Research ArticleCystic Fibrosis

In utero and postnatal VX-770 administration rescues multiorgan disease in a ferret model of cystic fibrosis

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Science Translational Medicine  27 Mar 2019:
Vol. 11, Issue 485, eaau7531
DOI: 10.1126/scitranslmed.aau7531
  • Fig. 1 Administration of VX-770 in utero enhances CFTRG551D-mediated intestinal fluid secretion and protects from development of MI.

    (A) Swelling of intestinal organoids in response to various forskolin doses, for the indicated genotypes, and measured as area under the curve (AUC) in the presence of DMSO or VX-770 (3 μM). Each point represents the AUC for five to seven independent experiments, each from independent donor animals. Data represent the means ± SEM. Significant differences were determined by two-way analysis of variance (ANOVA) and Bonferroni post hoc tests, **P < 0.01 and ***P < 0.001 for comparison of VX-770 to DMSO within a genotype, and #P < 0.1, ##P < 0.01, and ###P < 0.001 for comparison of VX-770 treatment groups across genotypes. There was no significant difference between any time points when comparing VX-770 to DMSO treatment groups for WT/WT and KO/KO groups. ANOVA P values for the significance of the VX-770 effect were WT/WT (P = 0.6471), KO/KO (P = 0.9569), G551D/KO (P < 0.0001), and G551D/G551D (P < 0.0001). (B) Percentage of the WT AUC swelling response for the indicated G551D genotypes and forskolin concentration. The two-way ANOVA P values for the significance of the genotype effect are given below the legend. The average KO/KO AUC over all time points was subtracted from WT and G551D genotypes before making this calculation. (C and D) Isc measurements on (C) freshly excised intestine at the level of the jejunum and (D) polarized PDE grown at an air-liquid interface. Shown are the changes in IscIsc) in response to sequential addition of amiloride, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX), and/or GlyH101 (CFTR inhibitor), as indicated for the given genotypes. Data represent the means ± SEM [n in graphs indicates independent measurements from three to four animals per genotype in (C) and independent cultures derived from three to four donor animals per genotype in (D)]. The order of genotypes in (D) from left to right is WT/WT, KO/KO, G551D/G551D + DMSO, and G551D/G551D + VX-770. Significant differences were determined by two-way ANOVA and Bonferroni post hoc tests; P > 0.05 [ns (not significant)], *P < 0.05, **P < 0.01, and **P < 0.001 for comparison between genotypes in (C) and for comparison to KO/KO in (D). (E) Representative images of intestine from neonatal kits of the indicated genotypes and treatment conditions with VX-770 or vehicle administration to jills at E28. Asterisks mark intestinal dilatation and arrows mark the point of intestinal obstruction. (F) Percentage of newborn kits passing meconium at birth. Starting at E28, pregnant jills were treated with VX-770, vehicle only, or left untreated. Numbers in parentheses represent births for each genotype evaluated. Fisher’s exact test was used to assess differences between the following groups: (i) treatment groups across all genotypes: no treatment, P = 0.3992; vehicle, P = 0.5398; and VX-770, P = 1.14 × 10−6; (ii) vehicle versus VX-770: KO/KO, P = 0.356; G551D/KO, P = 0.003; and G551D/G551D, P = 6.16 × 10−6; and (iii) genotypes with VX-770 treatment: KO/KO versus G551D/KO, P = 9.70 × 10−4; KO/KO versus G551D/G551D, P = 5.10 ×10−7; and G551D/KO versus G551D/G551D, P = 5.84 × 10−4. (G) Survival rates of kits after 1 week of age for the various genotypes. G551D genotypes were born to VX-770–treated jills and maintained on VX-770 after birth, whereas KO/KO genotypes were not treated with VX-770. Numbers in parentheses represent the number of kits in each group starting at 1 week of age. Log-rank (Mantel-Cox) test was used to assess significance of differences in survival between genotypes: KO/KO versus G551D/KO (P = 0.0062), KO/KO versus G551D/G551D (P < 0.0001), and G551D/KO versus G551D/G551D (P = 0.1186).

  • Fig. 2 In utero administration of VX-770 rescues vas deferens and epididymis development only in ferrets homozygous for the G551D allele.

