Figure 5A Alkaline gel Southern blot analysis of AAV DNA . AAV DNA was hybridized to a probe specific for the HLP promoter . Neg , AAV DNA treated with Dnase I ; CodopV3 , AAV - CodopV3 ; I , AAV - 5 ' N6 - N - intein ; II , AAV - C - intein - 3 ' N6 . B Chromogenic assay performed on plasma samples to detect F8 activity in AAV - treated mice compared to controls groups . Data are presented as mean ± SEM . The same animals were analysed at the various time - points . Each dot within the same group and within different groups of treatment corresponds to a single animal . The baseline includes all animals ( n = 20 ) before the treatment : CodopV3 : n = 8 ; N6 intein : n = 5 ; CodopN6 intein : n = 5 . The statistical difference between groups has been assessed at 4weeks post - injection ( wpi ) with the One - way Anova p value = 0 . 056 ; at 8 wpi with the Kruskal - Wallis test p value = 0 . 68 ; at 12wpi with the Kruskal - Wallis test p value = 0 . 45 ; at 16wpi with the Kruskal - Wallis test p value = 0 . 58 . C Activated partial thromboplastin time ( aPTT ) assay performed on plasma samples both at the baseline and at the last time - point of the analysis ( 16 wpi ) . Data are presented as mean ± SEM . Each dot within each group of treatment corresponds to different animals : The baseline includes all animals before the treatment n = 21 ; CodopV3 : n = 8 ; N6 intein : n = 5 ; CodopN6 intein : n = 5 . Significant differences between groups were assessed using the Kruskal - Wallis test . * * indicates the significant difference between the baseline and the N6 intein groups : p value ≤ 0 . 01 . * indicates the significant difference between the baseline and the CodopN6 intein groups : p value ≤ 0 . 05 . D Tail - clip assay reported as bleeding time in seconds . Significant differences between groups were assessed using One - way Anova test p value is = 0 . 11 . Data are presented as mean ± SEM . Each dot corresponds to a different animal . Knockout ( K . O . ) group : n = 4 ; CodopV3 : n = 4 ; N6 intein : n = 4 ; CodopN6 intein : n = E Haemoglobin content measured as Optical Density ( OD ) at 416 nm after the tail - clip assay . Significant differences between groups were assessed using One - way Anova test . Data are reported as mean ± SEM . Each dot corresponds to a single animal . Knockout ( K . O . ) group : n = 4 ; CodopV3 : n = 4 ; N6 intein : n = 4 ; CodopN6 intein : n = 6 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Alkaline gel Southern blot analysis of AAV DNA. AAV DNA was hybridized to a probe specific for the HLP promoter. Neg, AAV DNA treated with Dnase I; CodopV3, AAV-CodopV3; I, AAV-5'N6-N-intein; II, AAV-C-intein-3' N6.B Chromogenic assay performed on plasma samples to detect F8 activity in AAV-treated mice compared to controls groups. Data are presented as mean ±SEM. The same animals were analysed at the various time-points. Each dot within the same group and within different groups of treatment corresponds to a single animal. The baseline includes all animals (n=20) before the treatment: CodopV3: n=8; N6 intein: n=5; CodopN6 intein: n=5. The statistical difference between groups has been assessed at 4weeks post-injection (wpi) with the One-way Anova p value = 0.056; at 8 wpi with the Kruskal-Wallis test p value = 0.68; at 12wpi with the Kruskal-Wallis test p value = 0.45; at 16wpi with the Kruskal-Wallis test p value = 0.58.C Activated partial thromboplastin time (aPTT) assay performed on plasma samples both at the baseline and at the last time-point of the analysis (16 wpi). Data are presented as mean ±SEM. Each dot within each group of treatment corresponds to different animals: The baseline includes all animals before the treatment n=21; CodopV3: n=8; N6 intein: n=5; CodopN6 intein: n=5. Significant differences between groups were assessed using the Kruskal-Wallis test. **indicates the significant difference between the baseline and the N6 intein groups: p value ≤ 0.01.*indicates the significant difference between the baseline and the CodopN6 intein groups: p value ≤ 0.05.D Tail-clip assay reported as bleeding time in seconds. Significant differences between groups were assessed using One-way Anova test p value is = 0.11.Data are presented as mean±SEM. Each dot corresponds to a different animal. Knockout (K.O.) group: n=4; CodopV3: n=4; N6 intein: n=4; CodopN6 intein: n=E Haemoglobin content measured as Optical Density (OD) at 416 nm after the tail-clip assay. Significant differences between groups were assessed using One-way Anova test. Data are reported as mean±SEM. Each dot corresponds to a single animal. Knockout (K.O.) group: n=4; CodopV3: n=4; N6 intein: n=4; CodopN6 intein: n=6.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).


Figure 6A 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A 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.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.