Innate immune response after liver gene transfer with ssAAV2, -TM, or -8 vectors or with scAAV2 or -8 vectors to C3H/OuJ mice (n = 4 per experimental group). All vectors contained expression cassettes for hF.IX, except for scAAV8-GFP vector. (A) Fold-changes in hepatic mRNA levels (2 hours after vector administration at 1 × 10¹¹ vg) 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 administration at 1 × 10¹¹ vg. (C) Systemic IL-6 levels 2 hours after vector administration at 1 × 10¹¹ vg (in picograms per milliliter of plasma). Data are average per experimental group ± SD. (D-E) Fold-change in hepatic mRNA levels compared with PBS-injected mice as a function of time after vector administration shown for ssAAV2-hF.IX (D) and scAAV2-hF.IX (E) vectors was statistically different between vector groups at 2 hours, P < .001, and at 6 hours, P < .01, calculated by 2-way ANOVA of variance using “vectors delivered” and “genes up-regulated” as independent variables. Cytokine levels of TNF-α, IL-6, and MCP-1 in liver protein extract (F) or systemic IL-6 levels 2 hours after vector administration of either 2 × 10¹⁰, 1 × 10¹¹, or 6 × 10¹¹ vg (G); statistics were done using 2-tailed Student t test. *P < .05 in comparison with ssAAV2-transduced liver as calculated by 2-tailed Student t test.