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(Circulation. 1995;91:2687-2692.)
© 1995 American Heart Association, Inc.

Articles

Arterial Gene Therapy for Therapeutic Angiogenesis in Patients With Peripheral Artery Disease

Jeffrey M. Isner, MD, Principal Investigator; Kenneth Walsh, PhD, Co–Principal Investigator; James Symes, MD; Ann Pieczek, RN; Satoshi Takeshita, MD; Jason Lowry, BS; Susan Rossow, BS; Kenneth Rosenfield, MD; Lawrence Weir, PhD; Edi Brogi, MD; Robert Schainfeld, DO

From St Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Mass.

Correspondence to Jeffrey M. Isner, MD, St Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA 02135. E-mail jisner@opal.tufts.edu.

Key Words: genes • angiogenesis • peripheral vascular disease

	Background

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Peripheral Artery Disease: Primary Pharmacological Therapy Is Ineffective for Patients With Critical Limb Ischemia
The prognosis for patients with chronic critical leg ischemia, ie, rest pain and/or established lesions that jeopardize the integrity of the lower limbs, is often poor. Psychological testing of such patients has typically disclosed quality-of-life indexes similar to those of patients with cancer in critical or even terminal phases of their illness.¹ It has been estimated that in toto,² 150 000 patients per year require lower-limb amputations for ischemic disease in the United States. Their prognosis after amputation is even worse³ : the perioperative mortality for below-knee amputation in most series is 5% to 10% and for above-knee amputation 15% to 20%. Even when they survive, nearly 40% will have died within 2 years of their first major amputation; a major amputation is required in 30% of cases; and full mobility is achieved in only 50% of below-knee and 25% of above-knee amputees.

These grim statistics are compounded by the lack of efficacious drug therapy. As concluded in the Consensus Document of the European Working Group on Critical Leg Ischemia,³ ". . .there presently is inadequate evidence from published studies to support the routine use of primary pharmacological treatment in patients with critical leg ischemia. . . ." Evidence for the utility of medical therapy in the treatment of claudication is no better.^{4 5} Consequently, the need for alternative treatment strategies in such patients is compelling.

Therapeutic Angiogenesis Is a Novel Strategy for the Treatment of Critical Limb Ischemia
The therapeutic implications of angiogenic growth factors were identified by the pioneering work of Folkman⁶ and other workers more than two decades ago. Beginning a little more than a decade ago,⁷ a series of polypeptide growth factors were purified, sequenced, and demonstrated to be responsible for natural as well as pathological angiogenesis.

More recent investigations have established the feasibility of using recombinant formulations of such angiogenic growth factors to expedite and/or augment collateral artery development in animal models of myocardial and hindlimb ischemia. This novel strategy for the treatment of vascular insufficiency has been called therapeutic angiogenesis. The angiogenic growth factors first used for this purpose comprised members of the fibroblast growth factor (FGF) family. Baffour et al⁸ administered basic FGF (bFGF) in daily doses of 1 or 3 µg IM to rabbits with acute hindlimb ischemia; at the completion of 14 days of treatment, angiography and necropsy measurement of capillary density showed evidence of augmented collateral vessels in the lower limb compared with controls. Pu et al⁹ used acidic FGF (aFGF) to treat rabbits in which the acute effects of surgically induced hindlimb ischemia were allowed to subside for 10 days before a 10-day course of daily 4-mg IM injection was begun; at the completion of 30 days of follow-up, both angiographic and hemodynamic evidence of collateral development was superior to ischemic controls treated with IM saline. Yanagisawa-Miwa et al¹⁰ likewise demonstrated the feasibility of bFGF for salvage of infarcted myocardium, but in this case, growth factor was administered intra-arterially at the time of coronary occlusion, followed 6 hours later by a second intra-arterial bolus. More recently, administration of bFGF was shown to increase collateral flow¹¹ as well as improve myocardial function¹² in animal models of chronic myocardial ischemia.

