Innate immune response after liver gene transfer with scAAV2-hF.IX vector in TLR9^−/− (TLR9KO) or WT C57BL/6J mice (n = 5 per group). (A) Fold-changes in hepatic mRNA levels (2 hours after vector administration) compared with PBS-injected mice of same strain 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 administration. (C) Systemic IL-6 levels 2 hours after vector administration. Systemic hF.IX levels (D) and antibody titers (IgG2a; E) against AAV capsid as a function of time after vector administration (average ± SD). PBS, WT control mice injected with PBS. *P < .05 as calculated by 2-tailed Student t test (comparison of WT and TLR9^−/− mice).