Figure 4.
AAV-AnFIX gene therapy. (A) AAV vectors incorporating AAV2 inverted terminal repeats, either the HCB (146 nt) or HHS4 (294 nt) promoter, a minute virus of mice (MVM) intron (92 nt), a FIX transgene, and a synthetic β-globin polyadenylation signal are depicted. The predicted HCB- and HHS4-containing ssDNA AAV genome sizes are 2003 and 2151 nt, respectively. (B) Hemophilia B mice were injected IV with 5 × 1012 vg/kg AAV2/8 encoding An96 Padua-LCO (closed circles) or hFIX-Padua (open triangles) driven by the highly compact, liver-directed HCB promoter (n = 6 per group). Plasma was collected biweekly for 12 weeks, and FIX activity was determined by 1-stage clotting assay. (C) Hemophilia B mice were injected IV with a 37-fold lower dose (1.4 × 1011 vp/kg) of AAV2/8-HHS4-An96-Padua (closed circles), AAV2/8-HHS4-An96 (closed squares), or AAV2/8-HHS4-hFIX-Padua (open triangles; n = 4-6 per group). Plasma was collected biweekly for 12 weeks, and FIX activity was determined by 1-stage clotting assay (C) or chromogenic assay (Rox Factor IX; Diapharma, West Chester, OH) (D). (E) AAV vector copy number was determined from liver tissue genomic DNA using qPCR. No significant differences were observed among the groups (1-way analysis of variance [ANOVA]; P = .2). (F) Saphenous vein bleeding challenge was performed on mice from each group (from left to right, open diamonds represent wild-type C57Bl/6; closed diamonds, untreated hemophilia B; open triangles, hFIX-Padua transgene; closed circles, An96-Padua transgene; closed squares, An96 transgene). The average time to clot for each mouse was measured over 30 minutes. Comparisons among the groups were made by 1-way ANOVA and post hoc Holm-Sidak testing. All groups were significantly different to the untreated hemophilia B (negative control) group (P < .05). (G) Dose-response curves were generated by administration of log10 doses of AAV2/8-HHS4-An96-Padua at 1.4 × 109 (circles), 1.4 × 1010 (squares), or 1.4 × 1011 vp/kg (triangles) to hemophilia B mice (n = 5-7 per group). Plasma FIX activity was measured by 1-stage clotting assay at biweekly intervals.

AAV-AnFIX gene therapy. (A) AAV vectors incorporating AAV2 inverted terminal repeats, either the HCB (146 nt) or HHS4 (294 nt) promoter, a minute virus of mice (MVM) intron (92 nt), a FIX transgene, and a synthetic β-globin polyadenylation signal are depicted. The predicted HCB- and HHS4-containing ssDNA AAV genome sizes are 2003 and 2151 nt, respectively. (B) Hemophilia B mice were injected IV with 5 × 1012 vg/kg AAV2/8 encoding An96 Padua-LCO (closed circles) or hFIX-Padua (open triangles) driven by the highly compact, liver-directed HCB promoter (n = 6 per group). Plasma was collected biweekly for 12 weeks, and FIX activity was determined by 1-stage clotting assay. (C) Hemophilia B mice were injected IV with a 37-fold lower dose (1.4 × 1011 vp/kg) of AAV2/8-HHS4-An96-Padua (closed circles), AAV2/8-HHS4-An96 (closed squares), or AAV2/8-HHS4-hFIX-Padua (open triangles; n = 4-6 per group). Plasma was collected biweekly for 12 weeks, and FIX activity was determined by 1-stage clotting assay (C) or chromogenic assay (Rox Factor IX; Diapharma, West Chester, OH) (D). (E) AAV vector copy number was determined from liver tissue genomic DNA using qPCR. No significant differences were observed among the groups (1-way analysis of variance [ANOVA]; P = .2). (F) Saphenous vein bleeding challenge was performed on mice from each group (from left to right, open diamonds represent wild-type C57Bl/6; closed diamonds, untreated hemophilia B; open triangles, hFIX-Padua transgene; closed circles, An96-Padua transgene; closed squares, An96 transgene). The average time to clot for each mouse was measured over 30 minutes. Comparisons among the groups were made by 1-way ANOVA and post hoc Holm-Sidak testing. All groups were significantly different to the untreated hemophilia B (negative control) group (P < .05). (G) Dose-response curves were generated by administration of log10 doses of AAV2/8-HHS4-An96-Padua at 1.4 × 109 (circles), 1.4 × 1010 (squares), or 1.4 × 1011 vp/kg (triangles) to hemophilia B mice (n = 5-7 per group). Plasma FIX activity was measured by 1-stage clotting assay at biweekly intervals.

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