Optimized approach for the identification of highly efficient correctors of nonsense mutations in human diseases

Abstract

About 10% of patients with a genetic disease carry a nonsense mutation causing their pathology. A strategy for correcting nonsense mutations is premature termination codon (PTC) readthrough, i.e. incorporation of an amino acid at the PTC position during translation. PTC-readthrough-activating molecules appear as promising therapeutic tools for these patients. Unfortunately, the molecules shown to induce PTC readthrough show low efficacy, probably because the mRNAs carrying a nonsense mutation are scarce, as they are also substrates of the quality control mechanism called nonsense-mediated mRNA decay (NMD). The screening systems previously developed to identify readthrough-promoting molecules used cDNA constructs encoding mRNAs immune to NMD. As the molecules identified were not selected for the ability to correct nonsense mutations on NMD-prone PTC-mRNAs, they could be unsuitable for the context of nonsense-mutation-linked human pathologies. Here, a screening system based on an NMD-prone mRNA is described. It should be suitable for identifying molecules capable of efficiently rescuing the expression of human genes harboring a nonsense mutation. This system should favor the discovery of candidate drugs for treating genetic diseases caused by nonsense mutations. One hit selected with this screening system is presented and validated on cells from three cystic fibrosis patients.

2,6-Diaminopurine as a highly potent corrector of UGA nonsense mutations

“…DAP contributes to the PTC-correcting activity of H7 extract. An extract (H7) prepared from the mushroom Lepista inversa (also currently called Paralepista flaccida) has recently been shown to exhibit high UAA and UGA readthrough-promoting activity 33 . To identify the compounds responsible for this activity, a semipreparative HPLC fractionation protocol was used.…”

“…Nine fractions (F7-F15) were obtained ( Fig. 1a) and tested for PTC-readthrough activity in a system using a PTC-carrying firefly luciferase mRNA subject to NMD 33 (Fig. 1b).…”

“…In a recent report, we described a specific screening system for identifying compounds that efficiently correct nonsense mutations in human cells. It uses a firefly reporter gene carrying a nonsense mutation and an intron located more than 55 nucleotides downstream, making the corresponding mRNA subject to NMD 33 . This system, unlike other, previously described screening systems using PTC-containing reporter mRNAs immune to NMD, reproduces the fate of PTC-containing mRNAs in patient cells 21,[33][34][35] .…”

“…It uses a firefly reporter gene carrying a nonsense mutation and an intron located more than 55 nucleotides downstream, making the corresponding mRNA subject to NMD 33 . This system, unlike other, previously described screening systems using PTC-containing reporter mRNAs immune to NMD, reproduces the fate of PTC-containing mRNAs in patient cells 21,[33][34][35] . We have used it to show that an extract of the common mushroom Lepista inversa (H7) acts as a very efficient corrector of UGA and UAA nonsense mutations in human cells 33 .…”

Succinic Semialdehyde Dehydrogenase Deficiency: An Update

“…In cell culture, amlexanox was able to rescue the expression of functional proteins from mRNAs containing nonsense mutations for p53, dystrophin, aspartylglucosaminidase, and type VII collagen [127,157,159]. Further potentially efficacious read-through agents include the nucleoside analog clitocine and components of fungi extracts that were identified in a recent drug screen [160,161]. Since the efficacy of these drugs is also dependent on their capability to cross the BBB, further research will be required to test if these drugs might also provide a therapy option for SSADH-D. Interestingly, the findings of Akaboshi and colleagues suggested that nonsense variants, especially Trp204X and Arg412X substitutions, are frequently present in SSADH-D [31].…”