Characterization of two rat models of cystic fibrosis—KO and F508del CFTR—Generated by Crispr‐Cas9

Abstract

BackgroundGenetically engineered animals are essential for gaining a proper understanding of the disease mechanisms of cystic fibrosis (CF). The rat is a relevant laboratory model for CF because of its zootechnical capacity, size, and airway characteristics, including the presence of submucosal glands.MethodsWe describe the generation of a CF rat model (F508del) homozygous for the p.Phe508del mutation in the transmembrane conductance regulator (Cftr) gene. This model was compared to new Cftr−/− rats (CFTR KO). Target organs in CF were examined by histological staining of tissue sections and tooth enamel was quantified by micro‐computed tomography. The activity of CFTR was evaluated by nasal potential difference (NPD) and short‐circuit current measurements. The effect of VX‐809 and VX‐770 was analyzed on nasal epithelial primary cell cultures from F508del rats.ResultsBoth newborn F508del and Knock out (KO) animals developed intestinal obstruction that could be partly compensated by special diet combined with an osmotic laxative. The two rat models exhibited CF phenotypic anomalies such as vas deferens agenesis and tooth enamel defects. Histology of the intestine, pancreas, liver, and lungs was normal. Absence of CFTR function in KO rats was confirmed ex vivo by short‐circuit current measurements on colon mucosae and in vivo by NPD, whereas residual CFTR activity was observed in F508del rats. Exposure of F508del CFTR nasal primary cultures to a combination of VX‐809 and VX‐770 improved CFTR‐mediated Cl− transport.ConclusionsThe F508del rats reproduce the phenotypes observed in CFTR KO animals and represent a novel resource to advance the development of CF therapeutics.

Gene Therapy for Cystic Fibrosis: Progress and Challenges of Genome Editing

“…Recombination with the donor DNA, containing the three nucleotides deletion, produced a homozygous rat model for the ∆F508. Compared with knockout rats, they showed a residual CFTR activity and a consequently milder CF phenotype [97].…”

Advances in Genome Editing and Application to the Generation of Genetically Modified Rat Models

“…ssODNs containing a mutation observed in a human disease have been used to generate animal models (Figure 4B) such as for cystic fibrosis (Dreano et al, 2019; Table 7). The use of ssODNs will allow inclusion of specific features, such as restriction sites, to facilitate KI genotyping.…”

Treating Cystic Fibrosis with mRNA and CRISPR

“…However, the rats do not recapitulate damage in other organs at birth. 101 Second, mice harboring the G542X mutation, a nonsense mutation belonging to Class I, were also created with CRISPR Cas9. Given the lack of treatment options for patients harboring this mutation, this animal model proves timely to evaluate therapies such as readthrough molecules.…”

“…102 As described, there have been significant advances made in the development of CF animal models, which have been reviewed in detail. [93][94][95][96][97][98][99][100][101][102] There have been advances in the last decade that address the first three steps in the drug delivery process of RNA therapies for CF. First, there have been reports that densely coating nanoparticles with polyethylene glycol (PEG) seems to improve transport past different mucosal surfaces in humans.…”