title: Recombinant adeno-associated virus type 2-mediated gene delivery into theRpe65-/-knockout mouse eye results in limited rescue.
abstract: BACKGROUND
  Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10-15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of theRpe65-/-knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.
  
  
  METHODS
  rAAV.RPE65 was injected into the subretinal space ofRpe65-/-knockout mice and control mice. Histological and immunohistological analyses were performed to evaluate any rescue of photoreceptors and to determine longevity and pattern of transgene expression. Electron microscopy was used to examine ultrastructural changes, and electroretinography was used to measure changes in visual function following rAAV.RPE65 injection.
  
  
  RESULTS
  rAAV-mediated RPE65 expression was detected for up to 18 months post injection. The delivery of rAAV.RPE65 toRpe65-/-mouse retinas resulted in a transient improvement in the maximum b-wave amplitude under both scotopic and photopic conditions (76% and 59% increase above uninjected controls, respectively) but no changes were observed in a-wave amplitude. However, this increase in b-wave amplitude was not accompanied by any slow down in photoreceptor degeneration or apoptotic cell death. Delivery of rAAV.RPE65 also resulted in a decrease in retinyl ester lipid droplets and an increase in short wavelength cone opsin-positive cells, suggesting that the recovery of RPE65 expression has long-term benefits for retinal health.
  
  
  CONCLUSIONS
  This work demonstrated the potential benefits of using theRpe65-/-mice to study the effects and mechanism of rAAV.RPE65-mediated gene delivery into the retina. Although the functional recovery in this model was not as robust as in the dog model, these experiments provided important clues about the long-term physiological benefits of restoration of RPE65 expression in the retina.
authors.affiliation: Centre for Ophthalmology and Visual Science, The University of Western Australia, Perth, Western Australia, 6009, Australia.
authors.forename: Chooi-May
authors.initials: CM
authors.lastname: Lai
authors.affiliation: Department of Molecular Ophthalmology, Lions Eye Institute and The University of Western Australia, Perth, Western Australia, 6009, Australia.
authors.forename: Meaghan Jt
authors.initials: MJ
authors.lastname: Yu
authors.affiliation: Department of Molecular Ophthalmology, Lions Eye Institute and The University of Western Australia, Perth, Western Australia, 6009, Australia.
authors.forename: Meliha
authors.initials: M
authors.lastname: Brankov
authors.affiliation: Vision Touch and Hearing Research Centre, School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, 4072, Australia.
authors.forename: Nigel L
authors.initials: NL
authors.lastname: Barnett
authors.affiliation: Virus Core Facility, Gene Therapy Center, University of North Carolina, North Carolina, 27599, USA.
authors.forename: Xiaohuai
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland, 20892, USA.
authors.forename: T Michael
authors.initials: TM
authors.lastname: Redmond
authors.affiliation: Vision Science Group, Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri, 65211, USA.
authors.forename: Kristina
authors.initials: K
authors.lastname: Narfstrom
authors.affiliation: Centre for Ophthalmology and Visual Science, The University of Western Australia, Perth, Western Australia, 6009, Australia.
authors.forename: P Elizabeth
authors.initials: PE
authors.lastname: Rakoczy