F Western blot analysis of liver lysates ( 100 µg ) from either CodopN6 intein - treated mice ( n = 6 ) or untreated haemophilic mice ( n = 2 ) . A lysate from HEK293 cells transfected with the N6 full - length plasmid was used as positive control ( 50 µg ) . Arrows point at N6 full - length protein ( N6 ) , the 5 ' half of N6 ( 5 ' N6 ) and 3 ' half of N6 ( 3 ' N6 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Western blot analysis of liver lysates (100 µg) from either CodopN6 intein-treated mice (n=6) or untreated haemophilic mice (n=2). A lysate from HEK293 cells transfected with the N6 full-length plasmid was used as positive control (50 µg). Arrows point at N6 full-length protein (N6), the 5' half of N6 (5'N6) and 3' half of N6 (3'N6).


A The amount of anti - F8 antibodies analysed by indirect ELISA is reported in Arbitrary Units / milliliter ( AU / ml ) . Each numbered bar represents a single mouse . CodopV3 , AAV - CodopV3 injected group n = 8 ; N6 intein , AAV - N6 intein injected group n = 5 ; CodopN6 intein , AAV - CodopN6 intein injected group n = 5 . Significant differences were assessed as follow : Paired T - test has been used for CodopV3 n = 5 : p value ≤ 0 . 001 ; Wilcoxon test for CodopV3 n = 3 : p value ≤ 0 . 05 ; Paired T - test has been used for N6 intein n = 5 : p value ≤ 0 . 05 ; Paired T - test has been used for CodopN6 intein n = 5 p value ≤ 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A The amount of anti-F8 antibodies analysed by indirect ELISA is reported in Arbitrary Units/milliliter (AU/ml). Each numbered bar represents a single mouse. CodopV3, AAV-CodopV3 injected group n=8; N6 intein, AAV-N6 intein injected group n=5; CodopN6 intein, AAV-CodopN6 intein injected group n=5. Significant differences were assessed as follow: Paired T-test has been used for CodopV3 n=5: p value ≤ 0.001; Wilcoxon test for CodopV3 n=3: p value ≤ 0.05; Paired T-test has been used for N6 intein n=5: p value ≤ 0.05; Paired T- test has been used for CodopN6 intein n=5 p value ≤ 0.01.


B Bethesda assay performed on plasma samples mice injected with AAV - CodopV3 ; on the plasma of animals injected with AAV - N6 intein n = 3 and the plasma of animals injected with AAV - CodopN6 intein n = 2 ; plasma of a wild - type animal was used as a control . The dotted line indicates the threshold above which anti F8 - antibodies are considered inhibitors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Bethesda assay performed on plasma samples mice injected with AAV-CodopV3; on the plasma of animals injected with AAV-N6 intein n=3 and the plasma of animals injected with AAV-CodopN6 intein n=2; plasma of a wild-type animal was used as a control. The dotted line indicates the threshold above which anti F8-antibodies are considered inhibitors.


C Pro - inflammatory cytokines levels followed over - time and reported as percentage of the baseline levels ( time point before the injection ) . Data are represented as mean ± SEM . Five animals belonging to the CodopV3 group were analysed . Significant differences were assessed using the Kruskal − Wallis test ; for IP - 10 : * indicates the p value ≤ 0 . 05 between 0 and 4 wpi ; * indicates the p value ≤ 0 . 05 between 0 and 8 wpi ; * indicates the p value ≤ 0 . 05 between 0 and 16 wpi . ( n = 20 ) ; for eotaxin : * indicates the p value ≤ 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Pro-inflammatory cytokines levels followed over-time and reported as percentage of the baseline levels (time point before the injection). Data are represented as mean±SEM. Five animals belonging to the CodopV3 group were analysed. Significant differences were assessed using the Kruskal−Wallis test; for IP-10: * indicates the p value ≤ 0.05 between 0 and 4 wpi; * indicates the p value ≤ 0.05 between 0 and 8 wpi; * indicates the p value ≤ 0.05 between 0 and 16 wpi. (n=20); for eotaxin: * indicates the p value ≤ 0.05.