Long-term transgene expression in the RPE after gene transfer with a high-capacity adenoviral vector.
OBJECTIVE
To analyze duration of gene expression in the retinal pigment epithelium (RPE) in immunocompetent animals after gene transfer with a high-capacity adenoviral (HC-Ad) vector.


METHODS
An HC-Ad vector was constructed to express the enhanced green fluorescence protein (EGFP) from the human CMV promoter. This vector (HC-AdFK7) was used to transduce rat RPE cells in cell culture and after subretinal injection in vivo in adult immunocompetent Wistar rats. In cell culture, expression of EGFP was analyzed by fluorescence microscopy. In vivo expression was monitored by scanning laser ophthalmoscopy and stereo fluorescence microscopy. After enucleation of the eyes, immunohistochemical and morphologic analyses by fluorescence light microscopy and electron microscopy were performed.


RESULTS
In vitro, RPE cells were efficiently transduced with HC-AdFK7. Expression persisted for the observation time of 8 weeks. In vivo, the RPE was efficiently transduced with low doses of HC-AdFK7. EGFP synthesis was confirmed by antibody staining and found to be stable for the complete study period of 6 months. The neuroretina was well preserved over areas of subretinal vector administration, and significant morphologic changes were not detected. There was no accumulation of inflammatory T cells or macrophages.


CONCLUSIONS
In contrast to previous results with earlier generation adenoviral vectors, subretinal injection of an HC-Ad vector in immunocompetent rats resulted in long-term transgene expression without evidence of adverse immune reactions or significant toxicity, probably because of the absence of expression of the viral gene from this vector. Thus, HC-Ad vectors are suitable for the treatment of eye disorders that require durable gene expression in the RPE.
Kreppel
Florian
F
University Eye Hospital, Laboratory of Experimental Ophthalmology, Cologne, Germany.
Luther
Thomas T
TT
Semkova
Irina
I
Schraermeyer
Ulrich
U
Kochanek
Stefan
S
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002478:Cells, Cultured; D015870:Gene Expression / Q000502:physiology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008264:Macrophages / Q000276:immunology; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000276:immunology; D009887:Ophthalmoscopy; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D020318:Rats, Long-Evans; D017208:Rats, Wistar; D013601:T-Lymphocytes / Q000276:immunology; D013997:Time Factors; D019076:Transgenes
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins