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Sustained tetracycline‐regulated transgene expression in vivo in rat retinal ganglion cells using a single type 2 adeno‐associated viral vector
Author(s) -
Folliot Sebastien,
Briot Delphine,
Conrath Hervé,
Provost Nathalie,
Cherel Yan,
Moullier Philippe,
Rolling Fabienne
Publication year - 2003
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.367
Subject(s) - green fluorescent protein , transgene , biology , microbiology and biotechnology , retina , genetic enhancement , retinitis pigmentosa , viral vector , reporter gene , doxycycline , retinal degeneration , gene expression , gene , recombinant dna , genetics , neuroscience , antibiotics
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 ( d gfp) under a tet‐regulatable promoter and the tetracycline‐repressed transactivator (tTA) was generated (rAAVtetoff. d gfp) 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. d gfp 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. d gfp 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.