In vivo genome editing of the albumin locus as a platform for protein replacement therapy

Abstract

Site-specific genome editing provides a promising approach for achieving long-term, stable therapeutic gene expression. Genome editing has been successfully applied in a variety of preclinical models, generally focused on targeting the diseased locus itself; however, limited targeting efficiency or insufficient expression from the endogenous promoter may impede the translation of these approaches, particularly if the desired editing event does not confer a selective growth advantage. Here we report a general strategy for liver-directed protein replacement therapies that addresses these issues: zinc finger nuclease (ZFN) -mediated site-specific integration of therapeutic transgenes within the albumin gene. By using adeno-associated viral (AAV) vector delivery in vivo, we achieved long-term expression of human factors VIII and IX (hFVIII and hFIX) in mouse models of hemophilia A and B at therapeutic levels. By using the same targeting reagents in wild-type mice, lysosomal enzymes were expressed that are deficient in Fabry and Gaucher diseases and in Hurler and Hunter syndromes. The establishment of a universal nuclease-based platform for secreted protein production would represent a critical advance in the development of safe, permanent, and functional cures for diverse genetic and nongenetic diseases.

Figure 1

Hepatic gene targeting of the mouse albumin locus results in phenotypic correction of hemophilia B. (A) Schematic illustrating albumin targeting strategy. (B) Cel I nuclease assay from liver DNA measuring ZFN-induced indels within albumin intron 1. Lanes represent individual mice at day 7 after AAV8-ZFN treatment. (C) hFIX in mouse plasma after treatment with AAV8-hF9-donor and either AAV8-ZFN (blue circles) or AAV8-hF9-ZFN (green diamonds) with a target sequence not present in the mouse genome; n = 3 mice per group. (D) hFIX levels at week 2 after treatment are proportional to AAV dose (1:5 ZFN to donor). Gray bar: normal levels. Points represent individual mice. (E) hFIX levels in hemophilia B mice 2 weeks after treatment with AAV8-mAlb-ZFN and AAV8-hF9-donor (n = 4 mice per group). *P = .029, Fisher's exact test. (F) Clot formation in mice depicted in panel E, measured by aPTT prior to and 2 weeks after treatment. The aPTTs of wild-type mice are shown for comparison. **P < .01, 2-tailed Mann-Whitney test. HDR, homology directed repair; n.s., nonsignificant; SA, splice acceptor.

Figure 2

Targeting albumin supports production of therapeutic levels of FVIII and functional correction of hemophilia A phenotype. (A) FVIII activity as determined by chromogenic assay in hemophilia A/CD4-deficient mice 2 and 8 weeks after treatment with 1 × 10¹¹ vg of AAV8-mock (red circles), 5 × 10¹⁰ vg of each individual AAV8-ZFN (green circles), and 1 × 10¹¹ vg of donor 2 (see “Methods” section for details). **P = .008, Fisher's exact test. (B) Measurement of clot formation by aPTT prior to and 11 weeks after AAV administration. The aPTT of wild-type (▪) and untreated (●) mice are shown for comparison. **P < .01, 2-tailed Mann-Whitney test.

Figure 3

Expression of lysosomal enzymes deficient in Fabry and Gaucher diseases and Hurler and Hunter syndromes. Top panels of (A-D) Western blot detection of (A) α-galactosidase A, (B) α-l-iduronidase, (C) iduronate-2 sulfatase, and (D) acid β-glucosidase in liver lysates of mice 30 days after treatment with 3 × 10¹¹ vg of AAV8-ZFN and AAV8 of the appropriate donor at the indicated ratio of 1:1 or 1:5 (see “Methods” section for details). Middle panels (A-D) PCR detection of bands consistent with homology directed (HDR) and homology independent (NHEJ) integration of donor at the albumin locus. Lower panels (A-D) Indel formation as measured by MiSeq sequencing (n = 3 mice per group). Each lane represents an individual mouse. LSD, lysosomal storage disease.

Figure 4

Targeting of albumin locus promotes stable supraphysiological activity of GBA in mouse plasma. (A) GBA activity as determined by enzymatic activity assay in wild-type mice 3 weeks after treatment with either 1.2 × 10¹² vg AAV8-GBA donor alone (red circle) or in combination with 1.5 × 10¹¹ vg of each individual AAV8-ZFN (green circle). Untreated wild-type mice (▪) shown as controls. (B) Time course of GBA activity in the mice treated in (A). **P < .01, 2-tailed Mann-Whitney test comparing ZFN + donor group to untreated or donor only groups.