Engineering of a lysosomal-targeted GAA enzyme.

This article focuses on the rational engineering of a chimeric enzyme to improve enzyme replacement therapy (ERT) for Pompe disease, a genetic disorder caused by deficiency of the acid alpha-glucosidase (GAA) enzyme. The study describes the design of an N-terminal peptide tag (NTPT) derived from insulin-like growth factor II (IGF-II) that enhances binding to the IGF-II receptor (IGF2R) to facilitate lysosomal targeting while minimizing off-target binding to the insulin receptor (INS-R) and IGF-I receptor (IGF1R), which had previously caused adverse effects. Using computational modeling, library screening in CHO cells, and biochemical assays including surface plasmon resonance and cellular uptake tests, several NTPT-GAA variants were identified with improved specificity and preserved enzymatic activity. The lead variant, NTPTSeq44-GAA, demonstrated increased cellular uptake and selective IGF2R binding without detectable off-target receptor interactions, suggesting a promising approach to enhance lysosomal delivery of therapeutic enzymes in Pompe disease and potentially other lysosomal storage disorders.