Readthrough of nonsense mutations in Rett syndrome: evaluation of novel aminoglycosides and generation of a new mouse model

Abstract

Thirty-five percent of patients with Rett syndrome carry nonsense mutations in the MECP2 gene. We have recently shown in transfected HeLa cells that readthrough of nonsense mutations in the MECP2 gene can be achieved by treatment with gentamicin and geneticin. This study was performed to test if readthrough can also be achieved in cells endogenously expressing mutant MeCP2 and to evaluate potentially more effective readthrough compounds. A mouse model was generated carrying the R168X mutation in the MECP2 gene. Transfected HeLa cells expressing mutated MeCP2 fusion proteins and mouse ear fibroblasts isolated from the new mouse model were treated with gentamicin and the novel aminoglycosides NB30, NB54, and NB84. The localization of the readthrough product was tested by immunofluorescence. Readthrough of the R168X mutation in mouse ear fibroblasts using gentamicin was detected but at lower level than in HeLa cells. As expected, the readthrough product, full-length Mecp2 protein, was located in the nucleus. NB54 and NB84 induced readthrough more effectively than gentamicin, while NB30 was less effective. Readthrough of nonsense mutations can be achieved not only in transfected HeLa cells but also in fibroblasts of the newly generated Mecp2R168X mouse model. NB54 and NB84 were more effective than gentamicin and are therefore promising candidates for readthrough therapy in Rett syndrome patients.

Clinical and biological progress over 50 years in Rett syndrome

“…However, it may be possible to develop small molecules to act at the genomic level to reactivate the MECP2 allele on the inactive X chromosome 194 or at the level of RNA to enable read-through of nonsense mutations. 195,196 In contrast to targeting MECP2, pharmacological strategies targeting mechanisms downstream in the pathogenic process can make use of small molecules already developed or approved for other indications. Indeed, several drugs with proven efficacy in Mecp2 knockout mice have proceeded into clinical trials in patients with RTT (see Fig.…”

Designer Aminoglycosides That Selectively Inhibit Cytoplasmic Rather than Mitochondrial Ribosomes Show Decreased Ototoxicity

“…In this context, we have recently demonstrated the ability of some of our recent lead compounds of the NB series, including NB74 and NB84, to partially restore protein function in various clinically relevant cellular and animal models of genetic diseases, including cystic fibrosis (46), Rett syndrome (47,48), Usher syndrome (49 -51), and Hurler syndrome (52,53). These observations, together with the relatively low toxicity of NB74 and NB84, encourage the further testing of these novel designer AGs in animal models and human subjects to maximize the translational impact of our work.…”

Rett Syndrome: Reaching for Clinical Trials

“…Among different gene vectors, the serotype 9 of adeno-associated virus is the most promising owing to its ability to cross the blood-brain barrier (BBB) and to infect neurons efficiently, yielding long-term transgene expression in male Mecp2 knockout mice and in female Mecp2 heterozygous mice [67,68]. It should be noted that no deleterious consequences of the overexpression of Mecp2 in wild-type cells were noted in the mosaic female brain [69]. At present, there are no clinical trials of MECP2 gene therapy in individuals with RTT.…”

“…Such aminoglycosides and novel nonaminoglycoside Bread-through^compounds have been tested for therapeutic efficacy in Duchenne muscular dystrophy and cystic fibrosis [72]. Gentamycin or geneticin allowed translation of full-length MeCP2 protein in a lymphocyte cell line derived from an individual with a R255X nonsense mutation [73], and in fibroblasts from knock-in mice expressing an R168X nonsense mutation [69]. More recently, embryonic fibroblasts from a mouse model with the R255X nonsense mutation were treated with gentamycin and were able to express full-length MeCP2 [74].…”