been considered to be in a physiologic range - despite the fact that immunohistochemical examination of 
the transfected tissue disclosed evidence of successful transfection in <1% of cells in the transfected 
arterial segment 6. Thus, gene products which are secreted may have profound biological effects, even 
when the number of transduced cells remains low. In contrast, for genes such as bFGF which do not 
encode a secretory signal sequence, transfection of a much larger cell population might be required for that 
intracellular gene product to express its biological effects. 
We therefore applied 400 |ig of phVEGF^s, encoding the 165-amino acid isoform of VEGF, 
to the hydrogel polymer outside coating of an angioplasty balloon 3 and delivered the balloon catheter 
percutaneously to the iliac artery of rabbits in which the femoral artery had been excised to cause hindlimb 
ischemia. Site-specific transfection of phVEGFi 65 was confirmed by analysis of the transfected internal 
iliac arteries using reverse transcriptase-polymerase chain reaction (RT-PCR) (see mansucript in 
Appendix) and then sequencing the RT-PCR product. Augmented development of collateral vessels was 
documented by serial angiograms in vivo, and increased capillary density at necropsy. Consequent 
amelioration of the hemodynamic deficit in the ischemic limb was documented by improvement in the calf 
blood pressure ratio (ischemic/normal limb) to 0.70±0.08 in the VEGF-transfected group vs 0.50±0.18 in 
controls (p<0.05). These findings thus established the principle that site-specific arterial gene transfer can 
be used to achieve physiologically meaningful therapeutic modulation of vascular disorders, including 
therapeutic angiogenesis. 
Despite the encouraging results achieved with administration of the recombinant protein, we 
believe that transferring the gene encoding that protein (i.e. gene therapy) is preferable for two reasons. 
First, and perhaps most critical in the case of arterial gene therapy, is the potential requirement to maintain 
an optimally high and local concentration over time. In the case of therapeutic angiogenesis, for example, 
we believe that it is preferable to deliver a lower dose over a period of several days or more from an 
actively expressing transgene in the iliac artery, rather than a single or multiple bolus doses of recombinant 
protein. It is conceivable - though as yet unproven - that such continuous, local production of VEGF 
resulting from the transgene may be preferable, both from the standpoints of safety and efficacy, to a 
single, larger dose of the recombinant protein administered by any (intra-arterial, intra-venous, intra- 
muscular) route. 
Second, there is the matter of economics: namely, recombinant protein therapy would 
ultimately cost more to develop, implement, and reimburse, particularly for those indications requiring 
multiple or protracted treatments. Part of this cost is inherent in the production steps and corresponding 
analytical testing required to ensure that post-translational modifications - such as glycosylation and 
refolding - are consistently reproduced. The feasibility of a clinical trial of recombinant VEGF protein is 
currently limited by the lack of approved or available quantities of human-quality grade recombinant 
protein. Due principally to the extraordinary cost of scaling up from research grade to human quality 
recombinant protein and the associated uncertainty regarding reimbursement for recombinant protein 
therapies in the future, no for-profit company is to our knowledge currently planning a clinical trial of 
recombinant protein therapy with VEGF. 
These preliminary results have thus established the feasibility of arterial gene transfer as a 
novel approach for patients with rest pain and/or non-healing ischemic ulcers. The lack of available 
medical therapy implies that patients who are not satisfactory candidates for conventional revascularization 
will face amputation of a portion of their limb as their sole therapeutic option. The potential for achieving 
limb salvage in a selected group of patients with no alternative therapeutic option suggests to us that these 
patients represent good candidates for arterial gene therapy. 
I. C. Direct Gene Transfer with the Hydrogel Polymer Balloon Catheter Applied 
to an Angioplasty Catheter Balloon can be Used to Effect Direct Gene Transfer to the 
Arterial Wall. 
Direct arterial gene transfer has been previously achieved using double-balloon catheters and 
perforated balloons, in most cases facilitated by the use of cationic liposomes or viral vectors. These gene 
delivery systems, however, have been compromised by issues relating to efficacy and/or safety. We 
investigated the possibility that arterial gene transfer might be performed during balloon angioplasty by 
delivery of "naked" genetic material from the hydrogel polymer coating of a standard angioplasty balloon. 
The hydrogel polymer was originally designed to facilitate advancement of the balloon through tight 
vascular obstructions. We considered that it might be possible to use the hydrogel polymer as a "sponge" 
onto which highly concentrated, pure DNA could be applied ex vivo, and subsequently, at the time of 
balloon inflation, be delivered into the vessel wall, without the use of conventional vectors. Accordingly, 
DNA solution was applied to the surface of an angioplasty catheter balloon coated with a hydrogel polymer 
Recombinant DNA Research, Volume 20 
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