Nonsense mutations promote premature translational termination and represent the underlying cause of a large number of human genetic diseases. The aminoglycoside antibiotic gentamicin has the ability to allow the mammalian ribosome to read past a false-stop signal and generate full-length functional proteins. However, severe toxic side effects along with the reduced suppression efficiency at subtoxic doses limit the use of gentamicin for suppression therapy. We describe here the first systematic development of the novel aminoglycoside 2 (NB54) exhibiting superior in vitro readthrough efficiency to that of gentamicin in seven different DNA fragments derived from mutant genes carrying nonsense mutations representing the genetic diseases Usher syndrome, cystic fibrosis, Duchenne muscular dystrophy and Hurler syndrome. Comparative acute lethal toxicity in mice, cell toxicity and the assessment of hair cell toxicity in cochlear explants further indicated that 2 exhibits far lower toxicity than that of gentamicin.
Aminoglycosides are highly potent, broad-spectrum antibiotics that exert their bactericidal therapeutic effect by selectively binding to the decoding aminoacyl site (A-site) of the bacterial 16 S rRNA, thereby interfering with translational fidelity during protein synthesis. The appearance of bacterial strains resistant to these drugs, as well as their relative toxicity, have inspired extensive searches towards the goal of obtaining novel molecular designs with improved antibacterial activity and reduced toxicity. In the last few years, a new, aminoglycoside dependent therapeutic approach for the treatment of certain human genetic diseases has been identified. These treatments rely on the ability of certain aminoglycosides to induce mammalian ribosomes to readthrough premature stop codon mutations. This new and challenging task has introduced fresh research avenues in the field of aminoglycoside research. Recent observations and current challenges in the design of aminoglycosides with improved antibacterial activity and the treatment of human genetic diseases are discussed.
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.
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