Abstract
Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene, leading to deficient α-galactosidase A (α-Gal A) activity and pathological accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3) in various organs. To enhance α-Gal A expression and secretion for recombinant adeno-associated viral (rAAV) gene therapy, the signal peptide was engineered in HepG2 cells. Among 22 heterogeneous signal peptides, sp21 was identified and validated for this purpose. The enzyme activity of GLA with sp21 in conditioned media was 2.3 times higher than that with the wild-type signal peptide. Furthermore, the human GLA gene was codon optimized (hGLAco), aiming to achieve increased hepatic expression and prevent gene silencing. The secreted α-Gal A activity of hGLAco increased approximately 4-fold in vitro. Finally, the hGLAco transgene with sp21 driven by a liver-specific promoter was packaged in rAAV8 vectors and injected into Fabry mice. Compared with the wild-type cDNA, the engineered GLA gene resulted in a 6.9-fold increase in secreted α-Gal A activity in Fabry mice, and toxic Lyso-Gb3 accumulation in plasma and key tissues was effectively cleared. These findings demonstrate a promising strategy to develop a more efficient rAAV gene therapy for Fabry disease.