Appropriate control of ex vivo gene therapy delivering basic fibroblast growth factor promotes successful and safe development of collateral vessels in rabbit model of hind limb ischemia

Abstract

These findings suggest that 5 x 10(6) is a suitable number of cells to induce appropriate collateral vessel development and minimize potential side effects of this procedure. Despite use of ex vivo gene transfer, repeat administration of the cells was not feasible. Clinical relevance Since the present study determined the appropriate conditions for effective and safe stimulation of collateral vessels, the clinical relevance of the ex vivo therapy might be carried forward. However, the findings raised another issue that should be resolved before clinical application; that is, the number of gene-transduced cells able to be injected was strictly limited. To estimate the therapeutic range of cell number in humans, additional experiments using large animals are desirable.

Vascular Endothelial Growth Factor Gene Delivery by Magnetic DNA Nanospheres Ameliorates Limb Ischemia in Rabbits1

“…In the present study, the superficial femoral artery, including all its branches, was excised completely to create the limb ischemia model in rabbits, resulting in delayed spontaneous development of collateral and extensive necrosis of the hindlimbs compared with some published results, in which ligation of femoral artery [41], ligation of common iliac artery [11], or excision of femoral artery [42,43] was used to create the limb ischemia model and no visible necrosis was observed. Our results are in accord with one study [44] in which a similar method was used to establish the limb ischemia model, although some animals had to be amputated because of extensive necrosis.…”

Ex vivo gene delivery of ephrin-B2 induces development of functional collateral vessels in a rabbit model of hind limb ischemia

“…Male Japanese White rabbits weighing 2.7 to 3.4 Kg (Saitama Rabbitry, Tokyo, Japan) were anesthetized by an intramuscular injection of a mixture of ketamine (50 mg/ Kg) and xylazine (2.5 mg/Kg), and the left femoral artery was completely removed from its proximal origin to the bifurcation formed by the saphenous and popliteal arteries. 7,9,10 Before the wound was closed, a 10-ϫ 10-mm skin section was obtained for primary culture of fibroblasts.…”

“…6 We previously presented a new therapeutic strategy to achieve pinpoint delivery of an angiogenic growth factor to the peripheral vascular network of the donor artery, in which autofibroblasts, adenovirally transduced with an angiogenic growth factor gene, were selectively injected into the donor artery for blood inflow (ex vivo method). [7][8][9][10] Because the fibroblast is not a blood cell, the gene-transduced fibroblasts were mechanically trapped in peripheral small vessels of the donor artery after injection and released the growth factor for a certain period. In our previous studies, the gene expressing basic fibroblast growth factor (bFGF), a potent promoter of arteriogenesis, 3,11 was delivered by an ex vivo method in animal models of limb 7,9,10 and myocardial ischemia, 8 and favorable development of functional collateral vessels was augmented.…”

“…[7][8][9][10] Because the fibroblast is not a blood cell, the gene-transduced fibroblasts were mechanically trapped in peripheral small vessels of the donor artery after injection and released the growth factor for a certain period. In our previous studies, the gene expressing basic fibroblast growth factor (bFGF), a potent promoter of arteriogenesis, 3,11 was delivered by an ex vivo method in animal models of limb 7,9,10 and myocardial ischemia, 8 and favorable development of functional collateral vessels was augmented. Although the ex vivo method might be rather complex for clinical treatment, this method enables the delivery of membrane-bound ligands in the native form because auto cells were used as a delivery carrier, which might be a marked advantage compared with other delivery methods.…”

Gene Therapy for Fracture Repair

“…Its extracellular secretion is mediated by a highly inefficient, energy-dependent, non-ER/golgi pathway (Florkiewicz et al, 1995) and, as a result, the amount of secreted FGF-2 by this mechanism is low and inherently inconsistent (Moscatelli et al, 1986). These problems are largely responsible for the inconsistent efficacy of past FGF-2 gene therapy studies (Spencer et al, 2001;Hijjawi et al, 2004;Ishii et al, 2004). Accordingly, the human FGF-2 gene within our MLV-based vectors has been modified by: 1) adding the BMP2/4 hybrid secretion signaling sequence to the 5' end of the gene to enhance the transgene secretion; 2) adding an optimized Kozak sequence to promote protein translation; and 3) mutating two key cysteines (cys-70 and cys-88) to serine and asparagine respectively, to enhance protein stability.…”