[Transduction efficacy of recombinant type 1 and type 2 adeno-associated virus in the retinal cells].
OBJECTIVE
To investigate the transduction and gene expression of the recombinant adeno-associated viruses (rAAV) of the serotypes 1 and 2 in the retinal cells.


METHODS
rAAV vectors of type 1 and type 2 encoding EGFP were infected into the cultured retinal pigmentary epithelium (RPE) cells of the line CRL-2302 and primarily cultured retinal neural cells from normal SD rats, and primarily cultured RPE cells from an adult cornea donor. The cultured RPE cells transduced by rAAV2-EGFP or rAAV2/1-EGFP were harvested at the 7 th and 14 th day after infection to be detected by fluorescence-activated cell sorter. The onset of EGFP gene expression and EGFP positive rate were detected by flow cytometry and fluorescence microscopy. Then, rAAV2/1-EGFP and rAAV2-EGFP were injected into the subretinal spaces of 32 SD rats to investigate the onset of EGFP fluorescence and its distribution in the fundus in vivo via fluorescence stereoscope. HE staining and immunohistochemistry were used to observe the infected cell type and immune response in the retina.


RESULTS
The percentage of EGFP positive cells and mean intensity of EGFP fluorescence in the cells transduced by rAAV2-EGFP 7 and 14 days after transduction were 13.50% +/- 1.70% and 15.60% +/- 0.82%, and 2.75 +/- 0.12 and 3.80 +/- 0.72 respectively; and the EGFP positive cells and mean intensity of EGFP fluorescence in the cells transduced by rAAV2/1-EGFP were 1.09% +/- 0.5% and 1.98% +/- 0.45%, and 1.12 +/- 0.09 and 1.75 +/- 0.2 respectively. The EGFP fluorescence area in the retina were (5389 +/- 211) microm(2), (9832 +/- 364) microm(2), (14 454 +/- 446) microm(2), (20 528 +/- 648) microm(2), and (20 264 +/- 683) microm(2) respectively 3, 7, 14, and 75 days, and 4 month after transduction by rAAV2-EGFP in vivo; In the rat retina transduced by rAAV2/1-EGFP, the EGFP fluorescence areas were (9666 +/- 348) microm(2), (12 160 +/- 439) microm(2), (19 794 +/- 621) microm(2), (26 172 +/- 923) microm(2), and (26 022 +/- 965) microm(2) respectively 3, 7, 14, and 75 days, and 4 month after infection.


CONCLUSIONS
rAAV2 efficiently transduces retinal cells both in vitro and in vivo. rAAV2/1 is a more effective gene-transferring vector to be used in retinal cells in vivo than rAAV2.
Wu
Ji-hong
JH
Central Experimental Laboratory, First People's Hospital, Shanghai Jiaotong University, Shanghai 200080, China.
Zhang
Sheng-hai
SH
Liu
Yan
Y
Chen
Xia-fang
XF
Xu
Ping
P
Wu
Xiao-bing
XB
Huang
Qian
Q
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics*; D005434:Flow Cytometry; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000166:cytology; D014162:Transfection
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins