Gene therapy may offer a new treatment option, particularly for patients with severe hemophilia, based on recent research. However, individuals with pre-existing immunity to adeno-associated viruses (AAVs) may be less likely to benefit from AAV vector-based therapies. To study pre-existing AAV5 immunity in humans, we validated two complementary, sensitive, and scalable in vitro assays to detect AAV5 total antibodies and transduction inhibition (TI). Using these two assays, we found that 53% of samples from 100 healthy male individuals were negative in both assays, 18% were positive in both assays, 5% were positive for total antibodies but negative for TI and, of interest, 24% were negative for total antibodies but positive for TI activity, suggesting the presence of non-antibody-based neutralizing factors in human plasma. Similar findings were obtained with 24 samples from individuals with hemophilia A. On the basis of these results, we describe the development of a dual-assay strategy to identify individuals without total AAV5 antibodies or neutralizing factors who may be more likely to respond to AAV5-directed gene therapy. These assays offer a universal, transferrable platform across laboratories to assess the global prevalence of AAV5 antibodies and neutralizing factors in large patient populations to help inform clinical development strategies.

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the alpha-galactosidase A ( GLA ) gene, which encodes the exogalactosyl hydrolase, alpha-galactosidase A (α-Gal A). Deficient α-Gal A activity results in the progressive, systemic accumulation of its substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3), leading to renal, cardiac, and/or cerebrovascular disease and early demise. The current standard treatment for Fabry disease is enzyme replacement therapy, which necessitates lifelong biweekly infusions of recombinant enzyme. A more long-lasting treatment would benefit Fabry patients. Here, a gene therapy approach using an episomal adeno-associated viral 2/6 (AAV2/6) vector that encodes the human GLA cDNA driven by a liver-specific expression cassette was evaluated in a Fabry mouse model that lacks α-Gal A activity and progressively accumulates Gb3 and Lyso-Gb3 in plasma and tissues. A detailed 3-month pharmacology and toxicology study showed that administration of a clinical-scale-manufactured AAV2/6 vector resulted in markedly increased plasma and tissue α-Gal A activities, and essentially normalized Gb3 and Lyso-Gb3 at key sites of pathology. Further optimization of vector design identified the clinical lead vector, ST-920, which produced several-fold higher plasma and tissue α-Gal A activity levels with a good safety profile. Together, these studies provide the basis for the clinical development of ST-920.

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