Investigation of dose-exposure-response relationship for adeno-associated virus–mediated delivery of antibody genes

Adeno-associated virus (AAV)-based gene therapies have advanced significantly, yet the pharmacology of these therapies, particularly the relationship between dose and transgene expression, remains incompletely understood. Here, we investigated the dose-response dynamics of AAV8 vectors encoding an antibody (trastuzumab) gene in C57BL/6 mice to assess virus pharmacokinetics, transgene expression, liver toxicity, and immunogenicity across a broad dose range (1E8-1E13 vector genomes/mouse). Whole-body viral pharmacokinetics data revealed dose nonproportionality, with higher doses leading to less than proportional increases in AAV exposure across most tissues, except for the spleen, which exhibited a more than proportional increase. Plasma transgene concentrations demonstrated a sigmoidal dose-response relationship, with Emax of ∼ 8000 nM and EC50 of ∼8E9 vector genomes, indicating saturation at higher doses. Immune responses to AAV8 were dose-dependent, with IgM titers peaking on day 2 and IgG titers appearing by day 21, both escalating with increasing doses. Elevated aspartate aminotransferase and alanine aminotransferase levels at higher doses indicate a hepatotoxic effect. These findings suggest that the nonlinear dose-exposure relationship stems from saturable tissue distribution, nonlinear transgene production, or heightened immune activation at higher doses. Importantly, the nonlinearity in translational efficiency and an increased risk for immunogenicity and hepatotoxicity at higher dose levels underscore the critical need to optimize AAV dosing to balance efficacy and safety. These data provide valuable insights into the pharmacology of AAV gene therapies and offer a framework for determining optimal dosing strategies that maximize therapeutic benefit while minimizing toxicity and immunogenicity. SIGNIFICANCE STATEMENT: The research presented here demonstrates that there is an optimal dose for adeno-associated virus-based therapies and exceeding this dose might lead to more immunogenicity, more toxicity, and reduced transgene expression.