Complement Activation Within Minutes Following Systemic Administration of AAV in the Presence of AAV Immunity

A challenge of adeno-associated virus (AAV)–mediated gene therapy is the high prevalence of preexisting anticapsid antibodies, which can reduce vector efficacy and, after high-dose systemic administration, promote formation of antibody–vector complexes that activate complement. Complement-mediated toxicities reported after systemic AAV administration have generally been observed several days after dosing. However, observations in nonhuman primates (NHPs) suggested that complement activation may occur within minutes of vector exposure in the presence of anti-AAV antibodies. To characterize this response, we evaluated complement activation following repeat systemic administration of AAVrh.10 vectors in African Green monkeys and assessed early and delayed time points when NHPs were seronegative and seropositive for anti-AAV antibodies. Five NHPs received intravenous AAVrh.10 vectors (1 × 10 13 gc/kg) administered 42 days apart. Three animals received AAVrh.10 expressing human C1 esterase inhibitor and two received a transgene-null vector. Serum samples were collected from 10 min to 42 days after the first administration and from 10 min to 21 days after the second. Complement activation was assessed by measuring serum terminal complement complex (TCC) levels and anti-AAVrh.10 immunoglobulin G (IgG) titers were used to monitor humoral immunity. Vector administrations were well tolerated, with no treatment-related abnormalities. Following the initial dose, all animals developed robust anti-AAVrh.10 IgG responses that increased after readministration. No significant complement activation was observed after the first administration. In contrast, in the presence of anti-AAVrh.10 antibodies, all animals demonstrated rapid increases in TCC within 10 min of the second administration, reaching 10-fold above baseline and returning to baseline by 1 day. Complement activation was observed in animals with antivector titers >1/10 4 , whereas little or no response was detected at titers <1/10 3 . These findings identify rapid, transient complement activation as an underrecognized response to systemic AAV administration in the presence of anti-AAV antibodies and support complement monitoring during the first hour after vector dosing in clinical gene therapy studies.