Resolution of hepatic fibrosis after ZFN-mediated gene editing in the PiZ mouse model of human α1-antitrypsin deficiency

Abstract

Background: α1-antitrypsin deficiency is most commonly caused by a mutation in exon-7 of SERPINA1 (SA1-ATZ), resulting in hepatocellular accumulation of a misfolded variant (ATZ). Human SA1-ATZ-transgenic (PiZ) mice exhibit hepatocellular ATZ accumulation and liver fibrosis. We hypothesized that disrupting the SA1-ATZ transgene in PiZ mice by in vivo genome editing would confer a proliferative advantage to the genome-edited hepatocytes, enabling them to repopulate the liver.

Methods: To create a targeted DNA break in exon-7 of the SA1-ATZ transgene, we generated 2 recombinant adeno-associated viruses (rAAV) expressing a zinc-finger nuclease pair (rAAV-ZFN), and another rAAV for gene correction by targeted insertion (rAAV-TI). PiZ mice were injected i.v. with rAAV-TI alone or the rAAV-ZFNs at a low (7.5×1010vg/mouse, LD) or a high dose (1.5×1011vg/mouse, HD), with or without rAAV-TI. Two weeks and 6 months after treatment, livers were harvested for molecular, histological, and biochemical analyses.

Results: Two weeks after treatment, deep sequencing of the hepatic SA1-ATZ transgene pool showed 6%±3% or 15%±4% nonhomologous end joining in mice receiving LD or HD rAAV-ZFN, respectively, which increased to 36%±12% and 36%±12%, respectively, 6 months after treatment. Two weeks postinjection of rAAV-TI with LD or HD of rAAV-ZFN, repair by targeted insertion occurred in 0.10%±0.09% and 0.25%±0.14% of SA1-ATZ transgenes, respectively, which increased to 5.2%±5.0% and 33%±13%, respectively, 6 months after treatment. Six months after rAAV-ZFN administration, there was a marked clearance of ATZ globules from hepatocytes, and resolution of liver fibrosis, along with reduction of hepatic TAZ/WWTR1, hedgehog ligands, Gli2, a TIMP, and collagen content.

Conclusions: ZFN-mediated SA1-ATZ transgene disruption provides a proliferative advantage to ATZ-depleted hepatocytes, enabling them to repopulate the liver and reverse hepatic fibrosis.

FIGURE 1

Editing of the pathogenic mutation in the human SERPINA1 transgene (SA1-ATZ): (A) Gene correction schematic. (i) Recombinant AAV for correcting the pathogenic ATZ mutation in the human SA1-ATZ transgene in PiZ mice by homology-directed targeted insertion (rAAV-TI) was designed with the wild-type human SERPINA1 sequence. Left and right homology arms ~1 kb each were used to promote homology-directed repair at the ZFN cut site (between 25264 and 25277) near the PiZ mutation. Two silent mutations (SMS2 and SMS11) were introduced into 1 of the 2 ZFN binding sites (25264 and 25277) encoded within the AAV donor construct to prevent ZFN binding and recleavage after gene correction. These silent mutations do not change the amino acid coding sequence within exon-7 of the SA1-ATZ transgene, but alter the DNA sequence of the ZFN binding site, which prevents binding of the specific sequence-targeted 25264 ZFN. The rAAV-TI also consists of inverted terminal repeats (ITRs) at the 5′ and 3′ of the packaged viral genome. (ii) Human SA1-ATZ locus shown with ZFN binding sites and AAV gene correction donor homology arm sequences. (B) Genomic editing of human SA1-ATZ transgene in PiZ mouse livers 2 weeks and 6 months after injection of rAAVs. (i) rAAV-TI gene correction donor and/or rAAV-ZFN pair, encoding human SERPINA1-targeted ZFN expression constructs (25264 and 25277) were i.v. injected into PiZ mice at a constant dose of rAAV-TI (1.5×10^¹²vg) and/or high (HD, 1.5×10^¹¹vg) or low doses (LD, 7.5×10^¹⁰vg) of rAAV-ZFN. Although there was a trend of increasing indels from 2 weeks to 6 months for most ZFN-treated groups, differences in indels between the AAV-treated groups were not statistically significant. (ii) Targeted integration (TI) of the gene correction construct was observed only in groups receiving the donor rAAV-TI plus rAAV-ZFNs at 6 months after treatment. Gene correction occurred to a greater extent in the HD rAAV-ZFN group. p=0.0087 comparing TI at the 2-week versus 6-month time points in rAAV-TI + rAAV-ZFNs (high dose) groups. p=0.0303 comparing TI for low dose and high-dose Donor + ZFNs 6-month posttreatment groups. Green data points indicate animals from cohort 1; all other data points are from cohort 2. Error bars represent SE of the mean (SEM). Mann-Whitney unpaired nonparametric analysis was used to calculate statistical significance. (C) Genomic editing of human SA1-ATZ transgene in hepatocytes isolated from PiZ mouse livers at 2 weeks and 6 months after injection of rAAVs. (i) rAAV-TI and/or rAAV-ZFNs encoding ZFNs targeting the SA1-ATZ transgene (25264 and 25277) were injected i.v. into PiZ mice at a constant dose of rAAV donor (1.5×10^¹²vg/mouse) and/or a low or high dose of rAAV-ZFNs (7.5×10^¹⁰ vg/mouse or 1.5×10^¹¹ vg/mouse, respectively). Six months after treatment, hepatocytes were isolated by collagenase perfusion and purified by differential centrifugation for subsequent genome editing analysis. Difference in indel frequency observed between any of the rAAV-ZFN-treated groups was not statistically significant. (ii) Targeted integration (TI) of the gene correction construct was observed at higher levels only in the rAAV-TI plus rAAV-ZFN groups, although the difference in frequency of TI observed between groups did not reach statistical significance because of the limited sample size. Mann-Whitney unpaired nonparametric analysis was used to calculate statistical significance. Abbreviations: ZFN, zinc-finger nuclease.

