Liver Gene Therapy in Fabry Disease Mice With Low Doses of <scp>rAAV2</scp> /8 Expressing a Codon‐Optimized <scp>h</scp> <i>GLA</i> <scp>cDNA</scp> Results in Long‐Term Disease Correction

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene, which encodes for Alpha Galactosidase-A (α-Gal A). α-Gal A deficiency leads to glycosphingolipid accumulation, like globotriaosylceramide (Gb3) and its deacylated form, globotriaosylsphingosine (lyso-Gb3), resulting in systemic symptoms and reduced lifespan. Current treatments such as enzyme replacement therapy (ERT) and chaperone therapy are noncurative and have limitations. Gene therapy is an interesting alternative approach that may overcome most of these limitations, and different approaches are currently being tested. Here, we developed a gene therapy approach using rAAV2/8 vectors, delivered intravenously, that target the liver to produce and secrete functional α-Gal A into circulation. This enzyme is then captured by organs expressing mannose-6-phosphate receptors, reducing glycosphingolipid accumulation in affected tissues. We generated a codon-optimized GLA cDNA with enhanced translatability that was expressed under a strong liver-specific promoter. In a dose escalation study in juvenile Gla knock-out (ko) mice, the lowest dose (3.0E11vg/kg) resulted in 85%-95% clearance of lyso-Gb3 in plasma and tissues, while doses of 3.0E12 vg/kg and higher showed 98%-100% clearance of the glycosphingolipid. All AAV doses were more effective than systemic α-Gal A administration (ERT). Long-term treatment showed normal levels of lyso-Gb3 in plasma and tissues, and corrected neuropathic involvement, as shown in the hot plate test. This study provides a proof-of-concept showing that the tested liver-specific gene therapy vector is capable of preventing disease progression in juvenile Fabry mice at relatively low doses and shows potential in treating both early- and late-onset FD in patients.