![Figure 1. Evaluating codon-optimized and hyperfunctional FIX transgenes by LV delivery in hemophilic mice. Mice were intravenously administered matched doses (in transducing units, as measured on 293T cells) of LVs expressing the indicated FIX transgene. FIX expression and clotting activity were measured by ELISA (A,C,E) and activated partial thromboplastin time (B,D,F), respectively, on plasma samples collected at the indicated times after LV administration. Vector copies per diploid genome (vector copy number [VCN]) were measured at the end of the experiments in liver genomic DNA. (A-B) A total of 5 × 108 TU (filled line) of ET.cFIX.142T (squares, n = 4; VCN 1.3 ± 0.1) or ET.co-cFIX.142T (triangles, n = 4; VCN 0.9 ± 0.2); 1 × 109 TU (dashed line) of ET.cFIX.142T (squares, n = 4; VCN 1.6 ± 0.3) or ET.co-cFIX.142T (triangles, n = 4; VCN 1.4 ± 0.4). (C-D) A total of 7 × 108 TU of ET.cFIX.142T (squares, n = 4; VCN 1.1 ± 0.2) or ET.cFIXR338L.142T (diamonds, n = 6; VCN 1.3 ± 0.3) or ET.co-cFIXR338L.142T (triangles, n = 7; VCN 2.2 ± 0.2). (E-F) A total of 2.5 × 108 TU (black line, n = 3; VCN 1.4 ± 0.2) or 1.25 × 108 TU (gray line, n = 3; VCN 0.6 ± 0.1) of ET.co-cFIXR338L.142T. Data are mean ± SEM. *pGLOBAL < .05 (nonparametric combination statistics). ***pGLOBAL < .001 (nonparametric combination statistics). (G) Tail-clipping assay on hemophilia B mice treated with 2.5 × 108 TU of ET.co-cFIX.142T (n = 3) or ET.co-cFIXR338L (n = 3) as indicated. Blood loss (mean ± SEM) was determined by measuring the absorbance at 575 nm of hemoglobin content in the saline solution in which the tail was placed (black bars, left axis); cFIX activity (white bars, right axis). WT (n = 5) and untreated hemophilia B (HemoB) mice (n = 5) were used as controls. (H-I) FIX expression and clotting activity were measured by ELISA and chromogenic FIX assay, respectively, on plasma samples collected at the indicated times after 109 TU (n = 4) or 2 × 109 TU (n = 3) of ET.co-hFIXR338L.142T LV administration as indicated. Data are mean ± SEM ns indicates not significant. *P < .05 (t test or ANOVA). **P < .01 (t test or ANOVA). ***P < .001 (t test or ANOVA). (J) Analysis of immune tolerance induction in mice injected with 2 × 109 TU of ET.co-hFIXR388L.142T LV (n = 3). FIX-specific antibodies (mean ± SEM) were measured by ELISA at week 2 (w2) or week 4 (w4) after immunization with hFIX protein (ie, respectively, w8 and w10 after LV). FIX activity (mean ± SEM) was analyzed in parallel by chromogenic assay. Immunized PBS-injected hemophilia B mice (n = 3) were used as control.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/120/23/10.1182_blood-2012-05-432591/4/zh89991299580001.jpeg?Expires=1725024643&Signature=vAGIqIqAZxqK0xIeiS4AqLGfggNuX4eW4xKZZp4WGR3nNQd47BUdlYf6w2PiaTpUajdbIMzO9aSKS3PRCoU1LQH6-xtIvWFWEFFKg1hQp8xigDQnC0OT1MFh7hFKtP9tsMhS6fo1Qey9puvD0ezWfH~L1l2F4cJPhSyRRF9rbNNPPYkQ901PKvVMoNL49vDe0t2mJsAAtLks57KP3-9DT7beHi4XErD2mC5jKPIesWi6SuAycIldSrP9-I-8H-ijRP7eDTtqVcZfQRk4p-Fi8~rVkr-L-bP7phcs0ESaQHUPOVdWuMkH35VQj11EyUJ6xNLHHouSpuXmGm27kDUuMA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Evaluating codon-optimized and hyperfunctional FIX transgenes by LV delivery in hemophilic mice. Mice were intravenously administered matched doses (in transducing units, as measured on 293T cells) of LVs expressing the indicated FIX transgene. FIX expression and clotting activity were measured by ELISA (A,C,E) and activated partial thromboplastin time (B,D,F), respectively, on plasma samples collected at the indicated times after LV administration. Vector copies per diploid genome (vector copy number [VCN]) were measured at the end of the experiments in liver genomic DNA. (A-B) A total of 5 × 108 TU (filled line) of ET.cFIX.142T (squares, n = 4; VCN 1.3 ± 0.1) or ET.co-cFIX.142T (triangles, n = 4; VCN 0.9 ± 0.2); 1 × 109 TU (dashed line) of ET.cFIX.142T (squares, n = 4; VCN 1.6 ± 0.3) or ET.co-cFIX.142T (triangles, n = 4; VCN 1.4 ± 0.4). (C-D) A total of 7 × 108 TU of ET.cFIX.142T (squares, n = 4; VCN 1.1 ± 0.2) or ET.cFIXR338L.142T (diamonds, n = 6; VCN 1.3 ± 0.3) or ET.co-cFIXR338L.142T (triangles, n = 7; VCN 2.2 ± 0.2). (E-F) A total of 2.5 × 108 TU (black line, n = 3; VCN 1.4 ± 0.2) or 1.25 × 108 TU (gray line, n = 3; VCN 0.6 ± 0.1) of ET.co-cFIXR338L.142T. Data are mean ± SEM. *pGLOBAL < .05 (nonparametric combination statistics). ***pGLOBAL < .001 (nonparametric combination statistics). (G) Tail-clipping assay on hemophilia B mice treated with 2.5 × 108 TU of ET.co-cFIX.142T (n = 3) or ET.co-cFIXR338L (n = 3) as indicated. Blood loss (mean ± SEM) was determined by measuring the absorbance at 575 nm of hemoglobin content in the saline solution in which the tail was placed (black bars, left axis); cFIX activity (white bars, right axis). WT (n = 5) and untreated hemophilia B (HemoB) mice (n = 5) were used as controls. (H-I) FIX expression and clotting activity were measured by ELISA and chromogenic FIX assay, respectively, on plasma samples collected at the indicated times after 109 TU (n = 4) or 2 × 109 TU (n = 3) of ET.co-hFIXR338L.142T LV administration as indicated. Data are mean ± SEM ns indicates not significant. *P < .05 (t test or ANOVA). **P < .01 (t test or ANOVA). ***P < .001 (t test or ANOVA). (J) Analysis of immune tolerance induction in mice injected with 2 × 109 TU of ET.co-hFIXR388L.142T LV (n = 3). FIX-specific antibodies (mean ± SEM) were measured by ELISA at week 2 (w2) or week 4 (w4) after immunization with hFIX protein (ie, respectively, w8 and w10 after LV). FIX activity (mean ± SEM) was analyzed in parallel by chromogenic assay. Immunized PBS-injected hemophilia B mice (n = 3) were used as control.
