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.

Localized adenovirus gene delivery using antiviral IgG complexation

“…ery of antisense oligonucleotides [16][17][18][19][20] and plasmid DNA [21][22][23][24][25] have been shown to be both feasible and successful in experimental studies. Viral vectors have also been incorporated into microparticle sustained release systems that are formulated from polylactic polyglycolic acid, 26 and in separate studies, from collagen coacervates.…”

Molecular recognition elements - DNA/RNA-aptamers to proteins

“…Эти интересные результаты позволили группе Dzau провести испытания аптамера к E2F на человеке, чтобы определить, можно ли ограничить гиперплазию интимы при введении аптамера в вену [83,84]. E2F аптамер доставляли в паховую вену с помощью трансфекции.…”

Focused ultrasound (HIFU) induces localized enhancement of reporter gene expression in rabbit carotid artery

“…As a result, a variety of methods to increase the effectiveness of nonviral vectors have been investigated, such as electroporation, application of DNA to accelerated gold particles, and exposure to controlled pressure. [6][7][8][9] Although these methods have been demonstrated to enhance transfection efficiencies in a variety of tissues and cell types, widespread clinical application of many gene transfer strategies await further improvements on gene transfer methodology. In addition, many therapeutic strategies would benefit from a noninvasive means of directing gene transfer to a specific tissue site.…”

“…Mechanical disruption of the endothelium has been reported to enhance viral gene delivery into vascular smooth muscle cells of the vessel wall, 10 and the uptake of plasmid or oligonucleotide DNA in cardiovascular tissues has been shown to be enhanced by delivery in a pressurized environment. 9 Depending on the physical parameters, ultrasound as a mechanical wave transmission is in broad clinical use for both diagnostic and therapeutic medical purposes. for example, ultrasonic shockwaves are used for the nonthermal destruction of kidney stones in lithotripsy, and high-intensity focused ultrasound (HIFU) is clinically used for the thermal ablation of tumors.…”