FIGURE 2

Human SERPINA1 in liver and serum. Globules of polymerized mutant human SERPINA1 in PiZ mouse hepatocytes. Six-week-old PiZ mice were injected with the different combinations of rAAV as listed in Table 1 (6 mice per group) and sacrificed 2 weeks or 6 months later for histological and serological analysis. Age-matched PBS-injected PiZ mice were used as control. (A) Periodic acid-Schiff (PAS) staining: liver cryosections were treated with diastase to deplete glycogen and then polymerized PiZ globules were visualized by PAS staining (arrows). Inset shows a magnified globule-containing hepatocyte. (B) Quantification of PiZ globule-containing hepatocytes: percentage of globule-containing hepatocytes was determined by counting at least 1600 cells in each section. Bars represent means±SEM. Note that in PBS-treated control PiZ mice, the proportion of globule-containing cells decreased in the interval between 2 weeks and 6 months after injection. Similar degree of reduction of globule-containing cells occurred in mice receiving rAAV-TI only (group 1) between 2 weeks and 6 months after injection. In contrast, there was significantly greater reduction in the percentage of globule-containing cells between 2 weeks and 6 months in all groups receiving rAAV-ZFN at low (7.5×10^¹⁰ vg/mouse, LD) or high (1.5×10^¹¹vg/mouse, HD) dose without (groups 2 and 3) or with (groups 4 and 5) rAAV-TI. (*Significantly different from values in PBS-injected PiZ mice 6 months after injection, p<0.02). Addition of the rAAV-TI did not significantly enhance the reduction of the globule-containing cells over administration of rAAV-ZFN alone. (C) Serum total human AAT concentrations: AAT levels in the PiZ mouse sera were determined by ELISA using a human AAT-specific antibody, before rAAV administration and 2 weeks and 6 months after treatment. Injection of rAAV-TI alone (group 1) did not significantly reduce serum human PiZ levels between 2 weeks and 6 months after treatment. In contrast, serum human PiZ levels were significantly reduced (*p<0.02) at the 6-month time point compared with the 2-week levels in all recipients of rAAV-ZFN, both at the low (LD) or high (HD) dose, with or without rAAV-TI. Arrows: PiZ globules. Inset: magnifies view of PiZ globule-containing hepatocyte. Abbreviations: HR, homologous recombination; ZFN, zinc-finger nuclease.

