Real-time, noninvasive in vivo assessment of adeno-associated virus-mediated retinal transduction.
OBJECTIVE
To evaluate the efficiency, cell specificity, stability, and toxicity of recombinant adeno-associated virus (rAAV)-mediated retinal transduction in vivo in the adult immunocompetent mouse. To assess the usefulness of green fluorescent protein (GFP) for real-time, noninvasive monitoring of retinal transgene expression in vivo.


METHODS
Assessment of ocular GFP expression was performed in cohorts of mice for 11 weeks after subretinal injection of a recombinant adeno-associated virus carrying the complementary DNA (cDNA) for GFP. Examinations were performed in vivo by direct observation of fluorescence by ophthalmoscopy, using excitation-barrier filters. Histologic analyses of retinal tissue were used to identify transduced cells and to assess inflammation.


RESULTS
Retinal GFP expression can be monitored in vivo using real-time, noninvasive imaging. Recombinant AAV efficiently transduces a variety of cells of the neural retina and of the retinal pigment epithelium (RPE). Transgene expression was not observed until 1 week after infection. The number of GFP-expressing cells increased over 3 weeks, and expressing photoreceptors and RPE, cells persisted at least through 11 weeks (the termination of the experiment). There was no clinical or histologic evidence of inflammatory response.


CONCLUSIONS
Retinal gene transfer mediated by rAAV is stable and efficient and is associated with no clinically or histologically detectable toxicity or immune reaction. Green fluorescent protein allows noninvasive assessment of the extent and location of retinal transgene expression as a function of time and promises to be useful alone and as a tag for other transgenes delivered experimentally or therapeutically to the retina.
Bennett
J
J
Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania School of Medicine, Philadelphia 19104-6069, USA.
Duan
D
D
Engelhardt
J F
JF
Maguire
A M
AM
D000818:Animals; D018076:DNA, Complementary / Q000378:metabolism; D003673:Defective Viruses / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D010786:Photoreceptor Cells / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D015398:Signal Transduction; D019076:Transgenes
D018076:DNA, Complementary; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins