Abstract:Venous bypass grafts are useful treatments for obstructive coronary artery disease. However, their usefulness is limited by accelerated atherosclerosis. Genetic engineering of venous bypass grafts that prevented atherosclerosis could improve long-term graft patency and clinical outcomes. We used a rabbit model of jugular vein-to-carotid interposition grafting to develop gene therapy for vein-graft atherosclerosis. Rabbit veins were easily transduced in situ with a first-generation adenoviral vector; however, m… Show more
“…We delayed transduction until 4 weeks after grafting because transduction at the time of grafting leads to rapid loss of transgene expression, whereas delayed transduction yields durable transgene expression. 26 Figure 1 Study Protocols (A) Vein graft atherosclerosis study. Rabbits were begun on a 0.3% cholesterol diet.…”
Section: Resultsmentioning
confidence: 99%
“…In our previous work and in results reported by others, incubation of adenovirus in a vein lumen followed by grafting of the vein results in transgene expression that is largely confined to luminal endothelial cells. 26 , 38 , 39 , 40 , 41 Other groups have reported transgene expression in the venous media and adventitia, but this may be due to variability in technique. 42 , 43 , 44 Based on our previous work with vein grafts of chow-fed rabbits 26 and our finding that hyperlipidemia does not alter the endothelial predominance of transgene expression in adenovirus-infused arteries, 29 we assume that most HDAdNull genomes (if they are intracellular) are likely within luminal endothelial cells.…”
Section: Resultsmentioning
confidence: 99%
“…We delayed transduction until 4 weeks because our previous work showed that near-complete early (3 day) loss of vein-graft transgene expression could be avoided only by transducing grafted veins at a time point after the grafting surgery. 26 This earlier study was performed largely with FGAd, and we concede that we have not tested whether delivery of HDAd at the time of grafting would yield persistent transgene expression. However, we believe this is highly unlikely because our control experiments showed that loss of FGAd genomes within 3 days after grafting is due to exposure of the transduced veins to arterial flow and pressure, not to FGAd properties per se.…”
Section: Discussionmentioning
confidence: 99%
“…We recently reported that transduction of vein grafts of chow-fed rabbits with a helper-dependent adenoviral (HDAd) vector yielded expression of a transgene for at least 24 weeks, with stable expression from 4 weeks onward. 26 Transduction of arteries of chow-fed rabbits with the same HDAd achieved transgene expression for at least 48 weeks, also with stability after 4 weeks. 27 Because HDAd appears capable of mediating durable—if not indefinite—transgene expression in blood vessels, HDAd is a promising vector for developing gene therapy for vein graft atherosclerosis.…”
Coronary artery bypass vein grafts are a mainstay of therapy for human atherosclerosis. Unfortunately, the long-term patency of vein grafts is limited by accelerated atherosclerosis. Gene therapy, directed at the vein graft wall, is a promising approach for preventing vein graft atherosclerosis. Because helper-dependent adenovirus (HDAd) efficiently transduces grafted veins and confers long-term transgene expression, HDAd is an excellent candidate for delivery of vein graft-targeted gene therapy. We developed a model of vein graft atherosclerosis in fat-fed rabbits and demonstrated long-term (≥20 weeks) persistence of HDAd genomes after graft transduction. This model enables quantitation of vein graft hemodynamics, wall structure, lipid accumulation, cellularity, vector persistence, and inflammatory markers on a single graft. Time-course experiments identified 12 weeks after transduction as an optimal time to measure efficacy of gene therapy on the critical variables of lipid and macrophage accumulation. We also used chow-fed rabbits to test whether HDAd infusion in vein grafts promotes intimal growth and inflammation. HDAd did not increase intimal growth, but had moderate—yet significant—pro-inflammatory effects. The vein graft atherosclerosis model will be useful for testing HDAd-mediated gene therapy; however, pro-inflammatory effects of HdAd remain a concern in developing HDAd as a therapy for vein graft disease.
