Robust ZFN-mediated genome editing in adult hemophilic mice

Abstract

Monogenic diseases, including hemophilia, represent ideal targets for genome-editing approaches aimed at correcting a defective gene. Here we report that systemic adeno-associated virus (AAV) vector delivery of zinc finger nucleases (ZFNs) and corrective donor template to the predominantly quiescent livers of adult mice enables production of high levels of human factor IX in a murine model of hemophilia B. Further, we show that off-target cleavage can be substantially reduced while maintaining robust editing by using obligate heterodimeric ZFNs engineered to minimize unwanted cleavage attributable to homodimerization of the ZFNs. These results broaden the therapeutic potential of AAV/ZFN-mediated genome editing in the liver and could expand this strategy to other nonreplicating cell types.

Figure 1

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 described 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.

Figure 2

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.