Figure 4

GAGs accumulation and lysosomal mass evaluation. (A) Heat map showing the global behavior of GAGs accumulation (n = 4). GM00593 and GM01361 fibroblasts showed the best recovery after CRISPR/nCas9 system on this biomarker for both LP and MLPs. Notice an improvement on LP over MLPs for GM00593 (p = 0.0259), GM00958 (p = 0.0330) fibroblasts, and GM01259 (p = 0.0291) fibroblasts, when CRISPR/nCas9 and Donor AAVS1:GALNS plasmids were transfected. Fibroblasts were transfected either with the Donor AAVS1:GALNS plasmid (Donor) or the CRISPR/nCas9-Donor AAVS1:GALNS plasmids (CRISPR/nCas9:Donor). (B) Left histograms to correspond to representative findings of MFI for LysoTracker Deep Red observed on MPS IVA after CRISPR/nCas9:Donor AAVS1 treatment. The mean of relative MFI for each MPS IVA fibroblast to untreated cells (n = 4) is shown on the right. The horizontal dotted line represents the relative MFI of wild-type fibroblasts. Note that LP led a better improvement for GM00593 (p = 0.0284) and GM01259 (p = 0.0161) fibroblasts compared MLPs; while an opposite behavior was achieved for GM01361 fibroblasts (p = 0.0461). Fibroblasts were transfected either with the Donor AAVS1:GALNS plasmid (Donor) or the CRISPR/nCas9-Donor AAVS1:GALNS plasmids (CRISPR/nCas9:Donor). (C) Representative fluorescent images for LysoTracker Deep Red staining. Red fluorescence corresponds to lysosomal mass, while the blue signal to the nucleus. UT untreated, DG donor AAVS1:GALNS, CD CRISPR/Cas9 plus Donor AAVS1:GALNS. *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.001, ****p ≤ 0.0001. Two-tailed Student's t-test.