A G542X cystic fibrosis mouse model for examining nonsense mutation directed therapies

Abstract

Nonsense mutations are present in 10% of patients with CF, produce a premature termination codon in CFTR mRNA causing early termination of translation, and lead to lack of CFTR function. There are no currently available animal models which contain a nonsense mutation in the endogenous Cftr locus that can be utilized to test nonsense mutation therapies. In this study, we create a CF mouse model carrying the G542X nonsense mutation in Cftr using CRISPR/Cas9 gene editing. The G542X mouse model has reduced Cftr mRNA levels, demonstrates absence of CFTR function, and displays characteristic manifestations of CF mice such as reduced growth and intestinal obstruction. Importantly, CFTR restoration is observed in G542X intestinal organoids treated with G418, an aminoglycoside with translational readthrough capabilities. The G542X mouse model provides an invaluable resource for the identification of potential therapies of CF nonsense mutations as well as the assessment of in vivo effectiveness of these potential therapies targeting nonsense mutations.

Fig 1. Generation of the G542X mutation.

(A) To validate gRNA efficiency in guiding Cas9 nuclease to the desired site, an in vitro assay was performed. PCR amplified DNA containing exon 12 and surrounding region of mouse Cftr (1056 bp) is displayed on an agarose gel with no gRNAs (-) or with 1 of 4 different gRNAs (1–4). Cas9 nuclease activity results in the cleavage of the DNA into fragments of ~750 bp and ~300 bp. (B) Normal Cftr mouse DNA and amino acid sequence around the desired mutation site is shown with the gRNA sequence (in box) and the protospacer adjacent motif sequence recognized by Cas9 (in blue). A portion of the sequence for the G542X oligo is also shown with the substitution change shown in red. The G to T mutation corresponds to the glycine to stop mutation. A silent T to C mutation was also inserted to assist with genotyping and verify HDR had occurred.

Fig 2. Cftr expression in tissues from G542X mice.

Cftr expression in tissues from G542X and WT littermates were evaluated using qRTPCR. Airway (nasal epithelium, lung), intestine (duodenum, jejunum, ileum), and kidney Cftr expression as a percentage of WT Cftr expression is displayed. G542X expression was significantly reduced in all tissues compared to WT. (P<0.05 vs. WT. n≥5 per group).

Fig 3. CFTR function in the airway and intestine of G542X mice.

(A) NPD measurements and (B) change in intestinal short circuit current (ΔI_sc) measurements from the duodenum, jejunum, and ileum from WT and G542X mice are shown. (*P<0.05 vs. WT. n≥4 per group).

Fig 4. Survival and growth characteristics of G542X mice.

(A) Survival of G542X and WT mice up to 40 days of age. (B) Length of G542X and WT mice at 6 weeks of age. (C, D) Weight of G542X and WT mice sex-matched mice up to 40 days of age. Weight at every age was significantly different between G542X and WT littermates. (n≥10 per group. *P<0.05 vs. WT).

Fig 5. Intestinal organoids and tissue from G542X mice are used to test G418-mediated nonsense mutation readthrough.

Intestinal organoids were incubated with indicated doses of G418, or vehicle for 72 hours prior to swelling with 10 μM forskolin. (A) Representative images at T = 0 and T = 300 minutes of organoid swelling, conditions as indicated. (Scale bar = 100 μm) (B) Total change in area was measured over 300 minutes, and FIS was quantified by normalizing the total organoid area to T = 0. (n = 5 wells of organoids for each treatment, ± SE). (C) AUC at T = 300 for indicated treatment groups. (*P<0.05 compared to untreated G542X). (D) Expression of Cftr in WT and G542X organoids with and without G418 treatment as a percentage of untreated WT organoids. (E) Expression of Cftr in lung from untreated G542X mice or treated with G418 compared to expression from WT mice. (*P<0.05 compared to untreated WT. ^P<0.05 compared to untreated G542X.).

Fig 6. Intestinal organoids from G542X mice are used to test PTC124 mediated readthrough.

G524X organoids were treated with the indicated doses of PTC124 for 72 hours prior to the FIS assay. (A) Representative images at T = 0 and T = 300 minutes of organoid swelling, conditions as indicated. (Scale bar = 100 μm). (B) Normalized areas for PTC124 treated organoids are shown, compared to vehicle and 100 μM G418-treated organoids. (C) AUC measurements at 300 minutes. (*P<0.05 compared to untreated G542X.).