Virus-mediated gene transfer to induce therapeutic angiogenesis: where do we stand?

Abstract

The potential to induce therapeutic angiogenesis through gene transfer has engendered much excitement as a possible treatment for tissue ischemia. After 10 years of clinical experimentation, however, it now appears clear that several crucial issues are still to be resolved prior to achieving clinical success. These include the understanding of whether functional blood vessels might arise as a result of the delivery of a single angiogenic factor or require more complex cytokine combinations, the identification of the proper timing of therapeutic gene expression and, most notably, the development of more efficacious gene delivery tools. Viral vectors based on the adeno-associated virus (AAV) appear particularly suitable to address the last requirement, since they display a specific tropism for skeletal muscle cells and cardiomyocytes, and drive expression of the therapeutic genes in these cells for indefinite periods of time. In this review, I discuss the current applications of gene therapy for cardiovascular disorders, with particular attention to the possible improvements in the technologies involved in virus-mediated gene transfer.

Figure 1

Schematic representation of the genetic organization of AAV and adenovirus (left side) and of the respective vectors. The location at which the therapeutic gene cassette (promoter+gene+polyadenylation site) is inserted is indicated. TR, Terminal repeat sequence.

Figure 2

Production of AAV vectors. A plasmid containing the therapeutic gene cassette (promoter+gene+polyadenylation site) cloned between the AAV terminal repeats is co-transfected into epithelial HEK293 cells together with a plasmid containing the rep and cap AAV genes and some genes from adenovirus that provide helper function. After 48 hours, cells are lysed and recombinant vector particles are purified by cesium chloride gradient centrifugation. Fractions are collected from the gradient and the number of particles containing viral genomes are quantified by real-time PCR.

Figure 3

Sprouting angiogenesis. In adult organisms, angiogenesis is initiated upon the metabolic activation, proliferation and mobilization of endothelial cells lining pre-existing vessels. This process is triggered by various angiogenic cytokines, including VEGF and FGF2 and is accompanied by the destabilization of the extracellular matrix by matrix metalloproteases (MMP). At later time points, the newly formed capillary matures by the acquisition of pericytes and smooth muscle cells to form a new medial layer. The maturation process requires the action of several other cytokines, among which the angiopoietins, ephrins and semaphorins play a major role.