M6PR/IGF2R double-knockout supplies therapeutic sources for mucolipidosis II and Niemann-Pick C2.

Lysosomal storage disorders (LSDs) are intractable rare diseases caused by lysosomal dysfunction due mainly to defects in lysosomal enzyme genes. For many lysosomal enzymes to be transported correctly into lysosomes, their mannose-6-phosphate (M6P) labeling by GlcNAc-1 phosphotransferase is crucial. M6P-modified lysosomal enzymes are captured by M6P receptors and transported to lysosomes. The M6P-dependent pathway is utilized not only for newly synthesized lysosomal enzymes but also for the intracellular transport of exogenously administered lysosomal enzymes to lysosomes. In this study, we performed gene knockouts targeting the M6PR and IGF2R genes, which encode the M6P receptors. Cells with a double knockout of these genes secrete M6P-modified proteins into the culture supernatant double KO (dKO sup), and we investigated its potential for therapeutic application. I-cell disease (mucolipidosis II, ML-II) arises from GNPT deficiency and is a disorder in which dozens of lysosomal enzymes are deficient within the cell. Treating ML II cells with the dKO sup restored lysosomal enzyme activity, reduced inclusion bodies, and improved autophagic function. ML II patient-derived fibroblasts exhibited increased lysosomal enzyme activities, normalized morphology, and improved cryo-viability with the treatment. Niemann-Pick disease type C2 (NPC2) protein is not a lysosomal enzyme but functions cooperatively with NPC1 protein to mediate cholesterol transport in lysosomes. NPC2 deficiency causes NPC2, and the dKO sup restored the cellular function of the disease by supplementing the NPC2 protein. These results indicate that M6PR/IGF2R double-deficient cells provide a simple platform for supplying M6P-modified proteins that can be applied to the treatment of a broad range of LSDs.