Abstract

Lysosomal storage disorders (LSDs) are a group of inherited metabolic diseases characterized by deficiencies in lysosomal enzymes, leading to the accumulation of undegraded substrates and progressive cellular dysfunction. Patients often present with symptoms such as developmental delay, vision and hearing impairments, seizures, and neurological decline. Current enzyme replacement therapies (ERTs) offer treatment for some LSDs but are limited by poor bioavailability, poor efficacy, high cost and development of resistance. Neuronal Ceroid Lipofuscinosis type 10 (CLN10), caused by cathepsin D deficiency, exemplifies this challenge. Extracellular vesicles (EVs) have emerged as promising nanocarriers capable of crossing the blood brain barrier and effectively delivering functional cargo with low immunogenicity. In this study, we investigated the potential of EV-mediated delivery of Pep4, the yeast homolog of human cathepsin D, to restore lysosomal function in Saccharomyces cerevisiae models deficient in Pep4. I hypothesized that EVs containing Pep4 released from donor yeast cells would be endocytosed and delivered to vacuoles within target yeast lacking PEP4 (pep4∆). Here, Pep4 may cleave and activate the alkaline phosphatase Pho8, whose enzymatic activity was measured using an optimized colorimetric assay. Initial donor and target cell co-culture experiments with unmodified donor strains failed to restore enzymatic activity in target cells, indicating insufficient natural Pep4 packaging into EVs. To enhance Pep4 loading, I genetically modified donor cells to over-express Pep4 fused to the mammalian EV-sorting protein TSG101 and mNeonGreen (TSG101-Pep4-mNeonGreen). This strategy increased EV release but did not improve functional enzyme delivery. Characterization of engineered EVs revealed low TSG101-Pep4-mNeonGreen incorporation, with most tagged protein detected in soluble extracellular fractions rather than within EVs. Adding these EVs to pep4∆ target cells only marginally restored alkaline phosphatase activity. These findings highlight the challenges of efficient enzyme loading into EVs and suggest that additional engineering strategies are needed to improve cargo incorporation and therapeutic efficacy. This work establishes S. cerevisiae as a platform for exploring EV-based enzyme delivery and provides insight into the molecular constraints of EV cargo sorting, with implications for developing novel treatments for lysosomal storage disorders.