Promoting readthrough of nonsense mutations in CF mouse model: Biodistribution and efficacy of NV848 in rescuing CFTR protein expression

Abstract

Nonsense mutations, often resulting from single-nucleotide substitutions, produce mRNA harboring a premature termination codon (PTC), which causes the premature termination of protein synthesis. This produces truncated and non-functional proteins, which cause different genetic diseases, including cystic fibrosis (CF). This work aims to investigate the ability of NV848 (N-(5-methyl-1,2,4-oxadiazol-3-yl)acetamide), a translational readthrough-inducing drug (TRID), to rescue CF transmembrane conductance regulator (CFTR) protein expression in a murine model characterized by the G542X nonsense mutation in the CFTR gene. In vitro experiments assessed the drug's stability in human hepatic metabolism, and in vivo investigations on wild-type mice allowed us to clarify the distribution of the drug to the target organs. Moreover, its efficacy in recovering the CFTR protein after chronic treatment was assessed in G542X homozygous mice. Our results provide valuable insights into the biodistribution and therapeutic attributes of NV848, representing a promising therapeutic tool for enhanced clinical outcomes in individuals affected by CF with nonsense mutations.

Keywords: Mendelian diseases; cystic fibrosis; nonsense; oxadiazole; stop codons.

Graphical abstract

Figure 1

Percentage of tested compounds after 0, 5, 30, and 60 min incubated with human liver microsomes Samples concerning enzymatic activity (CYP or CYP/UGT) and phosphate buffers (DPBS and TRIS) have been distinguished. Data are expressed as mean values ± SEM. ∗p < 0.05 and ∗∗p < 0.01 (one-way ANOVA test) compared to the percentage of remaining analyte evaluated at time 0 (black bars) for every sample condition.

Figure 2

Plasma concentration of NV848 at established time points Data are expressed as mean values ± SEM. N = 5. ∗p < 0.05 (one-way ANOVA test) compared to NV848 levels at 45 min.

Figure 3

Concentration of NV848 in all organs for each interval (N = 5) ∗∗∗∗p < 0.0001 (two-way ANOVA test) compared to NV848 levels at 45 min.

Figure 4

CFTR^G542X/G542X mice express CFTR protein after 15 days of treatment with NV848 molecule. (A) Punnet’s mutant CFTR mouse colony production scheme shows animal matings to obtain G542X mice. (B) Example of genotyping and expected results: for each mouse in the gel, the first lane shows DNA amplified with primers recognizing the WT allele and the second lane with primers recognizing the G542X allele. (C) Real-time RT-PCR evaluated lung CFTR mRNA level expression in mutant mice treated for 15 days with H₂O and NV848; data are means ± SEM (in triplicate for each mouse), p < 0.05 (two-way ANOVA test) compared to the untreated (untr) mutant mouse. (D) CFTR protein expression was evaluated by western blot assay of lung protein extracted from mutant mice treated for 15 days with H₂O and NV848 and a histogram showing CFTR protein quantification normalized by tubulin and negative control; data are means ± SEM (in triplicate for each mouse), p < 0.05 (two-way ANOVA test) compared to the untr mutant mouse.

Figure 5

CFTR^G542X/G542X mice express CFTR protein( in lung sections after 15 days of treatment with NV848 molecule. (A) H&E staining and TTF1 expression in CFTR^WT and untreated (H₂O) CFTR^G542X/G542× lung sections (magnifications: 10×, 20×, 40×). (B) H&E staining and TTF1 expression in NV848-treated CFTR^G542X/G542× mouse lung sections (magnifications: 10×, 20×, 40×).

Figure 6

Expression of CFTR protein in different lung regions of CFTR^G542X/G542X mice. (A) Different regions of the CFTR staining (brown) in the CFTR^WT lung sections and (B) CFTR^G542X/G542X untreated (H₂O) control. (C) NV848-treated CFTR^G542X/G542X mouse lung sections show CFTR expression of the apical surface of the lung ducts (brown). All the images are shown with a 40× magnification.