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Protein Delivery by Peptide-Based Stealth Liposomes: A Biomolecular Insight into Enzyme Replacement Therapy
Melissa Santi1,2, Francesco Finamore3, Antonella Cecchettini4
1Center for Nanotechnology Innovation@NEST, Istituto Italiano di Tecnologia, Pisa 56127, Italy.
Insights
Researchers developed novel nanoparticles to deliver the palmitoyl-protein thioesterase-1 (PPT1) enzyme for infantile neural ceroid lipofuscinosis (INCL). This enzyme replacement therapy restored cellular function and corrected protein and palmitoylome dysregulation in patient cells.
Area of Science:
- Biochemistry
- Nanotechnology
- Neuroscience
Background:
- Infantile neural ceroid lipofuscinosis (INCL) is a genetic disorder caused by mutations in the CLN1 gene, leading to a deficiency in the enzyme palmitoyl-protein thioesterase-1 (PPT1).
- This deficiency results in progressive neurodegeneration, making enzyme replacement therapy (ERT) a promising but challenging treatment due to delivery obstacles to the central nervous system.
- Understanding the biochemical and proteomic impact of ERT beyond enzymatic activity is crucial for clinical translation.
Purpose of the Study:
- To develop and characterize a novel peptide-based stealth nanoparticle for delivering the PPT1 enzyme to treat INCL.
- To investigate the efficacy of enzyme-loaded nanoparticles in restoring PPT1 enzymatic activity and cellular function in CLN1 patient-derived fibroblasts.
- To analyze the impact of PPT1 enzyme delivery on the dysregulated proteome and palmitoylome in INCL models.
Main Methods:
- Design of peptide-based stealth nanoparticles that inhibit serum protein adsorption and utilize transferrin-driven internalization for enzyme delivery.
- Encapsulation of the PPT1 enzyme within the nanoparticles and assessment of its activity in CLN1 patient fibroblasts.
- Proteomic and palmitoylomic analyses to evaluate cellular pathway restoration after treatment with free or encapsulated PPT1 enzyme.
Main Results:
- The developed nanoparticles successfully delivered functional PPT1 enzyme into CLN1 patient fibroblasts, restoring enzymatic activity to levels comparable to the free enzyme.
- Encapsulation did not impede enzyme uptake or intracellular trafficking, demonstrating the nanocarrier's effectiveness.
- Treatment with the nanoparticles significantly reduced palmitoylated protein levels by half, restoring conditions similar to normal cells, and modulated various protein groups identified through proteomic analysis.
Conclusions:
- The innovative nanoparticle system effectively delivers functional PPT1 enzyme for INCL, restoring normal cellular conditions in a disease model.
- This study provides unprecedented molecular insights into the dysregulated palmitoylome and proteome in INCL and their correction via enzyme administration.
- The findings offer a foundation for validating future ERT strategies and other metabolic interventions for INCL and similar lysosomal storage disorders.
Abstract:
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.
