Pressure-mediated oligonucleotide transfection of rat and human cardiovascular tissues

Abstract

The application of gene therapy to human disease is currently restricted by the relatively low efficiency and potential hazards of methods of oligonucleotide or gene delivery. Antisense or transcription factor decoy oligonucleotides have been shown to be effective at altering gene expression in cell culture expreriments, but their in vivo application is limited by the efficiency of cellular delivery, the intracellular stability of the compounds, and their duration of activity. We report herein the development of a highly efficient method for naked oligodeoxynucleotide (ODN) transfection into cardiovascular tissues by using controlled, nondistending pressure without the use of viral vectors, lipid formulations, or exposure to other adjunctive, potentially hazardous substances. In this study, we have documented the ability of ex vivo, pressure-mediated transfection to achieve nuclear localization of fluorescent (FITC)-labeled ODN in approximately 90% and 50% of cells in intact human saphenous vein and rat myocardium, respectively. We have further documented that pressure-mediated delivery of antisense ODN can functionally inhibit target gene expression in both of these tissues in a sequence-specific manner at the mRNA and protein levels. This oligonucleotide transfection system may represent a safe means of achieving the intraoperative genetic engineering of failure-resistant human bypass grafts and may provide an avenue for the genetic manipultation of cardiac allograft rejection, allograft vasculopathy, or other transplant diseases.

Figure 1

(A) Efficiency of pressure-mediated transfection of FITC–ODN into human saphenous vein. FITC-labeled nuclei were counted after a transfection period of 10 min and are reported as a percentage of total vein wall nuclei. n = 4–6 at each point. (B) Total ³²P-labeled ODN delivery to human saphenous vein after pressure-mediated transfection for 10 min. n = 4 at each point. Transfection efficiency (C) and total ODN delivery (D) in human saphenous vein 2 hours after 10-minute, nondistending pressure-mediated transfection. n = 6 at each point.

Figure 2

Nuclear localization of FITC–ODN in human vein and rat heart. Fluorescent photomicrographs indicate concentrated green FITC–ODN signals (A, C, and E) that correspond to nuclei labeled with the blue intercalating chromatin dye, Hoechst 33342 (B, D, and F). FITC–ODN localization is seen in nuclei of the medial (arrow) and intimal (arrowhead) layers in human saphenous vein at ×100 (A and B) and ×400 (C and D) magnification. (E and F) Pressure-mediated FITC–ODN transfection of cells in rat myocardium (magnification ×400). (G and H) Control human saphenous vein pressure-treated with free, non-ODN-conjugated FITC. Note the absence of nuclear localization of FITC signal despite background green fluorescence (magnification ×400).

Figure 3

Ex vivo pressure-mediated transfection of rat myocardium. (A) Transfection efficiency increased with increasing pressure and with prolonged incubation in hearts both perfused with and surrounded on epicardial and endocardial surfaces by FITC–ODN solution (80 μM). (B) Optimum transfection was achieved with both coronary perfusion and submersion in a bath of FITC–ODN solution, the former contributing more significantly to total delivery. *, n = 4.

Figure 4

(A) Sequence-specific inhibition of IL-6 protein production determined by ELISA after pressure-mediated transfection of cultured human saphenous vein at 300 mmHg for 10 min with either sequence A or sequence B ODN (n = 4). (B) IL-6 mRNA measured by quantitative RT-PCR. One vein segment from each of three patients was either left untreated (U) or was treated with antisense (AS) or reverse antisense (RAS) ODN of IL-6 sequence A or sequence B (see text for details).

Figure 5

Relationship between transfection pressure and IL-6 protein inhibition by antisense ODN (10 μM) in human saphenous vein. n = 3 at each point.

Figure 6

Quantitative analysis of ICAM-1 inhibition by pressure-mediated transfection of allotransplanted rat hearts. (A) ICAM-1 mRNA levels measured by quantitative RT-PCR are similarly reduced in a sequence-specific manner after antisense ODN transfection. (B) Percentage of total myocardium stained positive with ICAM-1 immunohistochemistry is reduced by antisense (AS) but not reverse antisense (RAS) ODN transfection. ∗, P < 0.02; n = 3–4.