Ferret models of alpha-1 antitrypsin deficiency develop lung and liver disease
Nan He, … , John F. Engelhardt, Bradley H. Rosen
Nan He, … , John F. Engelhardt, Bradley H. Rosen
Published February 1, 2022
Citation Information: JCI Insight. 2022;7(5):e143004. https://doi.org/10.1172/jci.insight.143004.
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Research Article Pulmonology

Ferret models of alpha-1 antitrypsin deficiency develop lung and liver disease

Abstract

Alpha-1 antitrypsin deficiency (AATD) is the most common genetic cause and risk factor for chronic obstructive pulmonary disease, but the field lacks a large-animal model that allows for longitudinal assessment of pulmonary function. We hypothesized that ferrets would model human AATD-related lung and hepatic disease. AAT-knockout (AAT-KO) and PiZZ (E342K, the most common mutation in humans) ferrets were generated and compared with matched controls using custom-designed flexiVent modules to perform pulmonary function tests, quantitative computed tomography (QCT), bronchoalveolar lavage (BAL) proteomics, and alveolar morphometry. Complete loss of AAT (AAT-KO) led to increased pulmonary compliance and expiratory airflow limitation, consistent with obstructive lung disease. QCT and morphometry confirmed emphysema and airspace enlargement, respectively. Pathway analysis of BAL proteomics data revealed inflammatory lung disease and impaired cellular migration. The PiZ mutation resulted in altered AAT protein folding in the liver, hepatic injury, and reduced plasma concentrations of AAT, and PiZZ ferrets developed obstructive lung disease. In summary, AAT-KO and PiZZ ferrets model the progressive obstructive pulmonary disease seen in AAT-deficient patients and may serve as a platform for preclinical testing of therapeutics including gene therapy.

Authors

Nan He, Xiaoming Liu, Amber R. Vegter, T. Idil A. Evans, Jaimie S. Gray, Junfeng Guo, Shashanna R. Moll, Lydia J. Guo, Meihui Luo, Ningxia Ma, Xingshen Sun, Bo Liang, Ziying Yan, Zehua Feng, Lisi Qi, Arnav S. Joshi, Weam Shahin, Yaling Yi, Katherine N. Gibson-Corley, Eric A. Hoffman, Kai Wang, Christian Mueller, John F. Engelhardt, Bradley H. Rosen

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Figure 6

Generation of a PiZZ ferret model of AATD and characterization of hepatic disease.

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Generation of a PiZZ ferret model of AATD and characterization of hepati...
(A) Schematic depiction of CRISPR/Cas9-mediated Z-allele knockin (with donor oligo) in exon 5 of the SERPINA1 locus. Founders were confirmed by Sanger sequencing (at right). (B) Representative lanes from native PAGE gel performed on liver tissue lysates showing change in band migration of the PiZ protein. Arrows mark the PiMM (MM) control bands that are shifted in PiZZ (ZZ) samples and absent in the AAT-KO sample. PiMZ (MZ) shows a combined pattern. GAPDH is shown as loading control for the native PAGE. Denatured samples were separated by SDS-PAGE and probed for AAT at the bottom of the panel. (C) mRNA expression of SERPINA1 in PiZZ ferret liver tissue compared with PiMM controls and AAT-KO nulls (n = 3–5 ferrets/group as indicated). (D) Western blot of plasma from a representative PiZZ ferret and age-matched PiMM control showing change in circulating AAT over time (AAT band marked by arrowhead). Timed blood draws in PiZZ and PiMM ferrets at 2, 4, 6, and 13 months old; AAT-KO ferret drawn at 13 months old as negative control. Below is the Ponceau stain used for loading control. (E) Densitometric quantification of plasma AAT (normalized to Ponceau band) over time in 4 pairs of PiZZ and PiMM control ferrets. (F) Comparison of plasma AAT at each blood draw (n = 4 pairs at each time point; P value by paired Student’s t test, *P < 0.05). (G) Levels of plasma ALT over time in PiZZ and PiMM control ferrets (n = 2–5 ferrets at each time point; P value by mixed effects model, ***P = 0.00014). In C and E–G, blue = PiMM controls, yellow = PiZZ, and in C, red = AAT-KO. All graphs show mean ± SEM; some error bars are hidden by symbols. *P < 0.05; ***P < 0.001.