Lysosome-Specific Delivery of β-Glucosidase Enzyme Using Protein-Glycopolypeptide Conjugate via Protein Engineering and Bioconjugation.

Lysosomal enzyme replacement therapy (ERT) holds potential for treating lysosomal storage disorders, but achieving targeted delivery of deficient therapeutic enzymes remains a significant challenge. This study presents a novel approach for the lysosome-specific delivery of the β-glucosidase (B8CYA8) enzyme by covalently conjugating lysosome-targeting mannose-6-phosphate functionalized glycopolypeptides (M6P-GP). We used a protein-glycopolypeptide conjugate developed through advanced protein engineering and bioconjugation techniques. By conjugating β-glucosidase to M6P-GP that has a high affinity for the cation-independent mannose-6-phosphate receptors (CI-MPR) and lysosomal receptors, we enhance the enzyme's selective intracellular uptake and lysosome-specific localization. To attain maximum activity of the near-native enzyme after delivery, we have designed and synthesized an acetal linkage containing the pH-responsive linker maleimide-acetal-azide (MAA), which will cleave in the lysosomal acidic pH to detach the glycopolypeptide from the protein backbone. We demonstrated the efficient cellular uptake of the protein-glycopolypeptide conjugate and showed targeted lysosome delivery, leading to increased enzymatic activity compared to untreated cells. Our results proved that the approach mainly improves the specificity and efficiency of enzyme delivery, particularly into lysosomes, which may enable new methods for ERT. These findings suggest that protein-glycopolypeptide conjugates could represent a class of bioconjugates to design targeted enzyme therapies, offering a pathway to the effective treatment of Gaucher disease (GD) and potentially other related lysosomal storage disorders.