Extracellular Vesicle-Encapsulated AAV-GLA Enhances Transduction and Confers Resistance to Neutralization in vitro

Introduction:
Fabry disease is an X-linked lysosomal storage disorder caused by pathogenic mutations in the GLA gene, resulting in deficient α-galactosidase A activity and progressive accumulation of glycosphingolipid substrates across multiple organs. Enzyme replacement therapy, the current standard of care, requires lifelong periodic infusions and shows limited efficacy in addressing central nervous system involvement. Adeno-associated virus (AAV)-mediated gene delivery has therefore emerged as a potential long-term treatment strategy by enabling sustained endogenous expression and secretion of functional α-galactosidase A following a single systemic administration. Despite its promise, AAV-based gene delivery faces major translational barriers. A predominant limitation is the high prevalence of pre-existing neutralizing antibodies against AAV capsids, which can rapidly inactivate systemically administered AAV vectors, compromise transduction efficiency, and exclude a substantial proportion of patients from treatment. These challenges underscore the need for alternative delivery strategies capable of evading humoral immune neutralization. Extracellular vesicle-encapsulated AAV (EV-AAV) represents a promising strategy to address these limitations. By cloaking AAV particles within a natural EV membrane, EV-AAV can reduce exposure of the viral capsid to neutralizing antibodies and preserve transduction efficiency in antibody-rich environments. Moreover, EVs may provide opportunities to exploit endogenous trafficking and tissue-homing properties, potentially enhancing the versatility of AAV-based gene therapy for Fabry disease.
Methods:
The GLA gene was packaged into an AAV8 vector and transfected into suspension HEK293 producer cells using an optimized packaging system to improve EV-AAV yield. After 72 hours of culture, AAV-GLA and EV-encapsulated AAV-GLA (EV-AAV-GLA) were purified by iodixanol density gradient ultracentrifugation from cell pellets and culture supernatants, respectively. Viral genome titers were quantified by quantitative PCR and normalized for subsequent in vitro experiments. Huh7 cells were incubated with EV-AAV-GLA (MOI = 3 × 10⁵), conventional AAV-GLA (MOI = 3 × 10⁵), or high-dose AAV-GLA (MOI = 1 × 10⁶) in the presence of increasing concentrations of intravenous immunoglobulin (IVIg). At 48 hours post-transduction, both culture supernatants and cell lysates were collected, and α-galactosidase A enzymatic activity was measured to evaluate transduction efficiency and resistance to neutralizing antibodies among different treatment groups.
Results:
EV-AAV-GLA maintained substantially higher α-galactosidase A activity than conventional AAV-GLA at the same MOI (3 × 10⁵) across all IVIg concentrations tested. In contrast, conventional AAV-GLA exhibited a marked, dose-dependent reduction in enzymatic activity, approaching near-complete neutralization at higher antibody levels. Increasing the dose of conventional AAV-GLA (MOI = 1 × 10⁶) only partially improved transduction at low IVIg concentrations and failed to sustain activity under higher antibody conditions. Similar trends were observed for intracellular enzyme activity. These findings demonstrate that EV encapsulation significantly enhances resistance to antibody-mediated neutralization and preserves functional gene delivery efficiency.
Conclusion:
The EV-AAV platform significantly improves resistance to pre-existing neutralizing antibodies and enhances functional GLA gene delivery compared with conventional AAV vectors. This strategy represents a promising next-generation gene delivery approach to expand patient eligibility and improve therapeutic durability for Fabry disease gene therapy.