    (A to F) Whole-mount photomicrographs depicting gross morphology of the testis, vas deferens (Vas), and corpus region of the epididymis in newborn kits of the indicated genotypes and in utero treatment conditions (vehicle or VX-770). The middle row shows enlargements of boxed regions in photomicrographs in the top row. Black arrows mark intact vas deferens and corpus of epididymis; red arrows mark regions where the vas deferens and corpus of the epididymis should reside, but are absent. (G to L) Representative hematoxylin and eosin (H&E)–stained sections of the epididymis (corpus) in newborn kits for the above-listed genotypes and treatment conditions. Black arrows mark the intact epididymis; red arrows mark degenerated epithelium or location where the epididymis should reside. The number of animals within the indicated phenotype evaluated for each genotype and condition was as follows: WT/WT, n = 6; KO/KO, n = 4; G551D/KO vehicle, n = 3; G551D/KO VX-770, n = 5; G551D/G551D vehicle, n = 3; and G551D/G551D VX-770, n = 3.

  • Fig. 3 Administration of VX-770 to CFTRG551D ferrets in utero and postnatally enhances growth and partially preserves pancreatic exocrine function.

    In all experiments, treatment with VX-770 commenced at E28 and was sustained through the end of the experiment. (A) Body weight in animals of the indicated genotypes with and without VX-770 treatment. For each genotype, n = 6 to 11 animals. Significant differences in weight gain were observed between CFTRKO/KO versus CFTRG551D/KO and CFTRKO/KO versus CFTRG551D/G551D using two-way ANOVA and Bonferroni post hoc tests, P < 0.001. (B) EL-1 concentrations within the first 100 days of life in CFTRG551D/KO and CFTRG551D/G551D animals maintained on VX-770. The dotted lines correspond to 200 μg of EL-1 per gram of feces, below which is considered to indicate PI. Each unique symbol represents an independent animal (n = 9 CFTRG551D/KO; n = 8 CFTRG551D/G551D). (C) EL-1 concentrations for the indicated genotypes, treatment conditions, and age brackets. One animal in the CFTRG551D/G551D group exhibited PI after 2 months of age. For each genotype and age bracket, n = 6 to 20 animals. Significant differences in fecal EL-1 were observed between CFTRKO/KO versus CFTRG551D/G551D using two-way ANOVA and Bonferroni post hoc tests, **P < 0.01 and ***P < 0.001. In all graphs, data represent the means ± SEM. (D) Pancreas morphology in newborns of the indicated genotype and treatment, as assessed by H&E staining of sections. Arrows mark exocrine acini that are dilated and filled with eosinophilic secretory material. (E) Pancreas morphology in adult animals (>5 months) of the indicated genotype and treatment, as assessed by H&E staining. PI and PS, as judged by fecal EL-1 assay. The animal marked G551D/G551D + VX-770 (PI) is the single pancreatic insufficient CFTRG551D/G551D ferret shown in the bottom panel of (B). The animal marked G551D/G551D + VX (PS)➔−VX (PI) was an adult pancreatic sufficient CFTRG551D/G551D ferret removed from VX-770 for 67 days. Arrows mark islets. Scale bars, 100 μm (D) and 300 μm (E).

  • Fig. 4 Administration of VX-770 to CFTRG551D/G551D ferrets in utero and postnatally improves postprandial glucose tolerance only in animals with PS.

    (A) Blood glucose at baseline in nonfasted, actively nursing kits of the indicated genotypes at 1 to 2 months of age. All animals were treated with VX-770 in utero and maintained postnatally. (B) Blood glucose at 1 hour after gavage feeding an elemental diet for the animals shown in (A). Each data point in (A) and (B) represents the average of four to six measurements per kit on different days. (C) Blood glucose as assessed by MMTT in CFTRG551D/KO ferrets at 3 to 4 months of age. All animals were treated with VX-770 in utero; for the “On” group, treatment was sustained throughout the postnatal period and during testing, but for the “Off” group, treatment was terminated 3 weeks before testing. (D) Quantitation of blood glucose for animals in (C), by AUC analysis. (E) Blood glucose as assessed by MMTT in 3- to 4-month-old ferrets of the indicated genotypes. All animals were treated with VX-770 both in utero and throughout the postnatal period. For the CFTRG551D/G551D group, curves are provided for the entire cohort (n = 7), i.e., including both animals with PS (n = 6) and PI (n = 1), and the single CFTRG551D/G551D ferret that developed PI. (F) Quantitation of blood glucose excursions for animals in (E), by AUC analysis. Significant differences in (A), (B), and (F) were determined by one-way ANOVA and Bonferroni post hoc tests, *P < 0.05, ***P < 0.001, ns, for comparison to CFTRKO/KO, and for (E) two-way ANOVA and Bonferroni post hoc tests, **P < 0.01, ***P < 0.001 for comparison of the complete CFTRG551D/G551D and CFTRWT/WT cohorts to the CFTRKO/KO group. In all graphs, data represent the means ± SEM.

