Knowledge in the field of gene transfer to mammalian somatic cells is increasing, and a protocol for a pilot of gene transfer for the study of human disease has recently been approved. Major questions exist about effective ways to insure that products of transferred genes reach target organs to correct pathologic deficiencies. We propose to test three hypotheses regarding the role of genetically modified vascular endothelial cells (GMEC) as carriers for transferred genes, and as sources of gene product, in a model for development of a therapeutically applicable gene therapy delivery system. Hypothesis 1 That autologous canine external jugular vein endothelial cells (CEJVEC), transduced with retroviral vectors contain the gene sequence for human preproparathyroid hormone, synthesize and secrete human parathyroid hormone (hPTH) in cell culture and continue to do so after seeding on vascular grafts and subsequent implantation in canine carotid arteries. We will transfer genes into CEJVEC with either of two recombinant retroviral vectors: pMSV-hPTH-gpt, or pBGCP. Selection with mycophenolic acid (gpt) or by fluorescence-activated cell sorting (lacZ in pBGCP) will produce cultures containing high percentages of genetically modified cells. Hormones and related peptides secreted by GMEC will be characterized from culture medium by amino acid sequence analysis. Production of hPTH by cultures of GMEC will be calculated for each vector. GMEC seeded on vascular grafts will be reharvested after graft explantation, and characterized for endothelial-specific cytochemical markers, for gene transfer markers and for hPTH production in culture. Hypothesis 2 That the hPTH synthesized and secreted by transduced autologous CEJVEC exhibits biological activity in vitro and in vivo. hPTH isolated from culture medium will be assayed for its ability to stimulate adenylate-cyclase-coupled receptors for PTH on the surface of cultured ROS 17/2.8 osteosarcoma cells. In vivo activity of autologous GMEC culture medium will be evaluated by its ability to correct hypocalcemia in parathyroidectomized dogs. Vascular grafts seeded with GMEC will also be evaluated for the serum levels of hPTH they produce, and for their ability to correct surgically created hypocalcemia. Hypothesis 3 That the amount of gene product produced in vivo can be amplified by increasing the capacity of a carrier system from a single vascular tube with limited surface area to a device of greatly increased surface area such as a hollow fiber array. We will use the increased surface area to maximize the number of GMEC available for hPTH production. Conditions for optimum growth of GMEC on fiber material will be investigated. In anticipation of vascular implants, we will measure hPTH serum levels produced by GMEC-seeded devices in hypocalcemic dogs, using an ex vivo arteriovenous shunt preparation. Correction of hypocalcemia by an ex vivo, GMEC-seeded device will also be evaluated.
| Choi, E T; Callow, A D; Sehgal, N L et al. (1995) Halofuginone, a specific collagen type I inhibitor, reduces anastomotic intimal hyperplasia. Arch Surg 130:257-61 |
| Callow, A D; Choi, E T; Trachtenberg, J D et al. (1994) Vascular permeability factor accelerates endothelial regrowth following balloon angioplasty. Growth Factors 10:223-8 |
| Kamesaki, H; Zwiebel, J A; Reed, J C et al. (1994) Role of bcl-2 and IL-5 in the regulation of anti-IgM-induced growth arrest and apoptosis in immature B cell lines. A cooperative regulation model for B cell clonal deletion. J Immunol 152:3294-305 |
| Choi, E T; Engel, L; Callow, A D et al. (1994) Inhibition of neointimal hyperplasia by blocking alpha V beta 3 integrin with a small peptide antagonist GpenGRGDSPCA. J Vasc Surg 19:125-34 |