We used a rabbit model of hindlimb ischemia¹³ to investigate the therapeutic potential of a 45-kD dimeric glycoprotein, vascular endothelial growth factor (VEGF), isolated initially as a heparin-binding factor secreted from bovine pituitary folliculostellate cells.¹⁴ VEGF was also purified independently as a tumor-secreted factor that induced vascular permeability by the Miles assay^{15 16} ; thus its alternate designation, vascular permeability factor. Two features distinguish VEGF from other heparin-binding, angiogenic growth factors. First, the NH₂ terminus of VEGF is preceded by a typical signal sequence; therefore, unlike bFGF, VEGF can be secreted by intact cells.¹⁷ Second, its high-affinity binding sites, shown to include the tyrosine kinase receptors Flt1¹⁸ and Flk1/KDR,^{19 20} are present on endothelial cells but not other cell types; consequently, the mitogenic effects of VEGF—in contrast to aFGF and bFGF, both of which are known to be mitogenic for smooth muscle cells^{21 22} and fibroblasts as well as endothelial cells—are limited to endothelial cells.^{14 23}

We considered the fact that the VEGF gene encodes a secretory signal sequence that might be exploited as part of a strategy designed to accomplish therapeutic angiogenesis by arterial gene transfer. We had previously used the plasmid pXGH5 encoding the gene for human growth hormone, a secreted protein, to transfect rabbit aortic rings in vitro²⁴ and rabbit ear arteries in vivo²⁵ and obtained physiological levels of human growth hormone, even though immunohistochemical examination of the transfected tissue disclosed evidence of successful transfection in <1% of cells in the transfected arterial segment. Thus, gene products that are secreted may have profound biological effects, even when the number of transduced cells remains low. In contrast, for genes such as bFGF that 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 µg of phVEGF₁₆₅, encoding the 165–amino acid isoform of VEGF, to the hydrogel polymer outside coating of an angioplasty balloon^{26 27} 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 phVEGF₁₆₅ was confirmed by analysis of the transfected internal iliac arteries using reverse transcriptase–polymerase chain reaction (RT-PCR) 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 to normal limb) to 0.70±0.08 in the VEGF-transfected group versus 0.50±0.18 in controls (P<.05). These findings²⁸ 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 similarly encouraging results achieved in our laboratory with administration of the recombinant protein in the same animal model,^{29 30 31 32} we believe that transferring the gene encoding that protein (ie, gene therapy) is preferable, for two reasons. First, 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. Principally because of 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, to the best of our knowledge, is currently planning a clinical trial of recombinant protein therapy with VEGF.

Second is the potential requirement to maintain an optimally high and local concentration over time. In the case of therapeutic angiogenesis, for example, it may be preferable to deliver a lower dose over a period of several days or more from an actively expressing transgene in the circulation of the ischemic limb, rather than a single or multiple bolus doses of systemically recirculating recombinant protein. It is conceivable, although as yet unproven, that such continuous, local production of VEGF resulting from the transgene may be preferable, from the standpoints of both safety and bioactivity, to a single, larger dose of the recombinant protein administered by any (intra-arterial, intravenous, intramuscular) route.

	Experimental Design

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Objectives
Primary Objective
The primary objective of this investigation is to document the safety of the phVEGF₁₆₅ arterial gene therapy for therapeutic angiogenesis.

Secondary Objectives
The secondary objectives are (1) to determine the bioactivity of arterial gene therapy using phVEGF₁₆₅ to relieve rest pain and/or heal ischemic ulcers of the lower extremities in patients with peripheral artery disease and (2) to determine the anatomic and physiological extent of collateral artery development in patients receiving phVEGF₁₆₅ arterial gene therapy.

Selection of Patients
Patients will be selected for this protocol if they have rest pain and/or nonhealing ischemic ulcers and are not satisfactory candidates for nonsurgical or surgical revascularization. The lack of available medical therapy for patients with critical limb ischemia 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.

