Obligate heterodimer ZFNs reduce off-target cleavage in vivo while preserving equivalent levels of hF.IX secretion. (A) Cel-I cleavage at the 4 validated off targets identified by clustered integration site analysis 4 weeks after treatment with 5 × 10¹¹ vg AAV8-Donor and either 1 × 10¹¹ vg AAV8-ZFN^WT, 1 × 10¹¹ vg AAV8-ZFN^ELD:KKR, or 1 × 10¹¹ vg AAV8-Mock. (B) Plasma hF.IX levels following treatment. (C) On-target cleavage measured by Cel-I assay 4 weeks after AAV injection. (D) hF.IX expression in WT mice lacking ZFN target site 5 weeks after injection of 5 × 10¹¹ vg AAV8-Donor and either 1 × 10¹¹ vg AAV8-ZFN^WT or 1 × 10¹¹ vg AAV8-ZFN^ELD:KKR. hF.IX plasma levels were assayed by ELISA. A two-tailed t test was used. ***P < .0001 compared with ZFN^WT (n = number of mice in each cohort). Error bars denote standard error of the mean. Frequency of ZFN-induced insertions and deletions is indicated as “% of Indels” below each lane. Each lane represents an individual mouse. Plasma factor IX data are representative of at least 2 independent experiments.