Effect of a DNA nuclear targeting sequence on gene transfer and expression of plasmids in the intact vasculature.
Although the use of nonviral vectors for gene therapy offers distinct advantages including the lack of significant inflammatory and immune responses, the levels of expression in vivo remain much lower than those obtained with their viral counterparts. One reason for such low expression is that unlike many viruses, plasmids have not evolved mechanisms to target to the nucleus of the nondividing cell. In the absence of mitosis, plasmids are imported into the nucleus in a sequence-specific manner, and we have shown in cultured cells by transfection and microinjection experiments that the SV40 enhancer mediates plasmid nuclear import in all cell types tested (Dean et al., 1999, Exp Cell Res 253: 713-722). To test the effect of this import sequence on gene transfer in the intact animal, we have recently developed an electroporation method for DNA delivery to the intact mesenteric vasculature of the rat. Plasmids expressing luciferase or GFP from the CMV immediate-early promoter/enhancer and either containing or lacking the SV40 enhancer downstream of the reporter gene were transferred to the vasculature by electroporation. When transfected into actively dividing populations of smooth muscle or epithelial cells, the plasmids gave similar levels of expression. By contrast, the presence of the SV40 sequence greatly enhanced gene expression of both reporters in the target tissue. At 2 days post-transfer, plasmids with the SV40 sequence gave 10-fold higher levels of luciferase expression, and at 3 days the difference was over 40-fold. The presence of the SV40 sequence did not simply increase the rate of nuclear import and expression, since expression from the SV40-lacking plasmid did not increase beyond that seen at day 2, the time of maximum expression for either plasmid. In situ hybridization experiments confirmed that the increased gene transfer and expression was indeed due to increased nuclear localization of the delivered SV40 sequence-containing plasmid. Based on these findings, the ability to target DNA to the nucleus can increase gene transfer in vivo and inclusion of the SV40 sequence into plasmids will enhance nonviral gene delivery.
Young
J L
JL
Division of Pulmonary and Critical Care Medicine, Northwestern University Medical School, Chicago, IL 60611, USA.
Benoit
J N
JN
Dean
D A
DA
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000008:administration & dosage*; D018274:Electroporation / Q000379:methods*; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins; D008156:Luciferases / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D008638:Mesenteric Arteries / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D051381:Rats; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection / Q000379:methods*
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D008156:Luciferases