title: Whirlin replacement restores the formation of the USH2 protein complex in whirlin knockout photoreceptors.
abstract: OBJECTIVE
  Whirlin is the causative gene for Usher syndrome type IID (USH2D), a condition manifested as both retinitis pigmentosa and congenital deafness. Mutations in this gene cause disruption of the USH2 protein complex composed of USH2A and VLGR1 at the periciliary membrane complex (PMC) in photoreceptors. In this study, the adeno-associated virus (AAV)-mediated whirlin replacement was evaluated as a treatment option.
  
  
  METHODS
  Murine whirlin cDNA driven by the human rhodopsin kinase promoter (hRK) was packaged as an AAV2/5 vector and delivered into the whirlin knockout retina through subretinal injection. The efficiency, efficacy, and safety of this treatment were examined using immunofluorescent staining, confocal imaging, immunoelectron microscopy, Western blot analysis, histologic analysis, and electroretinogram.
  
  
  RESULTS
  The AAV-mediated whirlin expression started at two weeks, reached its maximum level at 10 weeks, and lasted up to six months post injection. The transgenic whirlin product had a molecular size and an expression level comparable to the wild-type. It was distributed at the PMC in both rod and cone photoreceptors from the central to peripheral retina. Importantly, the transgenic whirlin restored the cellular localization and expression level of both USH2A and VLGR1 and did not cause defects in the retinal histology and function in the whirlin knockout mouse.
  
  
  CONCLUSIONS
  Whirlin transgene recruits USH2A and VLGR1 to the PMC and is sufficient for the formation of the USH2 protein complex in photoreceptors. The combined hRK and AAV gene delivery system could be an effective gene therapy approach to treat retinal degeneration in USH2D patients.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Moran Eye Center, University of Utah, Salt Lake City, Utah 84132, USA.
authors.forename: Junhuang
authors.initials: J
authors.lastname: Zou
authors.forename: Ling
authors.initials: L
authors.lastname: Luo
authors.forename: Zuolian
authors.initials: Z
authors.lastname: Shen
authors.forename: Vince A
authors.initials: VA
authors.lastname: Chiodo
authors.forename: Balamurali K
authors.initials: BK
authors.lastname: Ambati
authors.forename: William W
authors.initials: WW
authors.lastname: Hauswirth
authors.forename: Jun
authors.initials: J
authors.lastname: Yang
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D000229:Dependovirus / Q000235:genetics; D004596:Electroretinography; D016326:Extracellular Matrix Proteins / Q000235:genetics* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000235:genetics; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000502:physiology*; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D019076:Transgenes; D052245:Usher Syndromes / Q000235:genetics
chemical_list: D016326:Extracellular Matrix Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D008565:Membrane Proteins; D043562:Receptors, G-Protein-Coupled; C485949:Adgrv1 protein, mouse; C491810:Ush2a protein, mouse; C477218:Whrn protein, mouse