Figure 1. AAV2/MFSD8 vector construct expressing human MFSD8 and its rescue of lysosomal function in primary fibroblasts from a CLN7 patient.

(A) Schematic diagram of AAV2/MFSD8 construct comprising a mutant AAV2 ITR with the D element deleted (ΔITR), the JeT promoter, the human MFSD8 codon-optimized coding sequence (hMFSD8opt), the synthetic polyadenylation SV40pA signal, and WT AAV2 ITR. (B) Lysosomal GCase activity (n = 4–5) was measured in fibroblasts from age-matched healthy control and a CLN7 patient. GCase activity was normalized to the cell volume. (C and D) Lysosomal and total GCase activity (n = 3–5) was measured following AAV2-mediated transduction of JeT-GAN (negative control), JeT-MFSD8 (therapeutic transgene at increasing doses), or UsP-MFSD8 (therapeutic transgene with stronger promotor). The fold differences in lysosomal (C) and total (D) GCase activity were normalized to the cell volume and to cohorts transfected with JeT-GAN. (E and F) MFSD8 mRNA and MFSD8 protein (n = 3–4) were assayed following AAV2-mediated transduction of CBh-GFP (negative control), JeT-MFSD8, or UsP-MFSD8. A ROUT test was used first to remove any outlier. All data in B–F are presented as mean ± SEM, with the scatter plot representing measurements from individual culture wells. Data sets that passed tests for normality or homogeneity of variance were analyzed using unpaired t test or 1-way ANOVA with α set at 0.05 and Dunnett’s correction for relevant pairwise comparisons. Data sets that did not pass tests for normality or homogeneity of variance were analyzed using the Kruskal-Wallis test with α set at 0.05 and Dunn’s correction for relevant pairwise comparisons. **P < 0.01; ****P < 0.0001, compared with control.