In this study, we described the first in vitro three-dimensional human skeletal muscle model of infantile-onset Pompe disease, determined an IOPD disease signature in GAA−/− vs. WT mice, and applied the developed IOPD myobundle model to study in vitro responses of human skeletal muscle to recombinant protein and AAV-based hGAA treatments. This evidence concerns the gene GAA and glycogen storage disease II.