Pompe disease is a rare, inherited, devastating neuromuscular disorder characterized by progressive skeletal weakness, cardiomyopathy and neuromotor dysfunction resulting from glycogen accumulation in striated and smooth muscle, as well as neurons. Gene therapy represents a promising therapeutic approach due to the multisystem nature of the disease and the need to address the central nervous system manifestations. However, effective therapy requires optimized transgene expression and targeted delivery for better enzyme secretion, while minimizing off-target liver expression.
Hypothesis: We hypothesized that engineering the acid-α-glucosidase (GAA) gene with optimized signaling peptides, combined with muscle-targeted and liver-detargeted adeno-associated virus (AAV) vector, would improve lysosomal processing and increase enzymatic activity in both brain and muscle, thereby improving the efficacy of Pompe disease gene therapy.
Methods: To test our hypothesis, we packaged three GAA variants into an engineered AAV capsid (Myotropic AAV4E). The control viral vector contained unmodified GAA, while the other two incorporated optimized signal peptides (E1E2-sp1-GILT2 and Int1-SP1-GILT-IGF2). Neonatal GAA knockout (KO) mice (model of Pompe Disease) received a single retro-orbital injection of the vector on postnatal day 1 (P1) at a 5E+13vg/kg dose. Mice were sacrificed one-month post-injection, and tissues (heart, quadriceps, diaphragm, liver, brain, spinal cord) were harvested to assess GAA enzymatic activity.
Results: Vectors incorporating signal peptide-modified GAA exhibited markedly increased enzymatic activity across the analyzed tissues, with up to a six-fold increase compared to unmodified GAA controls. The engineered myotropic AAV4E capsid demonstrated successful liver detargeting with preferential tropism for skeletal and cardiac muscle. Notably, heart tissue showed a significant increase (p<0.0001) in GAA activity in mice treated with signal peptide-modified vectors specially E1E2-GILT-IGF2.
Conclusion: Our study addresses two primary hurdles in Pompe disease gene therapy: tissue-specific delivery through capsid engineering and enhanced enzymatic potency via transgene optimization. Engineered AAV4E vectors expressing signal peptide-modified GAA, particularly E1E2-GILT-IGF2, demonstrated substantially improved GAA activity, supporting their potential in improving Pompe disease gene therapy outcomes.
Future: Next steps include testing enginee