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J. Zou, L. Luo, V. Chiodo, B. Ambati, W. Hauswirth, J. Yang; AAV-Mediated Gene Replacement Therapy in a Mouse Model of Usher Syndrome Type II Lacking Whirlin. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3102.
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Whirlin, USH2A and VLGR1 are the three causative genes of Usher syndrome type II, a condition with both retinitis pigmentosa and congenital deafness. It has been demonstrated that the proteins encoded by these three genes form a multi-protein complex at the periciliary membrane complex (PMC) in photoreceptors. Loss of any one of these three proteins causes disruption of this protein complex and retinal degeneration in mice. In this study, we evaluated the therapeutic effect of whirlin replacement using recombinant adeno-associated virus (AAV) in whirlin knockout mice.
Murine whirlin cDNA driven by a human rhodopsin kinase (hRK) promoter was packaged into an AAV5 vector (AAV5-hRK-whirlin) and delivered into whirlin knockout mice by subretinal injection at postnatal day 18. At 8 weeks post-injection, the localization and expression level of whirlin, USH2A and VLGR1 in the retina were examined by immunostaining and western blotting analyses. The DNA plasmid without whirlin cDNA packaged into the same AAV vector was used as a negative control.
In whirlin knockout mice injected with AAV5-hRK-whirlin, the transduced whirlin was expressed throughout the entire retina at its normal cellular location, the PMC, in photoreceptors. It had a molecular size and an expression level close to those in wildtype retinas. Importantly, while USH2A and VLGR1 were mislocalized and their expression decreased in whirlin knockout retinas, expression of whirlin delivered by the AAV5 vector was found to restore both the localization and expression levels of USH2A and VLGR1. No difference in the expression of whirlin, USH2A and VLGR1 was detected in whirlin knockout mice injected with the control AAV5 vector and in uninjected whirlin knockout mice.
This study further confirmed that whirlin, USH2A and VLGR1 form a multi-protein complex at the PMC in photoreceptors. The successful delivery of whirlin into photoreceptors by an AAV vector and the resulting expression of whirlin driven by the hRK promoter close to the endogenous level suggest that this AAV vector gene delivery system can be an effective gene therapy approach to treat retinal degeneration in patients with Usher syndrome type II caused by whirlin mutations and, possibly, other forms of retinal degeneration as well.
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