“…We delayed transduction until 4 weeks after grafting because transduction at the time of grafting leads to rapid loss of transgene expression, whereas delayed transduction yields durable transgene expression. 26 Figure 1 Study Protocols (A) Vein graft atherosclerosis study. Rabbits were begun on a 0.3% cholesterol diet.…”
Section: Resultsmentioning
confidence: 99%
“…In our previous work and in results reported by others, incubation of adenovirus in a vein lumen followed by grafting of the vein results in transgene expression that is largely confined to luminal endothelial cells. 26 , 38 , 39 , 40 , 41 Other groups have reported transgene expression in the venous media and adventitia, but this may be due to variability in technique. 42 , 43 , 44 Based on our previous work with vein grafts of chow-fed rabbits 26 and our finding that hyperlipidemia does not alter the endothelial predominance of transgene expression in adenovirus-infused arteries, 29 we assume that most HDAdNull genomes (if they are intracellular) are likely within luminal endothelial cells.…”
Section: Resultsmentioning
confidence: 99%
“…We delayed transduction until 4 weeks because our previous work showed that near-complete early (3 day) loss of vein-graft transgene expression could be avoided only by transducing grafted veins at a time point after the grafting surgery. 26 This earlier study was performed largely with FGAd, and we concede that we have not tested whether delivery of HDAd at the time of grafting would yield persistent transgene expression. However, we believe this is highly unlikely because our control experiments showed that loss of FGAd genomes within 3 days after grafting is due to exposure of the transduced veins to arterial flow and pressure, not to FGAd properties per se.…”
Section: Discussionmentioning
confidence: 99%
“…We recently reported that transduction of vein grafts of chow-fed rabbits with a helper-dependent adenoviral (HDAd) vector yielded expression of a transgene for at least 24 weeks, with stable expression from 4 weeks onward. 26 Transduction of arteries of chow-fed rabbits with the same HDAd achieved transgene expression for at least 48 weeks, also with stability after 4 weeks. 27 Because HDAd appears capable of mediating durable—if not indefinite—transgene expression in blood vessels, HDAd is a promising vector for developing gene therapy for vein graft atherosclerosis.…”
Coronary artery bypass vein grafts are a mainstay of therapy for human atherosclerosis. Unfortunately, the long-term patency of vein grafts is limited by accelerated atherosclerosis. Gene therapy, directed at the vein graft wall, is a promising approach for preventing vein graft atherosclerosis. Because helper-dependent adenovirus (HDAd) efficiently transduces grafted veins and confers long-term transgene expression, HDAd is an excellent candidate for delivery of vein graft-targeted gene therapy. We developed a model of vein graft atherosclerosis in fat-fed rabbits and demonstrated long-term (≥20 weeks) persistence of HDAd genomes after graft transduction. This model enables quantitation of vein graft hemodynamics, wall structure, lipid accumulation, cellularity, vector persistence, and inflammatory markers on a single graft. Time-course experiments identified 12 weeks after transduction as an optimal time to measure efficacy of gene therapy on the critical variables of lipid and macrophage accumulation. We also used chow-fed rabbits to test whether HDAd infusion in vein grafts promotes intimal growth and inflammation. HDAd did not increase intimal growth, but had moderate—yet significant—pro-inflammatory effects. The vein graft atherosclerosis model will be useful for testing HDAd-mediated gene therapy; however, pro-inflammatory effects of HdAd remain a concern in developing HDAd as a therapy for vein graft disease.
“…Here we tested several strategies aimed either at increasing HDAd-mediated apo A-I expression in EC at all time points or at stabilizing apo A-I expression between day 3 and day 28 after gene transfer (after day 28 HDAd appears to express apo A-I indefinitely in EC). [11][12][13] We first sought to increase translation of transgene mRNA by using species-specific codonoptimized transgene sequences. We next attempted to increase transgene mRNA stability by removing endogenous 3¢-untranslated (UTR) sequences.…”
Our long-term goal is to prevent or reverse atherosclerosis by delivering gene therapy from stably transduced endothelial cells (EC). We previously reported that EC-directed gene therapy with a helper-dependent adenovirus (HDAd) expressing apolipoprotein A-I (apo A-I) retarded development of atherosclerosis in rabbit carotid arteries over a 1-month interval. However, a 70% decline in apo A-I expression during this time raised concerns about long-term efficacy of this approach. Here we report use of several approaches aimed either at preventing this decline or at increasing apo A-I expression from HDAd at all time points: codon optimization, deletion of 3' untranslated sequences, substitution of a synthetic mammalian-based promoter (4XETE) for the cytomegalovirus (CMV) promoter, and co-transduction with an HDAd expressing interleukin-10. We tested these approaches using plasmid transfection of cultured EC and in vivo transduction of rabbit carotid artery EC. Codon optimization did not increase apo A-I expression. Deletion of 3' untranslated sequences extinguished apo A-I expression. Both substitution of 4XETE for the CMV promoter and expression of interleukin-10 stabilized apo A-I expression in vivo, although at the cost of lower early (3-day) expression levels. Surprisingly, both interventions stabilized apo A-I expression without altering the rate at which HDAd genomes were lost. These data establish that transgene expression from HDAd in EC is inherently stable in vivo and suggest that the early decline of CMV promoter-driven expression from HDAd-transduced EC is due neither to active downregulation of transcription nor to loss of HDAd genomes. Instead, apparent loss of expression from the CMV promoter appears to be a consequence of early (3-day) upregulation of CMV promoter activity via inflammatory pathways. Our results yield new paradigms to explain the early loss of genomes and transgene expression after in vivo gene transfer. These new paradigms will redirect strategies for achieving high-level, stable expression of transgenes in EC.
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