In vivo gene correction in adult mice results in stable circulating factor IX levels and correction of clotting times in hemophilic animals. (A-B) Levels of hF.IX in plasma of hF9mut (A) and hF9mut/HB (B) mice following intravenous injection at week 8 of life with 1 × 10¹¹ vg AAV8-ZFN alone, 1 × 10¹¹ vg AAV8-Mock and 5 × 10¹¹ vg AAV8-Donor, or 1 × 10¹¹ vg AAV8-ZFN and 5 × 10¹¹ vg AAV8-Donor. The previously described¹  hF9mut mouse model of hF.IX deficiency contains a liver-specific, promoter-driven human coagulation factor IX cDNA devoid of necessary catalytic exons 7 to 8 knocked into the Rosa26 locus. (C) Measurement of clot formation by activated partial thromboplastin time (aPTT) prior to, 2 weeks, and 80 weeks after AAV administration. The aPTT of WT mice is shown for comparison. A 2-tailed Mann-Whitney test was used to compare 2 groups. (D) Levels of hF.IX in treated mice remain stable following two-thirds partial hepatectomy. (E) hF.IX expression is more than 10-fold greater following AAV8-ZFN and AAV8-Donor treatment in hF9mut mice harboring the ZFN target site (hF9mut) compared with littermate controls that do not (WT). (F) PCR analysis showing successful gene targeting by both HDR and NHEJ 5 months after intravenous coinjection of ZFN and Donor vectors. PCR products were resolved by 5% polyacrylamide gel electrophoresis and autoradiographed. See supplemental Figure 2 for maps. Mice treated with ZFN alone or with Mock and Donor showed no evidence of targeting. Identity of the PCR products was confirmed by sequencing. The lower band (*) appears in all donor treated samples, which is an artifact apparently generated by the reverse primer and the AAV–inverted terminal repeat resulting in amplification from nonintegrated AAV genomes. hF.IX plasma levels were assayed by enzyme-linked immunosorbent assay (ELISA) and represent repeated measurements, obtained by serial bleeding, on the same group of animals over the time of the study (n = number of mice in each cohort). Error bars denote standard error of the mean. Plasma factor IX data are representative of at least 2 independent experiments.