Infantile neural ceroid lipofuscinosis (INCL) is a lysosomal storage disorder characterized by mutations in the CLN1 gene that leads to lack of the lysosomal enzyme palmitoyl-protein thioesterase-1 (PPT1), which causes the progressive death of cortical neurons. Enzyme replacement therapy (ERT) is one of the most promising treatments, but its translation toward a clinical use is hampered by the need to deliver the enzyme to the central nervous system and a more detailed understanding of its capability to restore physiologic conditions at the biochemical and protein level, beyond the simple regulation of enzymatic activity. Targeted nanoparticles can promote protein delivery to the central nervous system and affect biological pathways inside cells. Here, we describe an innovative peptide-based stealth nanoparticle that inhibits serum protein adsorption exploiting transferrin-driven internalization to convey the PPT1 enzyme to transferrin receptor-mediated pathways (endocytosis in this work, or transcytosis, in perspective, in vivo). These enzyme-loaded nanoparticles were able to restore stable levels of enzymatic activity in CLN1 patient's fibroblasts, comparable with the free enzyme, demonstrating that delivery after encapsulation in the nanocarrier does not alter uptake or intracellular trafficking. We also investigate, for the first time, dysregulated pathways of proteome and palmitoylome and their alteration upon enzyme delivery. Our nanoparticles were able of halving palmitoylated protein levels restoring conditions similar to the normal cells. From proteomic analysis, we also highlighted the reduction of the different groups of proteins after treatments with the free or encapsulated enzyme. In conclusion, our system is able to deliver the enzyme to a model of CLN1 disease restoring normal conditions in cells. Investigation of molecular details of pathologic state and enzyme-based correction reveals dysregulated pathways with unprecedented details for CLN1. Finally, we unveil for the first time the dysregulation landscape of palmitoylome and proteome in primary patient-derived fibroblasts and their modifications in response to enzyme administration. These findings will provide a guideline for the validation of future therapeutic strategies based on enzyme replacement therapy or acting at different metabolic levels.
Keywords: blood brain barrier (BBB); enzyme replacement therapy (ERT); liposomes; lysosomal storage disorders (LSDs); peptide aptamers.