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.
Folliot
Sebastien
S
Laboratoire de Thérapie Génique, CHU-Hotel DIEU, Bat. J. Monnet, 30 Avenue J. Monnet, 44035 Nantes Cedex 01, France.
Briot
Delphine
D
Conrath
Hervé
H
Provost
Nathalie
N
Cherel
Yan
Y
Moullier
Philippe
P
Rolling
Fabienne
F
D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D004318:Doxycycline / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D051381:Rats; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D013752:Tetracycline / Q000494:pharmacology*; D015533:Transcriptional Activation; D019076:Transgenes; D018523:Tropism
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D013752:Tetracycline; D004318:Doxycycline