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We developed a novel adeno-associated virus 5 gene therapy (AAV5-GLA) expressing human alpha-galactosidase A (GLA) under the control of a novel, small and strong, liver-restricted promoter. We assessed the preclinical potential of AAV5-GLA for treating Fabry disease, an X-linked hereditary metabolic disorder resulting from mutations in the gene encoding GLA that lead to accumulation of the substrates globotriaosylceramide and globotriaosylsphingosine, causing heart, kidney, and central nervous system dysfunction. Effects of intravenously administered AAV5-GLA were evaluated in (1) GLA-knockout mice aged 7–8 weeks (early in disease) and 20 weeks (nociception phenotype manifestation) and (2) cynomolgus macaques during an 8-week period. In both species, AAV5-GLA was observed as safe, generated detectable vector DNA and mRNA levels in liver, and produced stable enzyme activity in liver and plasma. In mice, dose-dependent transgene enzyme activity, cross-correction (substrate reduction) in kidney and heart, and improved nociception lasted over 6 months. Moreover, after delayed administration when animals displayed the nociception phenotype, target organ enzyme activity was present, and accumulated substrates were reduced. Given the strong, durable expression of active GLA with this promoter and favorable profile of adeno-associated virus 5-based gene therapy in humans, AAV5-GLA warrants further investigation in clinical trials for Fabry disease.

Gaucher disease (GD) is a lysosomal storage disorder (LSD) resulting from inherited glucocerebrosidase (GBA1) deficiency. GD diagnosis relies on GBA1 activity assays, typically employing 4-methylumbelliferyl-β-D-glucopyranoside (4MU-β-Glc) as fluorogenic substrate. However, these assays suffer from background 4MU release by the non-lysosomal GBA2 and cytosolic GBA3 enzymes. Here we developed GBA1-selective fluorogenic substrates by synthesizing a series of 6-O-acyl-4MU-β-Glc substrates with diverse fatty acid tails. Because of the chemical and enzymatic instability of the ester bonds, analogs of 6-O-palmitoyl-4MU-β-Glc (3) with different chemical linkages were synthesized. 6-O-alkyl-4MU-β-Glc 9, featuring an ether linkage, emerged as the most optimal GBA1 substrate, exhibiting both a low Km and compared to substrate 3 a high Vmax. Importantly, substrate 9 is not hydrolyzed by GBA2 and GBA3 and therefore acts as a superior substrate for GD diagnosis. Plants contain glycosyl phytosterols (campesterol, β-sitosterol, and sigmasterol) that may also be acylated at C-6. LC-MS/MS analysis revealed that 6-O-acylated and regular glycosylcholesterol (HexChol) tend to be increased in spleens of patients with GD. Moreover, significant increases in 6-O-acyl-glycosyl-phytosterols were detected in GD spleens. Our findings suggest uptake of (6-O-acyl)-glycosyl-phytosterols from plant food and subsequent lysosomal processing by GBA1, and comprise the first example of accumulation of an exogenous class of glycolipids in GD. Excessive exposure of rodents to glycosylated phytosterols has been reported to induce manifestations of Parkinson’s disease (PD). Further investigation is warranted to determine whether (6-O-acyl)-glycosyl-phytosterols could contribute to the enigmatic link between inherited defects in GBA1 and the risk for PD.