Effect of a DNA nuclear targeting sequence on gene transfer and expression of plasmids in the intact vasculature - PubMed (original) (raw)
Effect of a DNA nuclear targeting sequence on gene transfer and expression of plasmids in the intact vasculature
J L Young et al. Gene Ther. 2003 Aug.
Abstract
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.
Figures
Figure 1. Cartoon of plasmid constructs
Matched sets of GFP or luciferase reporter constructs were created. Reporter genes are driven by the CMV immediate early promoter which does not mediate plasmid nuclear import, and when present, the SV40 DTS is downstream of the reporter gene. All plasmids were prepared using Qiagen Gigaprep kits and greater than 80% were in the supercoiled form as determined by agarose gel electrophoresis.
Figure 2. Evaluation of cellular proliferation in electroporated rat mesenteric vessels
PCNA expression was detected by immunohistochemistry in naïve (A) and electroporated (B) rat mesenteric vessels or in rat small intestine (C). Vessels were electroporated as previously described. Briefly, male Sprague-Dawley rats (200–400 g) were anesthetized with isoflurane, a midline incision was made, and the small intestine was exteriorized. Approximately 1 cm of each neurovascular bundle was placed individually in the electrode and covered with a solution of 10 mM Tris, pH 8, 1 mM EDTA, 140 mM NaCl. Based on the design of the electrode, approximately 55 µl of solution were used to bathe the vessel. Vessels (8–10 per animal) were electroporated with 8 square wave pulses lasting 10 milliseconds each at a field strength of 200V/cm using a BTX830 electroporator (Genetronics, San Diego CA). After all vessels were electroporated within a given animal, the incision was closed and the animal was allowed to recover and returned to the vivarium. All experiments were conducted in accordance with institutional guidelines in compliance with the recommendations of the Guide for Care and Use of Laboratory Animals. One or two days post-transfer, vessels were removed from the animals which were then euthanized. Vessels were rinsed extensively with cold PBS, fixed in 10% buffered formalin, and embedded in paraffin for thin section preparation. Immunohistochemistry was performed on 6µm sections using a mouse monoclonal antibody directed against PCNA (Santa Cruz Biotechnology, Santa Cruz, CA), and detected with Alkaline Phosphatase ABC reagent and Vector Blue substrate (Vector Laboratories, Burlingame, CA). Sections were counterstained with eosin and photographed. Images are representative of multiple vessels (n= 3 animals, 6 naïve and 6 electroporated vessels from each). Bar = 100 µm.
Figure 3. Effect of the SV40 DTS on GFP gene expression in electroporated rat mesenteric arteries
Plasmid solutions containing DNA at 2 mg/ml in 10 mM Tris, pH 8, 1 mM EDTA, 140 mM NaCl were delivered to rat mesenteric vessels by electroporation as described in Figure 2. Vessels in panel A received pGFP and those in panel B received pGFP-DTS (or no DNA, “Control”). Two days post-transfer, vessels were removed from the animals which were then euthanized, rinsed extensively with cold PBS and the vessel was dissected away from the surrounding adipose tissue. GFP was excited at 488 nm and visualized at 515 nm using the appropriate filter cubes with a low power objective on an upright Leica DMRX fluorescence microscope. Fluorescent images were collected, all with the same exposure time and gain settings, using a Hamamatsu ORCA-2 cooled CCD camera and OpenLab3.0 software (Improvision, Lexington MA). The vessels shown are representative of those from multiple animals for each plasmid (n = 5 rats for pGFP; n = 11 rats for pGFP-DTS).
Figure 4. Quantitative evaluation of the effect of the SV40 DTS on vascular gene transfer
Rat mesenteric vessels were electroporated with pCMV-Lux (n = 65 vessels in 10 rats) or pCMV-Lux-DTS (n = 79 vessels in 10 rats), as described in Fig. 2. Two days post-transfer, vessels were removed, immediately snap frozen in liquid nitrogen on a prefrozen bed of Promega lysis buffer containing 1mM dithiothreitol (400 µl; Promega, Madison WI), and ground into a fine powder. Lysates were assayed for luciferase activity, standardized against purified recombinant luciferase protein (Promega) and expressed as pg luciferase per vessel. Mean expression levels were calculated and standard error of the mean (SEM) was determined using Instat software (GraphPad Inc., San Diego CA). Mann-Whitney U-test revealed a statistically significant difference in expression between the two groups, p<0.001 (*).
Figure 5. Time course of expression of plasmids containing or lacking import sequences
Plasmids were transferred to vessels using electroporation and at the indicated times, harvested for measurement of luciferase expression as described in Figs. 2 through 4. Between 2 and 10 animals were used at each time point (n ≥ 12 vessels per time point per construct). Time 0 represents vessels that received no DNA, and accounts for the level of sensitivity of the assay. * p<0.01 versus time 0; # p<0.05 versus pCMV-Lux at the same time point.
Figure 6. Plasmid localization in rat mesenteric vessels as analyzed by in situ hybridization
Plasmid solutions containing pCMV-Lux and pGFP (2mg/ml each; A, B, E, F, I, and J) or pCMV-Lux-DTS and pGFP-DTS (2 mg/ml each; C, D, G, H, and K – N) were transferred to the rat mesenteric vasculature by electroporation as described in Figure 2. Vessels were harvested at 8 (A – D), 24 (E – H), or 48 hours (I – N) post-electroporation, rinsed with cold PBS, fixed overnight in 10% buffered formalin and embedded in paraffin for thin sections. In situ hybridizations were performed on 10µm sections as described by Moorman, using biotin-labeled, nick translated pCMV-Lux-DTS and pGFP-DTS as probe. Following hybridization and washes, sections were treated with RNase H to eliminate detection of mRNA. Electroporated DNA was detected using the TSA Biotin System (Perkin-Elmer, Boston, MA) and Alkaline Phosphatase-Vector Blue ABC system (A, C, E, G, I, K, and M). Nuclei were counterstained with DAPI (B, D, F, H, J, L, and N). Panels A – L are at the same magnification; Bars = 100 µm.
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