Antisense oligonucleotide treatment ameliorates alpha-1 antitrypsin-related liver disease in mice

Abstract

Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disease that results from mutations in the alpha-1 antitrypsin (AAT) gene. The mutant AAT protein aggregates and accumulates in the liver leading to AATD liver disease, which is only treatable by liver transplant. The PiZ transgenic mouse strain expresses a human AAT (hAAT) transgene that contains the AATD-associated Glu342Lys mutation. PiZ mice exhibit many AATD symptoms, including AAT protein aggregates, increased hepatocyte death, and liver fibrosis. In the present study, we systemically treated PiZ mice with an antisense oligonucleotide targeted against hAAT (AAT-ASO) and found reductions in circulating levels of AAT and both soluble and aggregated AAT protein in the liver. Furthermore, AAT-ASO administration in these animals stopped liver disease progression after short-term treatment, reversed liver disease after long-term treatment, and prevented liver disease in young animals. Additionally, antisense oligonucleotide treatment markedly decreased liver fibrosis in this mouse model. Administration of AAT-ASO in nonhuman primates led to an approximately 80% reduction in levels of circulating normal AAT, demonstrating potential for this approach in higher species. Antisense oligonucleotides thus represent a promising therapy for AATD liver disease.

Figure 1. Characterization of an ASO that effectively reduces AAT levels in cells.

(A) Dose-dependent reduction of AAT mRNA in HepG2 cells. (B) Dose-dependent reduction of hAAT mRNA in primary hepatocytes isolated from PiZ mice. Cells were electroporated in growth medium in the presence of AAT-ASO at the indicated concentrations and plated. Twenty-four hours after transfection, total cellular RNA was isolated and the amount of AAT mRNA present was quantitated using a qRT-PCR assay (TaqMan). Results represent mean ± SD (n = 3) compared to untransfected control (UTC). (C) Dose-dependent reduction of hAAT mRNA levels in PiZ mice after AAT-ASO treatment. (D) Dose-dependent reduction of circulating hAAT protein in PiZ mice after AAT-ASO treatment. Six-week-old PiZ mice were treated for 4 weeks with the indicated doses of AAT-ASO via subcutaneous injection. hAAT mRNA levels in livers were quantified by qRT-PCR (TaqMan), and plasma AAT levels were determined by an immunoturbidimetric method. Results represent mean ± SD (n = 4). *P < 0.05, **P < 0.01 by 2-way ANOVA with Bonferroni’s post-hoc tests for A, B, and D; 1-way ANOVA with Tukey’s comparisons for C.

Figure 2. Soluble and aggregated AAT protein levels were reduced in PiZ mouse livers after AAT-ASO treatment.

(A) Six-week-old PiZ mice were treated for 8 weeks with PBS, 50 mg/kg/wk AAT-ASO, or control ASO via subcutaneous injection. hAAT mRNA levels in livers were quantified by qRT-PCR (TaqMan), and plasma AAT protein levels were determined by clinical chemistry analyzer. (B) IHC staining for AAT protein and PAS-D staining in liver. Scale bar: 50 μm. (C) Soluble and insoluble fractions from PiZ livers were analyzed by Western blot analysis with an anti-hAAT antibody. GAPDH was used as a loading control. BL, baseline. (D) Quantitation of soluble and (E) insoluble AAT protein from C. Results represent mean ± SD (n = 5–6 per group). **P < 0.01 by 1-way ANOVA with Tukey’s comparisons.

Figure 3. AAT-ASO treatment reversed liver globule accumulation and reduced hAAT in all hepatocytes in PiZ mice.

(A) Sixteen-week-old PiZ mice were treated for 20 weeks with 50 mg/kg/wk AAT-ASO via subcutaneous injection. Soluble and insoluble fractions from PiZ livers were analyzed by Western blot analysis with an anti-hAAT antibody. GAPDH was used as a loading control. (B) Quantitation of liver soluble AAT Western blot data shown in A. (C) Quantitation of liver insoluble AAT Western blot data shown in A. (D) ASO uptake by hepatocytes in PiZ mice. ASO was visualized by an antibody reactive to the ASO backbone. Arrows indicates perinuclear vesicle staining in hepatocytes; arrowheads indicates nonparenchymal cells. Scale bar: 50 μm; 20 μm (inset). (E) hAAT transcript visualized by in situ hybridization (QuantiGene ViewRNA, Affymetrix). The arrow indicates a globule-containing area; the arrowhead indicates a globule-devoid area. Scale bar: 100 μm; 20 μm (inset). Results represent mean ± range (n = 2 for baseline, n = 4 for treatment groups).

Figure 4. AAT-ASO treatment prevented Z protein aggregate formation and liver injury in young PiZ mice.

Two-week-old PiZ mice were treated for 8 weeks with PBS, 50 mg/kg/wk AAT-ASO, or control ASO via subcutaneous injection. (A) hAAT mRNA reduction in livers was quantified by qRT-PCR (TaqMan). (B) Plasma hAAT level reduction was determined using a clinical chemistry analyzer. (C) Plasma ALT and (D) plasma AST levels were monitored at indicated time points. (E) Liver AAT protein levels were measured by IHC before and after treatment. Liver PAS-D staining before and after treatment (representative pictures from male groups were shown). Scale bar: 50 μm. Results represent mean ± SD (n = 4–5, including both male and female mice). *P < 0.05, **P < 0.01 by 1-way ANOVA for A and repeated-measures 2-way ANOVA for B–D when AAT-ASO treatment group was compared with control ASO treatment group.

Figure 5. AAT-ASO treatment significantly reduced liver injury and fibrosis in PiZZ mice.

Five-week-old PiZZ mice were treated for 11 weeks with PBS, AAT-ASO, or control ASO via subcutaneous injection. (A) Plasma ALT levels and (B) plasma AST levels were monitored throughout the treatment period. Significant reduction of (C) Sirius red staining (quantitation in E), (F) hydroxyproline content, and (D) α-SMA staining (quantitation in G) in liver sections was observed after AAT-ASO treatment. Scale bar: 50 μm. (H) Liver fibrosis-related gene mRNA levels were quantified by qRT-PCR (TaqMan). Results represent mean ± SD in all panels except F, which is shown as mean ± SEM (n = 5–6). *P < 0.05, **P < 0.01 by repeated-measures 2-way ANOVA for A and B and 1-way ANOVA with Tukey’s comparisons for E–H when AAT-ASO treatment group was compared with control ASO treatment group.

Figure 6. Robust AAT reduction in cynomolgus hepatocytes and in vivo after AAT-ASO treatment.

(A) Dose-dependent reduction of AAT mRNA in primary hepatocytes isolated from cynomolgus monkeys. Cells were electroporated in growth medium in the presence of AAT-ASO at the indicated concentrations and plated. Twenty-four hours after transfection, total cellular RNA was isolated and the amount of AAT mRNA present was quantitated using a qRT-PCR assay (TaqMan). Results represent mean ± SD (n = 3) compared to untransfected control. (B) AAT mRNA levels in livers. (C) Plasma AAT protein levels during the treatment period. Cynomolgus monkeys were treated with AAT-ASO for 12 weeks at 25 mg/kg 3 times in the first week and twice a week in weeks 2 to 12. Liver total RNA was purified, and qRT-PCR was carried out to determine the levels of AAT mRNA in the liver. The plasma protein levels were measured by the immunoturbidimetry method using a clinical analyzer. Results represent the mean ± SD (n = 4). *P < 0.05, **P < 0.01 by 2-way ANOVA with Bonferroni’s post-hoc tests for A and C and Student’s t test for B.