THE LANCET 370 Vol 348 • August 10, 1996 Summary Background Preclinical findings suggest that intra-arterial gene transfer of a plasmid which encodes for vascular endothelial growth factor (VEGF) can improve blood supply to the ischaemic limb. We have used the method in a patient. Methods Our patient was the eighth in a dose-ranging series. She was aged 71 with an ischaemic right leg. We administered 2000 g human plasmid phVEGF 165 that was applied to the hydrogel polymer coating of an angioplasty balloon. By inflating the balloon, plasmid DNA was transferred to the distal popliteal artery. Findings Digital subtraction angiography 4 weeks after gene therapy showed an increase in collateral vessels at the knee, mid-tibial, and ankle levels, which persisted at a 12-week view. Intra-arterial doppler-flow studies showed increased resting and maximum flows (by 82% and 72%, respectively). Three spider angiomas developed on the right foot/ ankle about a week after gene transfer; one lesion was excised and revealed proliferative endothelium, the other two regressed. The patient developed oedema in her right leg, which was treated successfully. Interpretation Administration of endothelial cell mitogens promotes angiogenesis in patients with limb ischaemia. Lancet 1996; 348: 370–74 Introduction Among the growth factors that promote angiogenesis, vascular endothelial growth factor (VEGF), 2 also known as vascular permeability factor, 3 and vasculotropin, 4 is specifically mitogenic for endothelial cells. The first exon of the VEGF gene includes a secretory signal sequence that permits the protein to be secreted naturally from intact cells. 5 We have shown 6,7 that arterial gene transfer of naked DNA encoding for secreted protein yielded physiological levels of protein despite low transfection efficiency. Site-specific gene transfer of plasmid DNA encoding the 165-aminoacid isoform of human VEGF (phVEGF 165 ) applied to the hydrogel polymer coating of an angioplasty balloon, 8 and delivered percutaneously to the iliac artery of rabbits in which the femoral artery had been excised to cause unilateral hindlimb ischaemia led to development of collateral vessels and increased capillary density, improved calf blood-pressure ratio (ischaemic/normal limb) and increased resting and maximum vasodilator-induced blood flow. 9,10 We now use this strategy in the ischaemic limb of a patient. Patient and methods Patient A 70-year-old non-diabetic woman was referred for gangrene of the right great toe. About a year earlier, the patient had cramping right-foot pain; several corns were removed, she was given intramuscular cortisone, prescribed ibuprofen, and fitted with shoe inserts. Symptoms worsened and the patient received oxycodone, hydrocodone, and a fentanyl patch. The great toe lesion progressed to gangrene, and the second and third toes became compromised. She had no palpable pedal pulses of the right limb. Ankle-brachial index of the ischaemic limb was 0·26. Arteriography revealed a 40% stenosis of the proximal popliteal artery, and occlusion of the peroneal, anterior tibial, and posterior tibial arteries midway to the foot. Surgical exploration of the distal right limb failed to identify a suitable site for a bypass. The patient was suitable for arterial gene therapy according to a protocol 11 approved by the Human Institutional Review Board and Institutional Biosafety Committee of our centre, as well as the Recombinant DNA Advisory Committee of the National Institutes of Health and the US Food and Drug Administration. Plasmid DNA phVEGF 165 consists of a eucaryotic PUC 118 expression vector into which cDNA encoding the 165-aminoacid isoform of VEGF has been inserted. 11 A 763 basepair cytomegalovirus promoter/enhancer is used to drive VEGF expression. The PUC 118 vector includes an SV40 polyadenylation sequence, an Escherichia coli origin of replication, and the -lactamase gene for ampicillin resistance. The plasmid was prepared in the Human Gene Therapy Laboratory at our centre from cultures of phVEGF 165 -transformed E coli, purified with a Qiagen-tip 2500 column, precipitated with isopropanol, washed with 70% ethanol, and dried on a Speed Vac. The purified plasmid was reconstituted in sterile saline, stored in vials, and pooled for quality control analyses (absorbance at wavelengths of 260 and 280 mm to document ratio between 1·75 and 1·85; limulus amoebocyte lysate gel-clot assay [BioWhittaker] to establish bacterial endotoxin levels below 5 endotoxin units per kg bodyweight; microbial cultures; southern blot for level of contaminating genomic E coli DNA; and ethidium bromide staining after agarose-gel electrophoresis to confirm that over 90% of the nucleic acid was in the closed, circular supercoiled form). To confirm the identity of the prepared plasmid, the VEGF-coding region from each pooled batch was resequenced (Applied Biosystem 373A). Percutanous arterial gene transfer Arterial gene transfer was done with a hydrogel-coated balloon- angioplasty-catheter (Boston Scientific). 8 A sterile pipette was used to apply 2000 g plasmid DNA at 10·3 g/L in 194·2 L Departments of Medicine, Biomedical Research, Radiology, and Surgery, St Elizabeth’s M edical Center, Tufts University School of Medicine, Boston, Massachusetts, USA (Jeffrey M Isner MD, Ann Pieczek RN, Robert Schainfeld DO, Richard Blair MD, Laura Haley BS, Takayuki Asahara MD, Kenneth Rosenfield MD, Syed Razvi MD, Kenneth Walsh PhD, James F Symes MD) Correspondence to: Dr Jeffrey M Isner, St Elizabeth’s Medical Center, Boston, MA 02135, USA Clinical evidence of angiogenesis after arterial gene transfer of phVEGF 165 in patient with ischaemic limb Jeffrey M Isner, Ann Pieczek, Robert Schainfeld, Richard Blair, Laura Haley, Takayuki Asahara, Kenneth Rosenfield, Syed Razvi, Kenneth Walsh, James F Symes Early reports