Figure 2.

Functionality, safety, and specificity assessment of the vectors. (A-B) Phenotypic correction was assessed using a tail-clip model to measure the bleeding time and blood loss in the repeat experiment for experimental groups of R338L-Padua and CB 2679d-GT at the doses of 5 × 10⁹ vg per mouse (n = 4) (A) and 1 × 10¹⁰ vg per mouse (n = 4) (B) compared with the vehicle control (n = 5) and C57BL/6 normal controls (n = 10). For humane reasons, vehicle control mice were euthanized after 120 minutes, although they were still bleeding at that time. Results are presented as mean ± standard error of the mean. *P < .05; **P < .01; ***P < .001 (Student t test); NS, not significant (P > .1) (C-D) The FIX-specific antibodies were determined by ELISA in hemophilia B mice (FIX^−/−) injected with R338L-Padua and CB 2679d-GT at 3 doses (1 × 10⁹vg per mouse, 5 × 10⁹ vg per mouse and 1 × 10¹⁰ vg per mouse) at time points of 9 weeks (C) and 20 weeks (D) postinjection. *P < .05; **P < .01; ***P < .001 (Student t test); NS, not significant (P > .1). (E-H) AAV copy number in a panel of 8 organs from the mice injected with R338L-Padua or CB 2679d-GT at the 2 doses of 5 × 10⁹ vg per mouse (E) and 1 × 10¹⁰ vg per mouse (F) were analyzed at 20 weeks postinjection using quantitative real-time PCR. The same mice were also analyzed for RNA expression in a panel of 8 organs by quantitative reverse transcription PCR to confirm that the RNA expression for both the vector groups was specific to liver 5 × 10⁹ vg per mouse (G) and 1 × 10¹⁰ vg per mouse (H). The RNA expression of the transgene was normalized to mouse glyceraldehyde-3-phosphate dehydrogenase expression. Results are presented as mean ± standard error of the mean.