation (n = 2). d KI efficiency of LAL-AAV6 in HSPCs at day 9 (lines indicate mean; AAV n = 4, AAV + RNP n = 5). e Representative western blot of LAL in HSPC lysates, supernatants and BFU-E in untreated (UT), transduced (AAV) and KI-HSPCs (AAV + RNP). Anti-HA tag and anti-β tubulin antibodies were used. f Quantification of secreted LAL during erythroid differentiation. Anti-LAL antibody was used. Data are shown as fold increase over untreated cells (UT, donor=2). g LAL activity in HSPC supernatants during erythroid differentiation, data are shown as fold increase over untreated cells (UT, n = 2). h Integration pattern in single BFU-E: no integration (0), monoallelic (1) and biallelic (2). Mean ± SD, donor = 2). i Uptake of erythroid-expressed LAL by WD fibroblasts, measured by western blot or activity assay (mean; n = 2). j Cholesterol levels in WD fibroblasts after incubation with conditioned medium from untreated (UT) or LAL KI-erythroblasts. WT fibroblasts are shown as control (n = 4; p = 0.003 WD-UT vs WD-LAL; p < 0.001 WD-LAL vs WT; one-way ANOVA, Tukey’s test). k Nile Red staining in WD fibroblasts after incubation with conditioned medium from untreated (UT) or LAL KI-erythroblasts. WT fibroblasts are shown as control. Black lines indicate mean ± SD; number of fibroblasts analyzed is indicated. (***p < 0.001 one-way ANOVA, Tukey’s test). Source data are in the Source Data file.