Abstract
Generation of Alpha-1 Antitrypsin Knockout and PIZZ Ferrets Using Crispr/Cas9. A Genetic Model of Emphysema
Annals of the American Thoracic Society, Vol.15(Suppl 4), pp.S292-S293
12/2018
DOI: 10.1513/AnnalsATS.201806-429MG
PMCID: PMC6321996
PMID: 30758999
Abstract
Rationale:
The most prevalent genetic cause of chronic obstructive pulmonary disease is alpha-1 antitrypsin (A1AT) deficiency, a disorder that has yet to be widely modeled in animals because of species-specific differences between rodents and humans.
Objectives:
To address these challenges, we engineered two A1AT ferret models using zygote gene editing to test the hypothesis that unopposed protease activity within the lung leads to emphysema and bronchitis.
Methods:
Guide RNAs targeting exon 2 (for knockout) and exon 5 (for Z-allele mutation, Pi*Z) of the ferret A1AT gene were injected into ferret zygotes with
Cas9
mRNA. For PI*Z targeting, a short oligonucleotide carrying the mutation was included. Offspring were genotyped and plasma levels of A1AT determined by Western blot before entry into a longitudinal study. Adult A1AT ferrets underwent bronchoscopy and FlexiVent over time to characterize lung disease. Control animals were wild-type (WT) and age, sex, and size matched.
Results:
A1AT-deficient ferrets spontaneously develop hallmarks of emphysema as early as 3 months when compared with matched WT control ferrets. Over time, A1AT-KO ferrets reached an inspiratory capacity of 117% ± 5% (WT, 57.4 ml; A1AT, 67.5 ml) and compliance of 111% ± 4% (WT, 5.5 ml/cm H
2
O; A1AT, 6.1 ml/cm H
2
O;
N
= 8 pairs A1AT/WT,
P
< 0.05). Progression of these disease parameters and pressure–volume loop disturbances increased with age (oldest are ∼2 yr). The bronchoalveolar lavage (BAL) proteome of KO animals confirmed absence of A1AT protein along with elevated soluble Muc5AC (Log
2
fold change, 6.74;
P
< 0.001). Active neutrophil elastase was elevated in A1AT-KO versus WT BAL (292 ± 82 vs. 179 ± 17 ng/ml) but did not reach significance; in one experiment, active NE was sixfold higher in the upper lobe of the A1AT than the lower (1,240 vs. 209 ng/ml). Quantitative alveolar morphometry in one A1AT was 121% of its matched WT control. The PI*ZZ animals are entering studies at several months of age.
Conclusions:
Our findings suggest that A1AT-deficient ferrets spontaneously develop anatomic and physiologic aspects of progressive lung disease consistent with emphysema and chronic bronchitis.
Details
- Title: Subtitle
- Generation of Alpha-1 Antitrypsin Knockout and PIZZ Ferrets Using Crispr/Cas9. A Genetic Model of Emphysema
- Creators
- Nan He - Department of Anatomy and Cell Biology andBradley H Rosen - Department of Anatomy and Cell Biology andJaimie S Gray - Department of Anatomy and Cell Biology andIdil A Evans - Department of Anatomy and Cell Biology andMarina Zieger - Department of Pediatrics, University of Massachusetts Medical Center, Worcester, MassachusettsZiying Yan - Department of Anatomy and Cell Biology andFlorie Borel - Department of Pediatrics, University of Massachusetts Medical Center, Worcester, MassachusettsBo Liang - Department of Anatomy and Cell Biology andXingshen Sun - Department of Anatomy and Cell Biology andShashanna R Moll - Department of Anatomy and Cell Biology andMichael H Brodsky - Department of Pediatrics, University of Massachusetts Medical Center, Worcester, MassachusettsChristian Mueller - Department of Pediatrics, University of Massachusetts Medical Center, Worcester, MassachusettsJohn F Engelhardt - Department of Anatomy and Cell Biology and
- Resource Type
- Abstract
- Publication Details
- Annals of the American Thoracic Society, Vol.15(Suppl 4), pp.S292-S293
- DOI
- 10.1513/AnnalsATS.201806-429MG
- PMID
- 30758999
- PMCID
- PMC6321996
- NLM abbreviation
- Ann Am Thorac Soc
- ISSN
- 2329-6933
- eISSN
- 2325-6621
- Publisher
- American Thoracic Society
- Language
- English
- Date published
- 12/2018
- Academic Unit
- Roy J. Carver Department of Biomedical Engineering; Pulmonary, Critical Care, and Occupational Medicine; Anatomy and Cell Biology; Radiation Oncology; Internal Medicine
- Record Identifier
- 9984025303602771
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