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Katharine J. Liang, Kenton T. Woodard, Richard J. Samulski; AAV-Mediated Nrf2 Gene Replacement in the Nrf2 Knockout Mouse. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6506.
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© ARVO (1962-2015); The Authors (2016-present)
Reactive oxygen species (ROS) contribute to the pathogenesis of a wide variety of retinal diseases. In these pathological situations, normal redox homeostasis is overwhelmed by ROS levels increased beyond the capacity of endogenous antioxidant enzyme activity. Antioxidant enzyme expression is regulated by a common promoter region, the Antioxidant Response Element (ARE). The Nrf2 transcription factor binds the ARE and drives downstream expression of antioxidant genes. Accordingly, Nrf2 deficient mice harbor clinical and pathological features of Age-related Macular Degeneration (AMD). Adeno-associated virus(AAV)-mediated gene therapy has been shown to be a safe and long-lasting therapeutic strategy in three Leber’s Congenital Amaurosis clinical trials. Consequently, developing an AAV-Nrf2 vector to combat ROS in the retina is one potential therapeutic strategy for the prevention and treatment of AMD.
We have engineered AAV2.5 chimeric capsid vector selected for retinal transduction via intravitreal injection. AAV2.5 combines improved transduction properties of AAV1 with reduced antigenic cross-reactivity against antibodies directed at both parental serotypes while keeping receptor binding properties of AAV2. Nrf2 was cloned into a CBh promoter backbone and packaged into a single stranded AAV2.5 capsid viral vector. Nrf2 expression and downstream gene activation was assessed by western blot. Functional efficacy of Nrf2 was evaluated using fluorescent ROS indicator CM-H2DCFDA in a fluorescent platereader. AAV2.5-Nrf2 delivery to the retinas of Nrf2-/- mice via intravitreal injections is ongoing.
Preliminary data supports efficiency of AAV2.5 transgene delivery via intravitreal injection in the mouse retina. Vector yields were comparable to reporter genes suggesting the Nrf2 transgene is neither toxic nor inhibitory to AAV vector production. AAV-Nrf2 transduction of 293 cells showed a dose-dependent increase in expression at 1000 and 10,000 MOI. Downstream gene activation with and without sulfurophane was compared as an in vitro model of stressed vs. unstressed cells. In vivo analysis is ongoing and update of Nrf2 expression is being monitored by both western and histological analysis.
AAV vector development combined with gene replacement offers a unique opportunity to optimize and test both efficient transgene delivery as well as potent therapeutic potential. Future studies can expand upon Nrf2 gene replacement and determine whether Nrf2 overexpression in ROS deregulated systems can restore ROS homeostasis as potential therapeutic and preventative strategies.
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