Vector design and functional validation.Schematic representation of pAAV-TTR-co-hFIX-R338L (A), pAAV-HS-CRM8-TTR-co-hFIX-R338L (B) and pAAV-HS-CRM8-TTR-co-hFIX (C) plasmids used in this study. The liver-specific minimal transthyretin (TTR) promoter drives the codon-optimized human FIX (co-hFIX) complementary DNA (cDNA) with or without the Padua R338L mutation (co-hFIX-R338L). The HS-CRM8 is located upstream of the TTR promoter. The minute virus of mouse (MVM) mini-intron and bovine growth hormone polyadenylation site (pA) are also indicated. The expression cassettes were cloned in an scAAV backbone, flanked by the 5′ and 3′ AAV inverted terminal repeats (ITRs), as indicated, and were used to generate the cognate scAAV9-HS-CRM8-TTR-co-hFIX-R338L and scAAV9-HS-CRM8-TTR-co-hFIX vectors. The effect of the HSCRM8 element was assessed by hydrodynamic transfection of the pAAV-HS-CRM8-TTR-co-hFIX-R338L (indicated as +HS-CRM8) (B) and control pAAV-TTR-cohFIX-R338L (indicated as –HS-CRM8) (A) plasmids in C57BL/6 mice at doses of 2 µg/mouse and 5 µg/mouse (D-E). FIX expression was measured by using a validated hFIX-specific enzyme-linked immunosorbent assay (ELISA) (n = 4) on plasma samples collected at day 1 or 2 posttransfection. Similarly, to assess the impact of the Padua R338L mutation, hemophilic mice were hydrodynamically transfected with pAAV-HS-CRM8-TTR-cohFIX-R338L (indicated as co-hFIX-R338L) compared with the pAAV-HS-CRM8-TTR-co-hFIX control (indicated as co-hFIX) (F). The clotting factor activity was measured by using a functional chromogenic FIX assay. Subsequently, we injected the cognate scAAV9-HS-CRM8-TTR-co-hFIX-R338L (designated as co-hFIX-R338L) (G-I) and scAAV9-HS-CRM8-TTR-co-hFIX (designated as co-hFIX) (J-L) in FIX-deficient hemophilic mice at a dose of 1 × 10⁹ vg/mouse (5 × 10¹⁰ vg/kg), 5 × 10⁹ vg/mouse (2.5 × 10¹¹ vg/kg), and 2 × 10¹⁰ vg per mouse (10¹² vg/kg) (n = 3 per group) (G-L). FIX activity and antigen levels were determined at the indicated times after AAV administration by using a chromogenic FIX activity assay and hFIX-specific ELISA, respectively. Results are presented as mean ± standard error of the mean. *P < .05; **P < .01; ***P < .001 (Student t test); N.S., not significant (P > .1).