endpoints which will be used to measure the response to therapy in this study will be 
abolition of rest pain and/or healing of the ischemic ulcer. 
As indicated above, however, determination of the extent of collateral vessel augmentation which 
develops consequent to VEGF gene therapy constitutes a secondary objective of this study. 
Accordingly ,we have constructed an examination schedule which is intended to provide comprehensive 
data regarding both the clinical response of the ischemic limb, as well as anatomic and functional evidence 
of collateral vessel development 
IV. E. Potential Side Effects 
1 . Pain and bruising at the site of catheter entry in the groin. 
2. Complications related to the catheterization such as rupture of an artery, infection, 
embolization, or allergic reactions to the contrast media used to position the catheter. 
3. VEGF is known to be made by transformed cells of certain tumors, presumably to develop 
a blood supply that will allow the tumor to grow further. Although an extensive examination will be 
performed to exclude the possibility of a previously unrecognized neoplasm, a microscopic growth could 
be present that might be missed by the battery of screening tests. It is therefore theoretically possible that 
VEGF resulting from gene transfer could possibly promote the development of a tumor that is currently 
too small to be recognized. It should be noted, however, that previous laboratory studies have established 
that VEGF expression, although sufficient to promote a growth process, did not lead to malignant 
proliferation or to metastasis 66 ? a finding in agreement with the notion that stimulation of angiogenesis is 
necessary but not sufficient for malignant growth 67. 
4. It is also theoretically possible that VEGF may aggravate deteriorating eyesight due to 
diabetes mellitus. The risk of this complication will presumably be eliminated by exclusion of patients 
withType I diabetes and/or an opthalmologic examination which discloses evidence of diabetic eye 
changes. 
IV.F. Potential Risks of Gene Transfer 
Even though we have attempted to minimize that risks associated with gene transfer by 
eliminating the need for any viral, liposomal or other vectors, it is recognized that theoretical risks of gene 
transfer remain. Previous studies suggest that intravenous administration of DNA in the absence of the 
viral vectors does not elicit an autoimmune response 68. in the case of DNA complexed with liposomes, 
systemic (intravenous) administration has been observed (by PCR) to result in occasional distribution to 
lung, kidney, spleen, and liver, but not testes or ovary 68. Furthermore, direct injection of naked plasmid 
DNA into multiple organs and tissues of rats, confirmed by PCR, has yielded evidence of gene expression 
only in muscle 63. Though the DNA to be transferred to the arterial wall is considered to be harmless, 
events could occur within normal cells that take up the foreign DNA that allow them to be transformed. 
Laboratory studies suggest that this is unlikely, paraticularly in the case of VEGF where Chinese hamster 
ovary cells transfected with this gene failed to display any criteria of transformation in vitro 66. We 
nevertheless acknowledge that transfected cells could theoretically become abnormal after long periods of 
time. 
IV. G. Risk-Benefit Analysis 
In the patient with rest pain, the major benefit we anticipate is abolition of rest pain. It is 
conceivable that in certain patients, the augmentation of collateral blood flow might be of sufficient 
magnitude to also allow the patient to walk some distance free of claudication. 
In the patient with an ischemic ulcer, healing of the ulcer constitutes the anticipated benefit. 
If healing is incomplete but facilitates placement of a skin graft which was otherwise not feasible, or if the 
level of amputation is reduced compared to that anticipated prior to gene therapy (e.g. trans-metatarsal vs. 
below-knee), these may represent incomplete but nevertheless favorable responses to VEGF gene therapy. 
On the basis of pre-clinical animal studies, we do not anticipate adverse consequences. 
Because the patients to be recruited for this study are by definition patients who are not 
candidates for revascularization, in the absence of treatment by recombinant DNA transfer deleterious 
consequences for which the patient is at risk include persistent rest pain, progressive loss of tissue 
integrity, and/or amputation. 
IV. H. Necropsy Analysis 
Necropsy examination will be requested in all cases of death during, and following arterial 
gene transfer. The site of gene transfer will be retrieved and analyzed by RT-PCR for evidence of gene 
Recombinant DNA Research, Volume 20 
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