Ferret and pig models of cystic fibrosis: prospects and promise for gene therapy

Abstract

Large animal models of genetic diseases are rapidly becoming integral to biomedical research as technologies to manipulate the mammalian genome improve. The creation of cystic fibrosis (CF) ferrets and pigs is an example of such progress in animal modeling, with the disease phenotypes in the ferret and pig models more reflective of human CF disease than mouse models. The ferret and pig CF models also provide unique opportunities to develop and assess the effectiveness of gene and cell therapies to treat affected organs. In this review, we examine the organ disease phenotypes in these new CF models and the opportunities to test gene therapies at various stages of disease progression in affected organs. We then discuss the progress in developing recombinant replication-defective adenoviral, adeno-associated viral, and lentiviral vectors to target genes to the lung and pancreas in ferrets and pigs, the two most affected organs in CF. Through this review, we hope to convey the potential of these new animal models for developing CF gene and cell therapies.

FIG. 1.

Weight monitoring as a surrogate for lung infections in young cystic fibrosis (CF) ferrets. The weights of kits were measured every 6 hr and were compared between paired CF and non-CF kits born to the same jill. (A) Typical patterns of total daily weight gain for CF kits (blue bars) and non-CF kits (red bars). (B) The rolling average 6-hr delta weight gain over a 24-hr period (calculated as the average of five measurements over a 24-hr period) is plotted for a CF (blue bars)/non-CF (red bars) pair. A decline in this rolling average indicated early lung infection (yellow shaded region), and thus a second antibiotic (Baytril) was applied (at arrowhead). (C) A second set of CF and non-CF animals reared on Zosyn from birth, given Baytril on day 12 because of weight loss in the CF animal, and then removed from Baytril on day 15. The CF animal succumbed to lung infection at 19 days. (D and E) Lung histology and bacterial colony-forming units (CFU) in lung lysates from the 19-day-old (D) non-CF control and (E) CF animal shown in (C). (A) and (B) are reproduced with permission from Sun et al.

FIG. 2.

Gene transfer in the airways of 3- to 6-day-old ferrets. (A–E) Airways after intratracheal delivery of (A) vehicle or (B–E) helper-dependent Ad5 (i.e., gutted adenovirus) with X-Gal staining 8 days postinfection. This vector expresses nt-LacZ under the control of the K18 promoter. (A–D) are sections and (E) is a whole-mount preparation. The transgene is expressed predominantly in the small airways. (F–J) Airways after intratracheal delivery of (F) vehicle or (G–J) equine infectious anemia virus (EIAV) pseudotyped with influenza A virus subtype H7 hemagglutinin (HA) with X-Gal staining 8 days postinfection. This EIAV vector expresses nt-LacZ under the control of the CBA promoter. (F–H) are sections and (I and J) are whole-mount preparations. The transgene is expressed in the large and small airways as well as in alveolar regions. (K–N) Newborn ferrets were infected with an RSVmCherry-encoding FIV-GP64 virus. mCherry expression (red) was observed almost exclusively in bronchioles (Br) at 7 days postinfection. (L) shows only the mCherry channel of the boxed region in (K). (M) is the bright-field image of the fluorescence panel in (N). (K) and (N) are counterstained with DAPI to mark nuclei.

FIG. 3.

rAAV transduction of the pancreas in 3-day-old ferrets. Three-day-old ferrets were injected intraperitoneally with 200 μl of PBS (vehicle) or the indicated serotype rAAV vector encoding EGFP (2×10¹¹ particles/20 g body weight in 200 μl of PBS). Ferrets were sacrificed 2 weeks after infection. (A) Tissue homogenates of the various organs were used for Western blot detection of EGFP. The lane marked “EGFP” contained the same amount of EGFP protein (marked by arrows) on each blot. (B) Pancreatic tissue sections from animals injected with vehicle, rAAV2/5-EGFP, or rAAV2/8-EGFP were immunofluorescently stained for insulin (red) and EGFP (green) followed by mounting in the presence of DAPI (blue) to mark nuclei.