Patients will be considered to be "not satisfactory" candidates for nonsurgical revascularization (and therefore appropriate candidates for arterial gene therapy) if the lesions that require revascularization to restore pulsatile flow to the foot are classified as category 4 according to the Society of Cardiovascular and Interventional Radiology classification.³³

With regard to surgical revascularization, no such uniform, standardized classification system intended to define a patient's operative candidacy is available to draw on. Accordingly, we prefer to define patients who are satisfactory candidates for surgery, since there is typically more consensus regarding this characterization. Patients will therefore be considered to be satisfactory candidates for surgery if selective, digital-subtraction angiographic examination successfully identifies acceptable distal runoff, including angiographic evidence of at least one lower limb vessel that is of sufficient caliber and distal reconstitution to serve as a recipient graft site; if preoperative clinical assessment discloses no comorbid illness that would make the patient an unacceptably high risk for reconstructive surgery; and, in the event that surgical reconstruction requires distal bypass, if a sufficient length of autologous vein required to serve as the bypass conduit is available. Thus, patients will be considered candidates for the current gene therapy protocol if they do not satisfy these criteria or if a reconstructive procedure (at the same level) has already been attempted and failed.

The consequences of peripheral artery disease in the patient subset we have selected to study are sufficiently predictable to allow meaningful assessment of the results of gene therapy. Rest pain has an unrelenting course; ie, once the diagnosis has been established, it will not resolve spontaneously. For rest pain, a minimum of 4 weeks of dependence on narcotics will be required for patient selection. For nonhealing ulcers, patients will be included only if a minimum of 4 weeks of conservative measures has failed to improve the appearance of the ulcer. Published and/or clinical experience would suggest that spontaneous improvement in either rest pain and/or an ischemic ulcer following this duration of conservative therapy is unlikely.³⁴

We will recruit a total of 22 patients for this study; these patients will receive escalating doses of phVEGF₁₆₅ according to a schedule that will allow us to progressively achieve the primary objective—documentation of the safety of phVEGF₁₆₅ arterial gene therapy. As indicated below, the two initial patients will each receive 100 µg of the plasmid DNA, while the next two patients will each receive 300 µg. Provided that this proves safe, we then propose to treat subsequent groups of six patients each with escalating doses of 1000, 2000, and 4000 µg, respectively. In addition to documenting the safety of each incremental dose, a series of noninvasive and invasive tests designed to achieve the secondary objectives of this protocol—determination of bioactivity, including evidence of angiogenesis—will be performed as well.

While consideration has been given to the issue of a control group, the requirement for catheter manipulation in patients with marginal limb perfusion and extensive atherosclerosis mitigates against this option. For the patient undergoing gene transfer, we believe the risks associated with catheterization are offset by the potential relief from unremitting rest pain or healing of refractory ulcers; for the patient undergoing a sham transfection, the small risk is not offset by any potential benefit. The physician is thus placed in the uncomfortable position of persuading a patient and that patient's physician and family that the patient should undergo the inconvenience, discomfort, and small risk of an intervention from which the patient will derive no conceivable benefit.

Inclusion Criteria
Both men and women 40 years old are eligible. Patients must not be pregnant; if child-bearing capabilities are preserved, patients must agree to use barrier contraception for 3 months after gene transfer. Symptoms include rest pain typical of arterial insufficiency, with or without established ischemic ulcers (see below). A minimum of 4 weeks of rest pain with dependence on narcotics with no improvement will be required. Signs include nonhealing lesions of the lower extremities due to arterial insufficiency. A minimum of 4 weeks of conservative therapy without evidence of significant healing will be required.

Noninvasive findings will be as follows. (1) Resting ankle-brachial index in the affected limb must be <0.6 on two consecutive examinations performed at least 1 week apart. For patients with noncompressible ankle arteries (due to calcific deposits with or without associated diabetes), the great toe index must be <0.6. (2) Rest pain and/or ischemic ulcers may preclude exercise testing (graded-load protocol³⁵ ) in most patients. Each patient, however, will be asked to exercise; if the patient is unable to do so, this will be noted. The ability to successfully complete any portion of any exercise protocol after treatment will be evaluated in the event that rest pain is eliminated and/or ulcerations healed.

Diagnostic angiography must demonstrate occlusion in the affected limb of one or more of the following: the iliac, superficial femoral, popliteal, and/or one or more infrapopliteal arteries. The site of occlusion and associated anatomic findings must be such that selective arterial transfection is technically feasible.

Patients will be included only if they agree to and are judged appropriate to discontinue concomitant prostaglandin therapy and/or therapy with vasodilators, dextran, hetastarch, pentoxifylline, L-carnitine, and/or hyperbaric oxygen. Patients may continue to receive aspirin and coumarin, provided that these therapies have been used by the patient for a minimum of 6 months before entry into the study.

Exclusion Criteria
Exclusion criteria include (1) aortic or lower-extremity arterial surgery, angioplasty, or lumbar sympathectomy within 2 months; (2) radiographic and radioisotopic evidence of concomitant osteomyelitis in the ischemic extremity; (3) any concomitant disease process with a life expectancy of <1 year or sufficiently severe as to compromise clinical follow-up examinations; (4) significant history of alcohol or drug abuse within the past 3 months; (5) previous or current history of neoplasm; (6) clinically significant abnormality in liver function or other laboratory tests, including prostate-specific antigen and carcinoembryonic antigen, and/or signs by chest radiograph, abdominal CT scan, mammography in the case of women or prostate examination in the case of men of malignant neoplasm; (7) clinical evidence of type I diabetes mellitus, diabetic retinopathy, and/or other ophthalmologic complications of diabetes; (8) patients who are pregnant or refuse to use barrier contraception; and (9) refusal or inability to give informed consent.

	Construction of Plasmid

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Plasmid Structure
The cDNA to be used in this protocol encodes the 165–amino acid isoform of VEGF and has been described previously.^{17 36}

The plasmid into which the VEGF cDNA has been inserted, phVEGF₁₆₅, is a simple eukaryotic expression plasmid that uses the 763–base-pair cytomegalovirus (CMV) promoter/enhancer to drive VEGF expression. This promoter/enhancer has been used to express reporter genes in a variety of cell types and can be considered to be constitutive. Downstream from the VEGF cDNA is the SV40 polyadenylation sequence. Also included in this plasmid is a fragment containing the SV40 origin of replication that includes the 72-bp repeat, but this sequence is not functionally relevant (for autonomous replication) in the absence of SV40 T antigen. These fragments occur in the pUC118 vector, which includes an Escherichia coli origin of replication and the ß-lactamase gene for ampicillin resistance.

To confirm plasmid identity, the entire phVEGF₁₆₅ sequence (5651 bp) was determined (designated VEGF V1-V2) by use of 24 sequencing primers. This determined sequence was compared with the deduced sequence (designated 1. VEGF). The structure of the double-stranded DNA was determined by the cycle sequencing method using fluorescent dideoxy terminator nucleotides with an Applied Biosystem 373A automated sequencer. Sequences were analyzed on Macintosh Quadra computers with MacVector and Sequence Navigator software. The quality control for this sequencing analysis consists of parallel sequence analyses of Bluescript and M13 controls. Our sequencing found three regions that were missing from the predicted sequence: (1) -4 bp (of a repeat) at the junction of the SV40 early polyA and origin sequences; (2) -35 bp within the M13 region; and (3) -14 bp at the junction between M13 and pUC. Outside these regions, there was >98.2% sequence homology between the determined and predicted sequences.

Vectors
No vectors will be used to deliver the above-described plasmid. Instead, the plasmid will be applied to the hydrogel polymer coating of a standard angioplasty balloon, as was the case in our preclinical animal testing.^{26 28} The catheter and method used for this delivery mode are also described in detail below.

Plasmid Preparation
The plasmid DNA will be prepared at the time of gene transfer in the St Elizabeth's Medical Center's human gene therapy laboratory.

DNA will be prepared from cultures of phVEGF₁₆₅-transformed E. coli by the Qiagen method according to the directions of the manufacturer (Qiagen, Inc). Briefly, cultures will be grown from master glycerol stock in 500 mL of LB medium with 100 µg/mL ampicillin. Cells will be harvested at a density of 1.0 to 1.5 absorbance at 600 nm (A₆₀₀) U/mL and prepared with a Qiagen-tip 2500. After elution from the Qiagen column, the plasmid DNA will be precipitated with alcohol, dried on a Speed Vac, and stored in vials, reconstituted in sterile saline. We anticipate yields of 1.5 to 2.5 mg of plasmid DNA. These plasmid batches will be pooled before further analyses and, ultimately, administration to patients.

