title: Beneficial read-through of a USH1C nonsense mutation by designed aminoglycoside NB30 in the retina.
abstract: OBJECTIVE
  The human Usher syndrome (USH) is the most frequent cause of inherited combined deaf-blindness. USH is clinically and genetically heterogeneous, assigned to three clinical types. The most severe type is USH1, characterized by profound inner ear defects and retinitis pigmentosa. Thus far, no effective treatment for the ophthalmic component of USH exists. The p.R31X nonsense mutation in USH1C leads to a disease causing premature termination of gene translation. Here, we investigated the capability of the novel synthetic aminoglycoside NB30 for the translational read-through of the USH1C-p.R31X nonsense mutation as a retinal therapy option.
  
  
  METHODS
  Read-through of p.R31X by three commercial, clinically applied aminoglycosides and the synthetic derivative NB30 was validated in vitro, in cell culture, and in retinal explants. Restoration of harmonin functions was monitored in GST pull-downs (scaffold function) and by F-actin bundling analysis in HEK293T cells. Biocompatibility of aminoglycosides was determined in retinal explants by TUNEL assays.
  
  
  RESULTS
  In vitro translation and analyses of transfected HEK293T cells revealed a dose-dependent read-through by all aminoglycosides. In addition, gentamicin, paromomycin, and NB30 induced read-through of p.R31X in mouse retinal explants. The read-through of p.R31X restored harmonin protein function. In contrast to all commercial aminoglycosides NB30 showed good biocompatibility.
  
  
  CONCLUSIONS
  Commercial aminoglycosides and NB30 induced significant read-through of the USH1C-p.R31X nonsense mutation. However, the observed read-through efficiency, along with its significantly reduced toxicity and good biocompatibility, indicate that the novel derivate NB30 represents a better choice than commercial aminoglycosides in a read-through therapy of USH1C and other ocular diseases.
authors.affiliation: Institute of Zoology, Department of Cell and Matrix Biology, Johannes Gutenberg-University of Mainz, Mainz, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Goldmann
authors.forename: Annie
authors.initials: A
authors.lastname: Rebibo-Sabbah
authors.forename: Nora
authors.initials: N
authors.lastname: Overlack
authors.forename: Igor
authors.initials: I
authors.lastname: Nudelman
authors.forename: Valery
authors.initials: V
authors.lastname: Belakhov
authors.forename: Timor
authors.initials: T
authors.lastname: Baasov
authors.forename: Tamar
authors.initials: T
authors.lastname: Ben-Yosef
authors.forename: Uwe
authors.initials: U
authors.lastname: Wolfrum
authors.forename: Kerstin
authors.initials: K
authors.lastname: Nagel-Wolfrum
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D000617:Aminoglycosides / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D018929:Cell Culture Techniques; D018797:Cell Cycle Proteins; D018389:Codon, Nonsense / Q000187:drug effects*; D003598:Cytoskeletal Proteins; D004305:Dose-Response Relationship, Drug; D018274:Electroporation; D015870:Gene Expression / Q000502:physiology; D005839:Gentamicins / Q000494:pharmacology; D057809:HEK293 Cells / Q000378:metabolism; D006801:Humans; D020287:In Situ Nick-End Labeling; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D010303:Paromomycin / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D012160:Retina / Q000187:drug effects* / Q000378:metabolism; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000617:Aminoglycosides; D018797:Cell Cycle Proteins; D018389:Codon, Nonsense; D003598:Cytoskeletal Proteins; D005839:Gentamicins; C118970:USH1C protein, human; D010303:Paromomycin