Establishment of the method to efficiently transfer BDNF gene into IPE cells by adeno–associated virus vector

Abstract

Abstract: : Purpose: To establish a method to transfer BDNF gene effectively into iris pigment epithelial (IPE) cells by using recombinant adeno–associate virus type–2 (rAAV2). Methods: Human IPE cells were isolated by trypsinization and cultured in F12 supplemented with FBS. Cells were treated with DNA synthesis inhibitors such as hydroxyurea (HU) and sodium butyrate (SB) (HU–SB) or epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, and infected with purified rAAV2 (rAAV–LacZ or rAAV–BDNF). The transduction efficiencies were determined by counting cells stained with lac Z after 4 days of the infection with rAAV–LacZ. The expression of hBDNF in IPE cells transfected with rAAV–BDNF (AAV–BDNF–IPE) were measured by reverse transcription polymerase chain reaction (RT–PCR). The protein productions of hBDNF in AAV–BDNF–IPE were measured by sandwich ELISA and shown by western blotting analysis. Results: hIPE cells showed substantially lower infection than ARPE–19 and HT1080 cells, highly permissive cells for rAAV. The trasduction efficiency in hIPE cells increased to 2.598±0.111 from 0.680±0.273% by the treatment with HU and SB and that in ARPE–19 and HT1080cells increased to 10±2.381 % and 90±11.552 % respectively. Tyrphostin–1 enhanced AAV–mediated transgene expression up to 3.173±0.122%. The combination with HU–SB and tyrphostin–1 dramatically enhanced the transduction effeiciency up to 3.873±0.113%. We also demonstrated that the increase of transduction efficiency depend on the culture period. At 14 days of culture, the transduction efficiency in hIPE cells treated with HU–SB and tyrphostin–1 was 24.330±3.280%. In case of cells infected with rAAV–BDNF, the infection of high titration virus caused less transduction efficiency. Conclusions: We established a method for BDNF expressed hIPE cells by using rAAV–helper plasmid system. The combination treatment of HU–SB and tyrphostin–1enhanced the transduction efficiency. At a certain point in virus concentration, there is a maximum efficiency in the expression of hBDNF in AAV–BDNF–IPE. After reaching this point, there is a decrease in the efficiency of the expression of hBDNF in AAV–BDNF–IPE. Our results demonstrated that the rate of virus particle to cell is the key factor to transgene by using rAAV.

Keywords: adenovirus • gene transfer/gene therapy • iris