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.
Fig: Evaluation of assay sensitivity: Comparison of LODs between different capsid detection methods (A): cell-based transduction assay (grey bars), immunoassays (blue bars), iqPCR (pink bars), and conventional qPCR (green bars). Detectability (fixed binary variable) of individual sample replicates (black dots) in neat plasma (B) and semen (C) was plotted against the nominally spiked AAV5-FVIII-SQ concentration in vg/mL (continuous variable). Dots below the logistic regression curve (blue line) were detectable, dots above the curve were undetectable. Logistic regression analysis was performed to calculate an equation to fit the data, based on the probability of detection. The inverse prediction was used to determine LOD, defined as the AAV5-FVIII-SQ concentration with a 95% probability of detection.
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.
Sandza, K., Clark, A., Koziol, E. et al. Ultra-sensitive AAV capsid detection by immunocapture-based qPCR following factor VIII gene transfer. Gene Ther (2021). https://doi.org/10.1038/s41434-021-00287-1