Add like
Add dislike
Add to saved papers

Generation of Alpha-1 Antitrypsin Knockout and PI*ZZ Ferrets Using Crispr/Cas9. A Genetic Model of Emphysema.

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 H2 O; A1AT, 6.1 ml/cm H2 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 (Log2 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.

Full text links

We have located links that may give you full text access.
Can't access the paper?
Try logging in through your university/institutional subscription. For a smoother one-click institutional access experience, please use our mobile app.

For the best experience, use the Read mobile app

Mobile app image

Get seemless 1-tap access through your institution/university

For the best experience, use the Read mobile app

All material on this website is protected by copyright, Copyright © 1994-2024 by WebMD LLC.
This website also contains material copyrighted by 3rd parties.

By using this service, you agree to our terms of use and privacy policy.

Your Privacy Choices Toggle icon

You can now claim free CME credits for this literature searchClaim now

Get seemless 1-tap access through your institution/university

For the best experience, use the Read mobile app