Pompe Disease (OMIM: 232300) is a fatal neuromotor disorder caused by mutations in the human LYAG gene. The gene encodes lysosomal glucogenase (GAA), which releases stored lysosomal sugars upon cellular demand. The only approved treatment for Pompe Disease is enzyme replacement therapy (ERT), which involves periodic injections of recombinant human GAA (rhGAA). However, this therapy is only temporarily effective and does not stop disease progression. Hence, given the success of gene therapy in other lysosomal storage disorders, we aimed to develop gene therapy tools for Pompe patients.

Preliminar analysis of rhGAA revealed suboptimal peptide signals for trafficking, secretion, processing, and capture by CI-MP6. Therefore, we present revised GAA propeptides engineered to improve vectorization, intracellular trafficking to enhance secretion, and CI-M6P-independent interaction with target tissue cells. We codon optimized and conserved lisosomal processing and active enzymatic domains. To expedite screening and ease visualization, we substituted the GAA active peptide (76 kDa) with the eGFP reporter peptide (30 kDa) in our designs. Both peptides were provided with the same signal peptides, trafficking domains, and original GAA cleavage amino acidic sequences for lysosomal activation. We produced lentiviral vectors to test expression and secretion in cell lines using different promoters to provide a range of expression levels and evaluate secretion and localization under physiological or supraphysiological conditions.

Our findings pave the way for translational studies to identify feasible gene therapy candidates for improving the clinical outcome of Pompe patients.