Kinetics of efficient recombinant adeno-associated virus transduction in retinal pigment epithelial cells.
The aim of this study was to investigate the premise that retinal pigment epithelial (RPE) cells are more permissive to recombinant adeno-associated virus (rAAV) transduction than other cells. We investigated the kinetics and mechanisms of rAAV transduction in RPE cells and found that the transduction efficiencies of cultured RPE cells HRPE51 and ARPE19 were significantly higher than those of 293 (P < 0.008) and HeLa (P < 0.025) cells. In addition, RPE cells reached maximum transduction efficiency at a much lower m.o.i. (m.o.i. 10) than 293 cells (m.o.i. 25). Competition experiments using 1 microg/ml heparin inhibited the high level of transduction in RPE cells by 30%, but additional heparin failed to reduce rAAV transduction further. Southern hybridization of low-molecular-weight DNA from transduced RPE cells indicated that 42% of single-stranded rAAV DNA was translocated into the nucleus by 2 h postinfection. By 6 h postinfection, double-stranded rAAV DNA was observed, which coincided with the onset of transgene expression. Southern and fluorescence in situ hybridization of total genomic DNA indicated that long-term transgene expression in RPE cells was maintained by the integration of rAAV into the cellular chromosome. Together, these results suggest that the high permissiveness of RPE cells is not related to the presence of heparan sulfate receptors or nuclear trafficking but may be due to an enhanced rate of second-strand synthesis and that integration in RPE cells is responsible for long-term transgene expression.
Lai
Y K
YK
Centre for Ophthalmology and Visual Science, University of Western Australia, Nedlands, Western Australia, Australia.
Rolling
F
F
Baker
E
E
Rakoczy
P E
PE
D000925:Anticoagulants / Q000494:pharmacology; D002460:Cell Line; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics / Q000502:physiology*; D017930:Genes, Reporter; D005822:Genetic Vectors; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000502:physiology* / Q000821:virology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D019076:Transgenes
D000925:Anticoagulants; D011993:Recombinant Fusion Proteins; D006493:Heparin