Research

Recombinant adeno-associated virus type 2-mediated gene delivery into the Rpe65^-/- knockout mouse eye results in limited rescue

Chooi-May Lai^1* , Meaghan JT Yu^2* , Meliha Brankov² , Nigel L Barnett³ , Xiaohuai Zhou⁴ , T Michael Redmond⁵ , Kristina Narfstrom⁶ and P Elizabeth Rakoczy¹

¹  Centre for Ophthalmology and Visual Science, The University of Western Australia, Perth, Western Australia, 6009, Australia

²  Department of Molecular Ophthalmology, Lions Eye Institute and The University of Western Australia, Perth, Western Australia, 6009, Australia

³  Vision Touch and Hearing Research Centre, School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, 4072, Australia

⁴  Virus Core Facility, Gene Therapy Center, University of North Carolina, North Carolina, 27599, USA

⁵  Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland, 20892, USA

⁶  Vision Science Group, Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri, 65211, USA

author email corresponding author email* Contributed equally

Genetic Vaccines and Therapy 2004, 2:3doi:10.1186/1479-0556-2-3

Published:	27 April 2004

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 the Rpe65^-/- 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 of Rpe65^-/- 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 to Rpe65^-/- 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.

Conclusion

This work demonstrated the potential benefits of using the Rpe65^-/- 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.