miR-142-3p–regulated LV mediates stable hF.IX gene expression and correction of hemophilia B mice. (A) Measurement of hF.IX:Ag in hemophilia B mice treated with 5 × 10⁸ IU of LV.ET.FIX.142-3pT. Results are presented as the mean plus or minus SEM (n = 7/group). This includes evaluation of 2 different preparations of LV.ET.FIX.142-3pT and 3 different cohorts of hemophilia B mice. (B) Measurement of hF.IX:C by activated partial thromboplastin time (aPTT) in the same hemophilia B mice as shown in panel A. Results are presented as the mean plus or minus SEM (n = 7). (C) ELISA immunoassay of mouse plasma (1:100 dilution) to detect anti-hF.IX antibodies before (white bar) and after (gray bar) vaccination with hF.IX. Quantification was performed by analysis of absorbance at optical density (OD) 490 nm. Results are presented as the mean plus or minus SEM (n = 3/group). (D) Measurement of anti-OVA_257–264 CD8⁺ T cells. Untreated () and LV.PGK.FIX.142-3pT–treated () hemophilia B mice (n = 3/group) were immunized with the immunodominant epitope of OVA, OVA_257-264, in complete Freurd adjuvant. The frequency of anti-OVA_257-264 CD8⁺ T cells was measured by ELISPOT assay. Results are presented as the mean plus or minus SEM. (E) Tail transection was performed on LV.ET.FIX.142-3pT–treated and untreated hemophilia B mice (n = 3/group). Mice were monitored for 24 hours to assess survival. (F) Measurement of LV content in the liver of hemophilia B mice by qPCR at more than 280 days after injection (n = 7/group). Results are presented as the mean plus or minus SEM (*P < .05). (G) Measurement of LV content in the liver of hemophilia B mice by Southern blot analysis at more than 280 days after injection. Analysis was performed on the livers of 3 mice per treatment group. Untxt indicates untreated.