  • Fig. 5 Early postnatal administration of VX-770 to CFTRG551D/KO ferrets is required for marked improvements in growth, survival, and slowing of lung disease progression.

    (A) Growth of CFTRWT/WT and CFTRKO/KO kits during the first 3 weeks of life. (B) Growth of CFTRG551D/KO kits born to VX-770–treated jills starting at E28, with VX-770 treatment terminated at the indicated age in days (d). (C) Survival curves for CFTRG551D/KO kits receiving VX-770 in utero and postnatally for various lengths of time. Log-rank (Mantel-Cox) test was used to assess significance of differences in survival between days 0 and 7 versus day 14 (P = 0.0009 and P = 0.0088, respectively) and between days 0 and 7 versus day 25 (P = 0.0004 and P = 0.0010, respectively). (D) Quantification of bacterial taxa (left y axis) and CFU (right y axis, red dots) in lung homogenates from different CFTRG551D/KO ferrets shown in (C) at the time of euthanasia. Bacterial taxons were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) fingerprinting. Only the most abundant taxons are listed in the legend. (E to K) Representative lung images of a CFTRG551D/KO ferret removed from VX-770 at 14 days and euthanized at 174 days of age due to poor health, showing gross lung pathology (E to G) and H&E-stained sections (H to K). Boxed region in (E) is enlarged in (F) and shows air trapping (white arrow) and atelectasis (yellow arrow). (G) Thick mucus in the trachea. For histology of all five lobes from this CFTRG551D/KO animal, see fig. S4 (A to H). Boxed region in (J) is enlarged in (K) and shows mucus accumulation in submucosal glands (SMGs). (L) Representative SMG image from a wild-type ferret for comparison to (K). (M to Q) H&E-stained sections from another CFTRG551D/KO ferret removed from VX-770 at 25 days and euthanized at 177 days of age. Lung samples from this animal were not insufflated with fixative at the time of tissue collection. Boxed regions in (N) and (P) are enlarged in (O) and (Q), respectively. Histologic images (H to J, M, N, and P) are all different lobes from each animal with mucus accumulation in the airways marked by asterisks. Growth statistics (A and B): two-way ANOVA and Bonferroni post hoc tests were used to determine significant differences between groups when comparing the following dataset. For CFTRWT/WT (n = 11) versus (i) CFTRKO/KO (n = 11, days 11 to 21, P < 0.05), (ii) CFTRG551D/KO 0 days (n = 7, days 11 to 21, P < 0.05), (iii) CFTRG551D/KO 7 days (n = 5, days 15 to 21, P < 0.05), (iv) CFTRG551D/KO 14 days (n = 8, day 21, P < 0.01), and (v) CFTRG551D/KO 25 days (n = 6, all days were not significant). For CFTRKO/KO versus (i) CFTRG551D/KO 0 days (all days were not significant), (ii) CFTRG551D/KO 7 days (all days were not significant), (iii) CFTRG551D/KO 14 days (days 12 to 21, P < 0.05), and (iv) CFTRG551D/KO 25 days (days 10 to 21, P < 0.05).

  • Fig. 6 Withdrawal of VX-770 from adult CFTRG551D/KO ferrets leads to lung disease.

    (A) Timeline for analysis of CFTRG551D/KO ferrets. Bronchoalveolar lavage fluid (BALF) was collected before and after withdrawal of VX-770 to follow bacterial colonization of the lung. (B) Quantitation of bacterial load (measured as total CFU and CFU/ml) in the BALF from CFTRG551D/KO ferrets, before and after VX-770 withdrawal at 267 days (d) of age. The number of total bronchoscopies is indicated in parentheses. Data represent the means ± SEM, using the average CFU counts for each animal at each time frame (n = number of animals). One-way ANOVA using Kruskal-Wallis and Dunn’s post hoc test was used to assess significant differences in CFU counts between baseline (on VX-770) and the two time points off VX-770, **P < 0.01. (C to I) Representative histopathology images of the trachea (C) and lung (D to I) from an adult CFTRG551D/KO ferret in which VX-770 treatment terminated and the animal was euthanized 160 days later because of poor health. Separate lung lobes are provided for each low-power image. (E, H, and I) Enlargement of boxed regions in (D), (G), and (H), respectively. (J) Representative lung histology from a wild-type ferret. Asterisks: basophilic fibrillar material (mucus) and cellular debris.

  • Fig. 7 Withdrawal of VX-770 from adult CFTRG551D/G551Dferrets leads to lung disease.