The sealed vials containing aliquots of the plasmid DNA pool will be stored in a dedicated storage freezer/refrigerator in the human gene therapy laboratory. Approximately 1 hour before delivery, plasmid solution will be divided into aliquots in individual sterile vials mixed under sterile conditions as follows: 0.1 mL of plasmid DNA (5 mg/mL) is diluted to 250 µL with sterile water at room temperature. Approximately 15 minutes before delivery, vials of plasmid will be brought to the special procedures laboratory and, under sterile conditions, applied by pipette to the hydrogel polymer coating of the inflated angioplasty balloon, after which the balloon will be allowed to air-dry.

	Stages of Treatment

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Pretreatment Studies
The Table provides a schedule of pre–gene transfer studies.

View this table: [in this window] [in a new window]   Table 1. Schedule of Examinations for Year 1 Pre– and Post–Gene Transfer

Treatment
Gene transfer itself will be performed percutaneously in a fashion identical to that used for the preclinical animal studies. If the profunda is the intended site of gene transfer, then a retrograde, wire-guided approach from the contralateral common femoral artery will be used, according to standard techniques, to advance the angioplasty catheter/sheath to the site of gene transfer under fluoroscopic guidance. Once positioned, the sheath will be withdrawn and the balloon inflated for 5 minutes at nominal inflation pressures, after which the balloon will be deflated, drawn back into the sheath, and removed. An angiogram will then be recorded to document the anatomy and collateral circulation at the time of gene transfer and to confirm that no vessel trauma has occurred.

If the distal superficial femoral artery or popliteal arteries represent the site of gene transfer, the approach will be antegrade and ipsilateral but otherwise identical.

Posttreatment Studies
See the Table for a schedule of post–gene transfer studies.

Criteria for Response
The primary clinical end points that 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.

With regard to the secondary objective of this study, noninvasive and invasive studies are intended to provide comprehensive data regarding both the clinical response of the ischemic limb and anatomic and functional evidence of collateral vessel development.

Potential Side Effects
1. 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 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,³⁷ a finding in agreement with the notion that stimulation of angiogenesis is necessary but not sufficient for malignant growth.³⁸

2. It is also theoretically possible that VEGF may aggravate deteriorating eyesight due to diabetes mellitus.³⁹ The risk of this complication will presumably be reduced by exclusion of patients with type I diabetes and/or an ophthalmologic examination that discloses evidence of diabetic eye changes.

Potential Risks of Gene Transfer
Even though we have attempted to minimize the 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.⁴⁰ This empirical experience is consistent with the observation that double-stranded DNA is minimally antigenic because the bases are in essence "shielded" from immunologic surveillance by the double helices.⁴¹ Administration of supercoiled double-stranded DNA is thus unlikely to elicit an immunologic response. DNA that is subsequently denatured is likely to be removed by the reticuloendothelial system. It is further worth noting that recent studies suggest that histones may account for a significant proportion of the antigenicity associated with DNA⁴² ; in the case of plasmids generated from E. coli, however, histones are absent from the recombinant DNA, thus further reducing the likelihood of an immunologic response.

	Acknowledgments

 
The authors gratefully acknowledge the assistance of Mickey Neely in the preparation of the manuscript, and Dr Joachim Schorr (Qiagen GmbH, Hilden, Germany) for helpful discussions and aid in determining plasmid quality.

	Footnotes

 
This article is an abridged version of a protocol approved by the Food and Drug Administration, the Recombinant DNA Advisory Committee of the National Institutes of Health, and the principal investigator, co–principal investigator, and coinvestigators.

Received December 7, 1994; accepted December 28, 1994.

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Top Background Experimental Design Construction of Plasmid Stages of Treatment References

 

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