Innate and adaptive immune response after liver gene transfer with scAAV2-hF.IX vector in WT C57BL/6J mice (n = 4 per group). Vector was coadministered with TLR9 inhibitory (TLR9i) or passive (contr) ODN. (A) Fold-changes in hepatic mRNA levels (2 hours after vector/ODN administration) compared with PBS-injected mice are shown. A change > 2.5-fold (horizontal line) was considered greater than the variability of the assay. (B) Cytokine levels of TNF-α, IL-6, and MCP-1 in liver protein extract 2 hours after vector/ODN or PBS administration. (C) Systemic IL-6 levels 2 hours after vector/ODN or PBS administration. (D) IgG2a antibody titers against AAV2 capsid 2 and 4 weeks after injection, measured by ELISA. (E) Systemic hFIX levels 2 and 4 weeks after delivery, measured by ELISA. (F) CD8⁺ T cell responses to AAV2 capsid 1 week after gene transfer, measured as IFN-γ spot-forming units (SFU) per 10⁶ cells using ELISpot. *P < .05, **P < .01 calculated by 2-tailed Student t test for comparison of TLR9i or control ODN.