Abstract
Adeno-associated virus (AAV)-based gene therapy vectors are replication-incompetent and thus pose minimal risk for horizontal transmission or release into the environment. In studies with AAV5-FVIII-SQ (valoctocogene roxaparvovec), an investigational gene therapy for hemophilia A, residual vector DNA was detectable in blood, secreta, and excreta, but it remained unclear how long structurally intact AAV5 vector capsids were present. Since a comprehensive assessment of vector shedding is required by regulatory agencies, we developed a new method (termed iqPCR) that utilizes capsid-directed immunocapture followed by qPCR amplification of encapsidated DNA. The limit of detection for AAV5 vector capsids was 1.17E+04 and 2.33E+04 vg/mL in plasma and semen, respectively. Acceptable precision, accuracy, selectivity, and specificity were verified; up to 1.00E+09 vg/mL non-encapsidated vector DNA showed no interference. Anti-AAV5 antibody plasma concentrations above 141 ng/mL decreased AAV5 capsid quantification, suggesting that iqPCR mainly detects free capsids and not those complexed with antibodies. In a clinical study, AAV5-FVIII-SQ capsids were found in plasma and semen but became undetectable within nine weeks after dose administration. Hence, iqPCR monitors the presence and shedding kinetics of intact vector capsids following AAV gene therapy and informs the potential risk for horizontal transmission.