Abstract
Abstract: :
Purpose:Normal human lens epithelial cells (HLECs) show very limited growth in vitro. To overcome this limitation we introduced hTERT into dividing and non-dividing lens epithelial cells. Telomerase has been reported to overcome in vitro senescence, maintain the diploid state and permit retention of in vivo characteristics. Methods: First we introduced a DNA expression construct encoding the catalytic subunit of hTERT driven by the MPSV LTR promoter using Fugene. This strategy was only effective for dividing cells. Growth was compared to controls without the hTERT. Second, since the vast majority of lens epithelial explants show little mitosis, we used a feline immunodeficiency virus (FIV) vector system developed by the Nolan laboratory (In: "Methods In Molecular Medicine Gene Therapy Protocols", Humana Press 2001). It has the ability to transfer recombinant genes across the nuclear envelope and to transduce non-dividing cells. It consists of three plasmids. A plasmid carrying the FIV structural genes, a second plasmid containing the VSV-G envelope gene, and a transfer vector termed pPanther. We modified pPanther to carry a bicistronic gene expression cassette composed of hTERT and EGFP driven by a CMV promoter. The 3 plasmids were cotransfected into 293T producer cells to assemble a replication deficient virus that was used to carry hTERT into young and old HLECs. Results: hTERT was introduced into dividing fetal HLECs. The subsequent growth of cells containing hTERT and control (untransfected) cells was compared. Control cells at 52 population doublings (pds), were enlarged and stopped dividing. Cells transfected with hTERT completed 102 pds and showed no signs of senescence. Next, we determined if the FIV system could introduce hTERT into HLECs. Expression of the bicistronic cassette was confirmed by the presence of GFP. Conclusions: Introduction of hTERT into dividing HLECs overcame cell senescence. The data show that the FIV vector system can be used to transduce hTERT into HLECs from young and old donors. This system may permit the introduction of hTERT into human primary capsule-epithelial outgrowths, which exhibit little mitosis. This approach may facilitate the routine establishment of long-term lines from young, old and cataractous lenses and permit experiments on differences in the epithelium associated with aging and cataract formation. The FIV system should also permit introduction of any gene of choice into HLECs. We thank GERON corp. for generously supplying hTERT, and Drs. Nolan and Curran for their generous gifts of the FIV vectors.
Keywords: proliferation • gene transfer/gene therapy • gene/expression