Nonviral gene transfer strategies for the vasculature

Abstract

Major attention has been focused on the development of gene therapy approaches for the treatment of vascular diseases. In this review, we focus on an alternative use of gene therapy: the use of genetic means to study vascular cell biology and physiology. Both viral and nonviral gene transfer strategies have limitations, but because of the overwhelming inflammatory responses associated with the use of viral vectors, nonviral gene transfer methods are likely to be used more abundantly for future applications in the vasculature. Researchers have made great strides in the advancement of gene delivery to the vasculature in vivo. However, the efficiency of gene transfer seen with most nonviral approaches has been exceedingly low. We discuss how to circumvent and take advantage of a number of the barriers that limit efficient gene delivery to the vasculature to achieve high-level gene expression in appropriate cell types within the vessel wall. With such levels of expression, gene transfer offers the ability to alter pathways at the molecular level by genetically modulating the activity of a gene product, thus obviating the need to rely on pharmacological agents and their foreseen and unforeseen side effects. This genetic ability to alter distinct gene products within a signaling or biosynthetic pathway or to alter structural interactions within and between cells is extremely useful and technologically possible today. Hopefully, with the availability of these tools, new advances in cardiovascular physiology will emerge.

Figure 1

Barriers to gene transfer. Cartoon depicting the various barriers encountered by DNA between administration and protein production.

Figure 2

Electrode design for vascular electroporation.

Figure 3

Use of electroporation for vascular gene expression. (A) Electroporation does not damage the vessel. Untreated or electroporated vessels 2 days after gene transfer were fixed, embedded in parafin, sectioned, and stained with hematoxylin and eosin. As can be seen, there are no apparent histological differences between the vessels. (B) High-level gene expression is reproducible. Plasmids expressing green fluorescent protein (GFP) were transferred by electroporation to vessels within multiple animals. Two days after gene transfer, vessels were removed and viewed by fluorescence microscopy for GFP expression. As can be seen, gene transfer is reproducible and is highly efficient. (C) Gene transfer and expression occurs in all cell layers of the vasculature. A set of serial sections from a vessel electroporated with a GFP-expressing plasmid were prepared 2 days after gene transfer. Gene expression can be detected in the adventitial, smooth muscle, and endothelial cell layers.

Figure 4

Methods for cell-specific gene delivery. (A) Delivery route. Plasmids can be administered via the lumen with or without damage (e.g., balloon angioplasty) to transfer genes to the endothelium or smooth muscle cells, respectively. Alternatively, plasmids can be delivered to the adventitial surface to transfect adventitial cells. (B) Cell-specific promoters. Smooth muscle or endothelial cell specific promoters can be used to drive gene expression in plasmids specifically in smooth muscle or endothelia cells, respectively. (C) Nuclear targeting. Plasmids containing a nuclear localizing DNA sequence from cell type X (e.g., smooth muscle or endothelial cells) can bind to transcription factors present only in cell type X to form a protein-DNA complex that can be imported into the nucleus. By contrast, in all other cell types, these transcription factors are absent; consequently, complexes cannot form, the DNA is not transported into the nucleus, and no gene expression occurs.

Figure 5

Dominant negative mutants. In the case of a protein that must dimerize for formation of its active site, if high levels of a dominant negative mutant are expressed, the mutant will compete for binding to the wild type protein, producing inactive complexes.

Figure 6

DNAzymes. The 15 nucleotide 10–23 catalytic domain is sandwiched between two 7–9 nucleotide arms that are designed to be complementary to the desired target mRNA. Once delivered to cells, the DNAzyme hybridizes to its target mRNA and then cleaves the target, releasing two pieces of the RNA. Because the mRNA has been cleaved, it is no longer capable of directing protein translation; consequently, the levels of product decline on the basis of the half-life of the target protein.