system is <0.5%. As outlined above, additional in vitro and in vivo studies performed in our laboratory 
suggest that this low level of transfection efficiency may be sufficient to achieve a biologically meaningful 
effect when the transgene encodes for a secreted protein. 
II. EXPERIMENTAL DESIGN 
II. A. Objectives 
II. A. 1 Primary Objective. The primary objectives of this investigation are a) to 
determine the efficacy of arterial gene therapy using ph.VEGF to relieve rest pain and/or heal ischemic 
ulcers of the lower extremities in patients with PAD; and b) to document the safety of the ph.VEGF arterial 
gene therapy for therapeutic angiogenesis. 
II. A. 2 Secondary Objective. The secondary objective is to determine the anatomic 
and physiologic extent of collateral artery development in patients receiving ph.VEGF arterial gene 
therapy. 
II. B. Selection of Patients 
Patients will be selected for this protocol if they have rest pain and/or non-healing ischemic ulcers; and 
are not satisfactory candidates for non-surgical or surgical revascularization. As indicated above, the lack 
of available medical therapy for such patients implies that many of these patients may face amputation of a 
portion of their limb as the 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 as outlined below. It should also be noted in this regard that patients 
with critical leg ischemia - as defined in this study - have a poor prognosis in general, probably as the 
result of multifocal atherosclerosis 2; in a Scandanavian study of patients with rest pain alone, the mortality 
rate was 50% after 5 years 57. 
In our view, the consequences of PAD in the patient subset we have selected to study are sufficiently 
predictable to allow for meaningful assessment of the results of gene therapy. Rest pain has an unrelenting 
course; i.e. once the diagnosis has been established it will not resolve spontaneously. As a means of 
substantiating the otherwise subjective symptom of rest pain, a minimum- of four weeks or greater of 
dependence on narcotics will be required for patient selection. For non-healing ulcers, patients will be 
included only if a minimum of 8 weeks of conservative measures have failed to improve the appearance of 
the ulcer. Published and/or clinical experience would suggest that spontaneous improvement in an 
ischemic ulcer following this duration of conservative therapy is unlikely. 
We propose to recruit 12 patients for the first phase of this study. Our intention would be to seek 
permission to recruit additional patients after evaluating the safety and efficacy of therapy in this initial 
cohort. 
We initiated a prospective registry in our own institution approximately one year ago, designed to 
identify those patients referred to or otherwise seen by us who might be appropriate candidates for this 
protocol. During this time we prospectively identified 45 patients from our institution alone who would be 
eligible for this study. Thus, including patients from other institutions or geographic areas, the total 
number of patients whom we would anticipate being able to identify each year would be several times this 
number. 
II. B.l. Inclusion Criteria 
a. Sex. Both males and females will be eligible. 
b. Age. Patients must be 40 years or older. 
c. Child-bearing status. Patients must not be pregnant. Women in whom child- 
bearing capabilities are preserved must have a negative pregnancy test. Men and women in whom child- 
bearing capabilities are preserved must agree to employ barrier contraception for 3 months post-gene 
transfer. 
d. Symptoms. Patients will be candidates for this study if they have rest pain considered 
by at least two physicians to be typical of arterial insufficiency, with or without established ischemic ulcers 
(vide infra). Such pain is typically characterized as calf cramping, or a burning sensation involving the 
heel, toes, and sole of the foot, and is often relieved by assuming the upright position or placing the 
affected limb in the dependent position when supine. To reduce the likelihood that such pain represents 
acute exacerbation of limb ischemia, due, for example, to an embolic etiology, a minimum duration of four 
weeks of rest pain with dependence on narcotics with no improvement will be required for inclusion. 
e. Signs. Patients will be candidates for this study if they have established ulcerations or 
other non-healing lesions of the lower extremities which are judged by two physicians to be due to arterial 
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