Figure 1.
Vector design and functional validation. (A-B) Schematic representation of the pAAV-AAT-co-hFIX-R338L-Padua (A), pAAV-AAT-co-hFIX-CB2679d-GT (B) plasmids used in this study. The liver-specific promoter (AAT) drives the (co-hFIX) complementary DNA with the Padua variant (ie, R338L-Padua) or a CB 2679d-GT transgene that encodes a hFIX with 3 amino acid substitutions (ie, R318Y, R338E, T343R). The minute virus of mouse (MVM) mini-intron and bovine growth hormone polyadenylation site bghpA) are also indicated. The expression cassettes were cloned into an scAAV backbone, flanked by the 5′ and 3′ AAV inverted terminal repeats (ITRs), as indicated. (C-E) The R338L-Padua and CB 2679d-GT vectors were injected into hemophilia B mice (FIX−/−) at doses of 1 × 109 vg per mouse (5 × 1010 vg/kg) (n = 6), 5 × 109 vg per mouse (2.5 × 1011 vg/kg) (n = 8), and 1 × 1010 vg per mouse (5 × 1011 vg/kg) (n = 8). The FIX antigen levels were determined at the indicated times after AAV administration by using a hFIX-specific ELISA at 1 × 109 vg per mouse (C), 5 × 109 vg per mouse (D), and 1 × 1010 vg per mouse (E). To measure CB 2679d-GT and R338L-Padua antigen levels by ELISA and exclude any potential bias, FIX standard curves were constructed with known amounts of the purified recombinant CB 2679d-GT or R338L-Padua, respectively. (F-H) The FIX activity was determined as a measure of clotting time using an aPTT assay for the doses 1 × 109 vg per mouse (n = 6) (F), 5 × 109 vg per mouse (n = 8) (G), and 1 × 1010 vg per mouse (n = 8) (H). Vehicle-injected hemophilia B mice (FIX−/−) (n = 7) and C57BL/6 normal control (n = 4) (dotted line) were used as controls. (I-L) Mouse plasma samples were also used to determine the FIX activity (units per mL) and specific activity (units per mg) calculated based on a single-stage, aPTT-based factor IX clotting assay with WHO FIX standard (09/172) and the FIX antigen levels shown above. Two vector doses were used 5 × 109 vg per mouse (I,K) and 1 × 1010 vg per mouse (J,L). Results are presented as mean ± standard error of the mean. *P < .05; **P < .01; ***P < .001 (Student t test); NS, not significant (P > .1).

Vector design and functional validation. (A-B) Schematic representation of the pAAV-AAT-co-hFIX-R338L-Padua (A), pAAV-AAT-co-hFIX-CB2679d-GT (B) plasmids used in this study. The liver-specific promoter (AAT) drives the (co-hFIX) complementary DNA with the Padua variant (ie, R338L-Padua) or a CB 2679d-GT transgene that encodes a hFIX with 3 amino acid substitutions (ie, R318Y, R338E, T343R). The minute virus of mouse (MVM) mini-intron and bovine growth hormone polyadenylation site bghpA) are also indicated. The expression cassettes were cloned into an scAAV backbone, flanked by the 5′ and 3′ AAV inverted terminal repeats (ITRs), as indicated. (C-E) The R338L-Padua and CB 2679d-GT vectors were injected into hemophilia B mice (FIX−/−) at doses of 1 × 109 vg per mouse (5 × 1010 vg/kg) (n = 6), 5 × 109 vg per mouse (2.5 × 1011 vg/kg) (n = 8), and 1 × 1010 vg per mouse (5 × 1011 vg/kg) (n = 8). The FIX antigen levels were determined at the indicated times after AAV administration by using a hFIX-specific ELISA at 1 × 109 vg per mouse (C), 5 × 109 vg per mouse (D), and 1 × 1010 vg per mouse (E). To measure CB 2679d-GT and R338L-Padua antigen levels by ELISA and exclude any potential bias, FIX standard curves were constructed with known amounts of the purified recombinant CB 2679d-GT or R338L-Padua, respectively. (F-H) The FIX activity was determined as a measure of clotting time using an aPTT assay for the doses 1 × 109 vg per mouse (n = 6) (F), 5 × 109 vg per mouse (n = 8) (G), and 1 × 1010 vg per mouse (n = 8) (H). Vehicle-injected hemophilia B mice (FIX−/−) (n = 7) and C57BL/6 normal control (n = 4) (dotted line) were used as controls. (I-L) Mouse plasma samples were also used to determine the FIX activity (units per mL) and specific activity (units per mg) calculated based on a single-stage, aPTT-based factor IX clotting assay with WHO FIX standard (09/172) and the FIX antigen levels shown above. Two vector doses were used 5 × 109 vg per mouse (I,K) and 1 × 1010 vg per mouse (J,L). Results are presented as mean ± standard error of the mean. *P < .05; **P < .01; ***P < .001 (Student t test); NS, not significant (P > .1).

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