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. Copyright 2003 John Wiley & Sons, Ltd.
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. Copyright 2003 John Wiley & Sons, Ltd.
Authors: S Goverdhana; M Puntel; W Xiong; J M Zirger; C Barcia; J F Curtin; E B Soffer; S Mondkar; G D King; J Hu; S A Sciascia; M Candolfi; D S Greengold; P R Lowenstein; M G Castro Journal: Mol Ther Date: 2005-08 Impact factor: 11.454
Authors: Xuyang Liu; Carol A Rasmussen; B'ann T Gabelt; Curtis R Brandt; Paul L Kaufman Journal: Surv Ophthalmol Date: 2009 Jul-Aug Impact factor: 6.048