Protein Delivery by Peptide-Based Stealth Liposomes: A Biomolecular Insight into Enzyme Replacement Therapy

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.