Phase Ib Safety, Two-Dose Study of MultiGeneAngio in Patients with Chronic Critical Limb Ischemia.

Critical limb ischemia (CLI) is the most severe presentation of peripheral arterial disease. We developed cell-based therapy entailing intra-arterial injection of autologous venous endothelial cells (ECs) modified to express angiopoietin 1, combined with autologous venous smooth muscle cells (SMCs) modified to express vascular endothelial growth factor. This combination promoted arteriogenesis in animal models and was safe in patients with limiting claudication. In an open-label, phase Ib study, we assessed the safety and efficacy of this therapy in CLI patients who failed or were unsuitable for surgery or intravascular intervention. Of 23 patients enrolled, 18 with rest pain or non-healing ulcers (Rutherford categories 4 and 5) were treated according to protocol, and 5 with significant tissue loss (Rutherford 6) were treated under compassionate treatment. Patients were assigned randomly to receive 1 × 107 or 5 × 107 (EC-to-SMC ratio, 1:1) of the cell combination. One-year amputation-free survival rate was 72% (13/18) for Rutherford 4 and 5 patients; all 5 patients with Rutherford 6 underwent amputation. Of the 12 with unhealing ulcers at dosing, 6 had complete healing and 2 others had >66% reduction in ulcer size. Outcomes did not differ between the dose groups. No severe adverse events were observed related to the therapy.

Phase I study of multi-gene cell therapy in patients with peripheral artery disease.

UNLABELLED: Alternative treatment strategies for claudication are needed and cell-based therapies designed to induce angiogenesis are promising. The purpose of this report was to conduct a Phase I safety, dose-escalating, non-randomized, open-label study of autologous, fully differentiated venous endothelial and smooth muscle cells called MultiGeneAngio (MGA) for claudication due to peripheral artery disease. Twelve subjects, at two centers, received a single intra-arterial infusion of a suspension of equal amounts of transduced autologous venous smooth muscle cells expressing vascular endothelial growth factor (VEGF165) and endothelial cells expressing angiopoietin-1 (Ang-1) (Cohort 1: 1 × 10(7), Cohort 2: 2 × 10(7), Cohort 3: 5 × 10(7), Cohort 4: 7 × 10(7)). The treatment was given unblinded and in the more symptomatic lower extremity. Transduced cells were tested for in vitro doubling time, telomerase activity, and gene expression. The main outcomes were clinical safety and tolerability. Other safety measures included ankle-brachial index (ABI) and walking time on a treadmill. All subjects were male (mean age 60 ± 5 years) including 25% with diabetes mellitus. At 1-year follow-up, there was one serious adverse event possibly related to MGA. Safety endpoints including VEGF and Ang-1 plasma protein levels were within normal ranges in all subjects. The mean maximal walking time increased from baseline to 1 year and the index limb ABI was unchanged, indicating no safety concerns. MGA, an autologous, transduced, cell-based therapy was well tolerated and safe in this Phase I study. Further evaluation is warranted in randomized human studies. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00390767.

Endothelial cells are activated by angiopoeitin-1 gene transfer and produce coordinated sprouting in vitro and arteriogenesis in vivo.

RATIONAL AND OBJECTIVES: Activation of fully differentiated vascular cells using angiogenic genes can lead to phenotypic changes resulting in formation of new blood vessels. We tested whether Ang-1 gene transfer to endothelial cells (EC) activates these cells. METHODS AND RESULTS: EC and SMC were transduced using retroviral or adenoviral vectors to produce Ang-1 or vascular endothelial growth factor (VEGF). EC Tie-2 receptor was phosphorilated by autologous secretion of Ang-1. Transduced EC and SMC sprouting capacity was tested using collagen embedded spheroids assay and capacity to produce arteriogenesis was tested in a hind limb model of ischemia. EC expressing Ang-1 in the presence of SMC expressing VEGF exhibited high levels of sprouting of the two cell types. Flow and numbers of arteries were increased after transduced cells implantation in vivo. CONCLUSIONS: Autologous secretion of Ang-1 by transduced EC resulted in Tie-2 activation and in the presence of SMC expressing VEGF resulted in coordinated sprouting in vitro and increase in flow and number of arteries in vivo.

Efficient transduction and seeding of human endothelial cells onto metallic stents using bicistronic pseudo-typed retroviral vectors encoding vascular endothelial growth factor.

BACKGROUND: Stents seeded with genetically modified endothelial cells (EC) may provide an attractive therapeutic modality for treating vascular diseases by combining the mechanical properties of the metallic stent with the biologic activity of native or genetically engineered ECs. The clinical feasibility of implanting seeded stents depends on the ability to achieve adequate stent coverage within a clinically applicable time frame. We tested the hypothesis that this goal could be achieved by seeding stents with human ECs overexpressing vascular endothelial growth factor (VEGF) and by using an efficient gene transfer system. METHODS AND RESULTS: Efficiency of gene transfer to human ECs using an amphotropic retroviral vector and a gibbon ape leukemia virus (GALV) pseudo-typed retroviral vector was examined and compared. For assessment of transduction rates, LacZ-encoding vectors were used and beta-galactosidase activity was determined 48 h after gene transfer. The transduction rate of primary human ECs using the amphotropic retroviral vector encoding the LacZ gene was low (2.9+/-2% of cells). Under the same conditions, the GALV pseudo-typed vector encoding LacZ transduced 94+/-2% of cells (P<.001). To test the effect of VEGF gene transfer on stent coverage, we transduced ECs using a bicistronic GALV pseudo-typed retroviral vector encoding either GFP alone or both VEGF and GFP. Since all transduced cells expressed GFP, stent coverage by ECs could be assessed by fluorescent inverted microscopy, which demonstrated that stent coverage by ECs overexpressing VEGF was more rapid and effective than coverage by ECs overexpressing GFP. Progressively increasing quantities of VEGF protein were detected in the conditioned medium of stents seeded with endothelia cells expressing VEGF 2, 3, and 5 days after seeding. CONCLUSIONS: High-rate gene transfer to human primary ECs was observed 48 h after transduction with GALV pseudo-typed retroviral vectors, eliminating the need for the time-consuming process of cell selection. Seeding with ECs overexpressing VEGF improved stent coverage and was associated with continuing secretion of the protein. The findings provide support for the feasibility of implanting genetically engineered biologically active cellular-coated stents.

Viral vectors for vascular gene therapy.

Vascular gene therapy is the focus of multiple experimental and clinical research efforts. While several genes with therapeutic potential have been identified, the best method of gene delivery is unknown. Viral vectors have the capacity to transfer genes at high efficiency rates. Several viral-based vectors have been used in experimental vascular gene therapy for in vivo and ex vivo gene transfer. Adenoviral-based vectors are being used for the induction of angiogenesis in phase 1 and 2 clinical trials. In the present review, the characteristics of the 'ideal' viral vector are discussed and the major types of viral vectors used in vascular gene transfer are reviewed. Basic knowledge of the use of viral vectors for direct in vivo gene transfer (adenoviral-based vectors, etc) and for ex vivo gene transfer (retroviral-based vectors) is provided. New developments in the field of viral vectorology, such as pseudotyping of retroviral vectors and targeting of other viral vectors to a specific cell type, will enhance the more rapid transition of vascular gene therapy from the experimental arena to the clinical setting.