Abstract
Abstract: :
Purpose: To develop a safer method for retinal gene therapy without systemic infection of the virus vector, a method of transplantation of iris pigment epithelium (IPE) with gene transfer by the recombinant adenovirus (rAV) vector is assessed by the detection of the virus DNA in the systemic organs. Methods: We prepared a rAV vector to express LacZ (rAV-LacZ; 3.43x109PFU/ml). The rat IPE cells were transduced with the rAV-LacZ (IPE-rAV; 5x104 cells/µl). A group of SD rats was treated with subretinal injection with 2µl of the rAV-LacZ. Another group was transplanted with 2µl of the IPE-rAV. After 7 days postoperation, their optic nerve, brain, lung, liver, kidney and testis were removed. The genomic DNA was extracted from these organs, and was analyzed to detect the LacZ gene using polymerase chain reaction (PCR). In addition,ß-Gal staining was done in the ocular tissues of each group. Resuls: In the experiment of the rAV-LacZ injection, the LacZ DNA was detected not only the eye but also the other systemic organs by PCR analysis. In contrast, the LacZ DNA was not detected from the systemic organs in the experiment of transplantation of IPE-rAV.Histological analysis showed that the LacZ gene was widely expressed in the rat ocular tissues including the retinal pigment epithelium, the choroidal vessels and the epithelial tissue of the ciliary body and the iris in the sample of the rAV-LacZ injection. In contrast, no ß-Gal staining was not observed in the ocular tissues of IPE-rAV transplantation except for the transplanted IPE cells. Conclusions: Our results demonstrated that the subretinal transplantation of the gene transferred IPE cells using rAV could prevent the systemic dissemination of this vector and express the transgene only at the targeted region. Therefore, transplantation of transduced IPE cells into the subretinal space can avoid organic infection of the virus vector, reducing its side effect.
Keywords: adenovirus • gene transfer/gene therapy • transplantation