    CFTRG551D/G551D ferrets were born to E28 VX-770–treated jills and maintained on VX-770 until adulthood, at which time pancreatic function and glucose tolerance testing was performed. VX-770 was then tempered off over a month. (A to F) Lung histopathology from a CFTRG551D/G551D ferret (animal 3) euthanized 187 days after VX-770 cessation. All low-power images represent different lobes. The pathology in this animal was characterized by distal airway disease (A, B, and D to F) and pneumonia in some but not all lobes (C). (B and F) Enlargement of boxed regions in (A) and (E), respectively. Insets in (B) and (F) are taken at the region of the black arrows and demonstrate neutrophil and accumulation in the distal airways. (G to P) Lung histopathology from an adult CFTRG551D/G551D ferret (animal 2) euthanized 67 days after VX-770 cessation. (G) Breath-hold computed tomography image on the day before sacrifice demonstrating an occluded lobe of the lung (asterisk). (H) Gross lung pathology showing the atelectatic lobe (asterisk) and abscesses in the distal lung [boxed region with enlargement shown in (I)]. (J) Bronchoscopy at 47 days after VX-770 cessation demonstrating thick viscous mucus with the indicated CFU titer of bacteria found in BALF. (K to P) Lung histopathology from animal 2 with (K) being the atelectatic lobe. (L and M) Enlargement of boxed regions in (K). All low-power images represent different lobes. Airway mucus. Yellow arrows mark bronchial-associated lymphoid tissue expansion.

  • Fig. 8 Withdrawal of VX-770 from adult CFTRG551D/KO ferrets leads to enhanced lung inflammation.

    BALF was collected from three CFTRG551D/KO ferrets before and 1 month after withdrawal of VX-770. The BALF proteome was then examined in a paired fashion using quantitative proteomics. (A) Volcano plot of 239 BALF proteins detected with the log2 (fold change) representing (OFF VX-770)/(ON VX-770) [black circles: not statistically significant or log2 (fold change) was <1.5 or >−1.5; red circles: statistically significant P < 0.05 with a log2 (fold change) ≥1.5 or ≤−1.5; n = 3 paired samples]. Proteins (gene names) found in neutrophil granules are marked in blue, whereas proteins secreted by airway epithelium and submucosal glands are marked in black. (B) Volcano plot of 63 BALF proteins enriched (P = 3.2 × 10−35) in the ReactomePA neutrophil degranulation pathway. (C) List of selected significant disease pathways discovered using MeSH analysis. (D and E) List of selected significant (D) canonical pathways and (E) diseases or functions pathways discovered using IPA. The number of proteins found in each pathway is given in parentheses, and pathways with near-significant (>1.9) or significant (>2) positive z scores are highlighted in red.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/485/eaau7531/DC1

    Materials and Methods

    Fig. S1. Generation of G551D-CFTR ferrets using AAV-directed mutagenesis and somatic cell nuclear transfer.

    Fig. S2. Forskolin-induced swelling of intestinal organoids and Isc of intestinal epithelia and PDE.

    Fig. S3. Expression of CFTR mRNA in the fetal intestine and pancreas.

    Fig. S4. Lung histology of all lobes from a CFTRG551D/KO ferret removed from VX-770 and a CFTRG551D/G551D ferret maintained on VX-770.

    Fig. S5. Withdrawal of VX-770 from adult CFTRG551D/G551D ferrets leads to PI and variable degrees of glucose intolerance.

    Fig. S6. Lung histology of CFTRG551D/KO and CFTRG551D/G551D ferrets maintained on VX-770 for life.

    Table S1. Primary data (provided as a separate Excel file).

    Data file S1. Proteomics data (provided as a separate Excel file).

    Data file S2. RNA tissue quantification and smFISH probe maps.

    References (3035)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Generation of G551D-CFTR ferrets using AAV-directed mutagenesis and somatic cell nuclear transfer.
    • Fig. S2. Forskolin-induced swelling of intestinal organoids and Isc of intestinal epithelia and PDE.
    • Fig. S3. Expression of CFTR mRNA in the fetal intestine and pancreas.
    • Fig. S4. Lung histology of all lobes from a CFTRG551D/KO ferret removed from VX-770 and a CFTRG551D/G551D ferret maintained on VX-770.
    • Fig. S5. Withdrawal of VX-770 from adult CFTRG551D/G551D ferrets leads to PI and variable degrees of glucose intolerance.
    • Fig. S6. Lung histology of CFTRG551D/KO and CFTRG551D/G551D ferrets maintained on VX-770 for life.
    • Legend for table S1
    • Legends for data files S1 and S2
    • References (3035)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Primary data (provided as a separate Excel file).
    • Data file S1. Proteomics data (provided as a separate Excel file).
    • Data file S2 (.docx format). RNA tissue quantification and smFISH probe maps.

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