FIGURE 3

Histological evaluation of liver fibrosis: The extent of liver fibrosis was evaluated by staining liver paraffin sections with 2 different methods: Sirius red and Masson trichrome. The experimental and control groups are as in Table 1. (A) Sirius red staining: representative liver sections from PBS-treated PiZ mice and mice from each of the 5 experimental groups at indicated time points after injection are shown. Note that without treatment, liver fibrosis increases with age in PiZ mice, as shown in the PBS-stained control. (B) left panel: Sirius red–stained liver section from a 30-wk old PBS-treated control PiZ mouse showing a regenerative nodule consisting of groups of hepatocytes surrounded by collagen strands. Right panel: liver section from 30-week-old PiZ mouse from group 5, which had received treatment 6 months before the analysis, showing no evidence of regenerative nodule formation. (C) Quantification of the sirius red–stained collagen fibers by ImageJ analysis. Gray bars, 2 weeks after treatment; solid black bars, 6 months after treatment. Liver sections from untreated normal C57/Bl6 mice were analyzed as normal controls. *Significantly different from values in PBS-injected PiZ mice 6 months after injection, p<0.02. (D). Masson trichrome staining: representative liver sections from values in PBS-treated PiZ mice and mice from experimental groups 1 and 5 at indicated time points after injection are shown. (E) Quantification of the Masson trichrome-stained collagen fibers (blue) by ImageJ analysis. Gray bars, 2 weeks after treatment; solid black bars, 6 months after treatment. Liver sections from untreated normal C57/Bl6 mice were analyzed as normal controls. *Significantly different from values in PBS-injected PiZ mice 6 months after injection, p<0.02). Abbreviations: HD, high dose; HR, homologous recombination; rAAV, recombinant adeno-associated viruses; WT, wild type; ZFN, zinc-finger nuclease.

FIGURE 4

Western blot analysis of hepatic collagen. (A) Western blot analysis for collagen-1: Western blot analysis was performed on liver homogenates of PiZ mice in all 5 experimental groups and PBS-injected PiZ mouse controls 2 and 6 weeks after injections. Livers from age-matched wild-type C57/Bl6 mice were also analyzed. Representative western blots for collagen-1 are shown along with β-actin as loading control. B. Quantification of collagen-1: hepatic collagen-1 contents were determined by scanning the western blot bands and are shown as percentage of the collagen-1 content in 30-week-old wild-type C57/Bl6 mouse livers (means±SEM, 6 mice in each group). *Significantly different from values in PBS-injected PiZ mice 6 months after injection, p<0.02). Abbreviations: rAAV, recombinant adeno-associated viruses.

FIGURE 5

Hepatic Ihh, Shh, and TAZ (WWTR1) content: representative western blots for Ihh (A), Shh (C), and TAZ/WWTR1 (E), along with β-actin as loading control are shown for group 1 (rAAV-TI only), group 3 (rAAV-ZFN HD), group 5 (rAAV-ZFN HD + rAAV-TI), and PBS-injected control PiZ mice at the 6 month time point after injection. Liver from an age-matched wild-type C57/Bl6 mouse is also shown. Hepatic contents of Ihh (B), Shh (D), and TAZ/WWTR1 (F), as well as β-actin were determined by scanning the western blot bands and are shown as percentage of the protein content in 30-week-old wild-type C57/Bl6 mouse livers (means±SEM, 6 mice in each group). *Significantly different from values in PBS-injected PiZ mice 6 months after injection, p<0.02). Abbreviations: Ihh, Indian hedgehog; rAAV, recombinant adeno-associated viruses; Shh, sonic hedgehog.

FIGURE 6

Hepatic Gli2 and TIMP content: representative western blots for Gli2 (6A) and TIMP (6C), along with β-actin as loading control are shown for group 1 (rAAV-TI only), group 3 (rAAV-ZFN HD), group 5 (rAAV-ZFN HD + rAAV-TI), and PBS-injected PiZ mice 6 months after injection. Liver from an age-matched wild-type C57/Bl6 mouse is also shown. Hepatic Gli2 (6B) and TIMP (6D) contents were determined by scanning the western blot bands and are shown as percentage of the Gli2 and TIMP content in 30-week-old wild-type C57/Bl6 mouse livers (means±SEM, 6 mice in each group). *Significantly different from values in PBS-injected PiZ mice 6 months after injection, p<0.02). Abbreviation: Gli2, glioblastoma-2; rAAV, recombinant adeno-associated viruses.