Beneficial Read-Through of aUSH1CNonsense Mutation by Designed Aminoglycoside NB30 in the Retina

6533b7dafe1ef96bd126f5b7

RESEARCH PRODUCT

Beneficial Read-Through of aUSH1CNonsense Mutation by Designed Aminoglycoside NB30 in the Retina

Uwe Wolfrum Tamar Ben-yosef Kerstin Nagel-wolfrum Valery Belakhov Annie Rebibo-sabbah Timor Baasov Nora Overlack Igor Nudelman Tobias Goldmann

subject

Paromomycin Usher syndrome Blotting Western Nonsense mutation Cell Culture Techniques Gene Expression Cell Cycle Proteins Paromomycin Biology Pharmacology Transfection Retina Mice 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Retinitis pigmentosa In Situ Nick-End Labeling otorhinolaryngologic diseases medicine Animals Humans Adaptor Proteins Signal Transducing 030304 developmental biology Genetics 0303 health sciences Retina Dose-Response Relationship Drug Aminoglycoside Retinal medicine.disease 3. Good health Mice Inbred C57BL Cytoskeletal Proteins Aminoglycosides Electroporation HEK293 Cells medicine.anatomical_structure Microscopy Fluorescence chemistry Codon Nonsense Protein Biosynthesis Gentamicin Gentamicins 030217 neurology & neurosurgery medicine.drug

description

PURPOSE. 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. (Invest Ophthalmol Vis Sci. 2010;51:6671‐6680) DOI:10.1167/iovs.10-5741

year	journal	country	edition	language
2010-12-01	Investigative Opthalmology & Visual Science

https://doi.org/10.1167/iovs.10-5741