Sustained tetracycline‐regulated transgene expression in vivo in rat retinal ganglion cells using a single type 2 adeno‐associated viral vector

BACKGROUND: Viral vector delivery of neurotrophic-expressing transgenes in the retina may retard or prevent the onset of blindness associated with photoreceptor degeneration. A key safety issue is to achieve regulated expression of these genes in the retina. The purpose of our study was to evaluate whether a single recombinant AAV-2 (rAAV) encoding for a tetracycline (Tet)-regulated destabilized reporter gene could provide quantitative profiles of gene regulation targeted to the rat neuroretina. METHODS: A rAAV vector carrying a destabilized green fluorescent protein (dgfp) under a tet-regulatable promoter and the tetracycline-repressed transactivator (tTA) was generated (rAAVtetoff.dgfp) and administered intravitreally in nine Wistar rats. Retinas were monitored for 6 months using noninvasive fluorescence imaging and the animals were subjected to two cycles of doxycycline (Dox), a tetracycline analog. Eyes were ultimately examined by histology. RESULTS: Intravitreal injection of rAAVtetoff.dgfp resulted in effective transduction of ganglion cells. Following full expression of the transgene in the absence of Dox, 95% of the GFP signal was shut down 48 h post Dox administration and the signal was undetectable 7 days later. Initial levels of GFP expression were restored 21 days after Dox administration ceased. This pattern of expression was repeated twice over a period of 6 months. CONCLUSIONS: This report demonstrates that rAAVtetoff.dgfp intravitreally injected rats displayed tight and sustained long-term regulation of the reporter gene in ganglion cells. These findings may have important implications regarding rAAV-mediated gene therapy using neuroprotective approaches for retinitis pigmentosa and glaucoma.