Samer A. Kanaan

Intramuscular Gene Transfer of Interleukin‐10 Reduces Neutrophil Recruitment and Ameliorates Lung Graft Ischemia‐Reperfusion Injury

Interleukin‐10 (IL‐10) has potent anti‐inflammatory properties but its direct effects on neutrophil trafficking in lung transplant ischemia‐reperfusion (I/R) injury are unknown. This study was performed to determine if recipient intramuscular IL‐10 gene transfer reduces neutrophil infiltration in lung isografts and ameliorates I/R injury. Twenty‐four hours before transplantation, recipient rodents received intramuscular injection with 1 × 1010 plaque‐forming units (pfu) adenovirus encoding human IL‐10 (hIL‐10), 1 × 1010 pfu adenovirus control encoding β‐galactosidase, or saline. Gene expression in muscle and plasma was confirmed. Lung grafts were harvested, stored at 4 °C for 18 h, and assessed 24 h after transplantation. Peak muscle and plasma expression of hIL‐10 was achieved 24 h after gene transfer and returned to baseline by 7 days (p < 0.05 vs. controls). Gene transfer of hIL‐10 reduced neutrophil sequestration and emigration in lung grafts as measured by morphometry and myeloperoxidase activity (p < 0.03 vs. controls). Furthermore, hIL‐10 improved graft oxygenation and reduced lung edema (p < 0.01 vs. controls). Intramuscular gene transfer of hIL‐10 releases hIL‐10 protein into plasma and reduces neutrophil sequestration and emigration in lung isografts. This is associated with a reduction in I/R injury with improved isograft oxygenation and reduced tissue edema. Intramuscular gene transfer may be a useful strategy to reduce clinical I/R injury.

Tumor necrosis factor inhibitor gene transfer ameliorates lung graft ischemia-reperfusion injury.

OBJECTIVE Tumor necrosis factor is an important mediator of lung transplant ischemia-reperfusion injury, and soluble type I tumor necrosis factor receptor binds to tumor necrosis factor and works as a tumor necrosis factor inhibitor. The objectives of this study were to demonstrate that gene transfer of type I tumor necrosis factor receptor-IgG fusion protein reduces lung isograft ischemia-reperfusion injury and to compare donor endobronchial versus recipient intramuscular transfection strategies. METHODS Three donor groups of Fischer rats (n = 6/group) underwent endobronchial transfection with either saline, 2 x 10(7) plaque-forming units of control adenovirus encoding beta-galactosidase, or 2 x 10(7) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein. Left lungs were harvested 24 hours later. Two recipient groups (n = 6/group) underwent intramuscular transfection with 2 x 10(7) plaque-forming units or 1 x 10(10) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein 24 hours before transplantation. All donor lung grafts were stored for 18 hours before orthotopic lung transplantation. Graft function was assessed 24 hours after reperfusion. Transgene expression was evaluated by means of enzyme-linked immunosorbent assay and immunohistochemistry of type I tumor necrosis factor receptor. RESULTS Endobronchial transfection of donor lung grafts with 2 x 10(7) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein significantly improved arterial oxygenation compared with the saline and beta-galactosidase donor groups (366.6 +/- 137.9 vs 138.8 +/- 159.9 and 140.6 +/- 131.4 mm Hg, P =.009 and.010, respectively). Recipient intramuscular transfection with 1 x 10(10) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein improved lung graft oxygenation compared with that seen in the low-dose intramuscular group (2 x 10(7); 320.3 +/- 188.6 vs 143.6 +/- 20.2 mm Hg, P =.038). Type I tumor necrosis factor receptor-IgG fusion protein was expressed in endobronchial transfected grafts. In addition, intramuscular type I tumor necrosis factor receptor-IgG fusion protein expression was dose dependent. CONCLUSIONS Donor endobronchial and recipient intramuscular adenovirus-mediated gene transfer of type I tumor necrosis factor receptor-IgG fusion protein improved experimental lung graft oxygenation after prolonged ischemia. However, donor endobronchial transfection required 500-fold less vector. Furthermore, at low vector doses, it does not create significant graft inflammation.

Adenovirus encoding soluble tumor necrosis factor α receptor immunoglobulin prolongs gene expression of a cotransfected reporter gene in rat lung

OBJECTIVE Because almost all pulmonary diseases are not caused by one gene, multiple gene transfection is required for current gene therapy. Adenovirus is an important gene therapy vector, but a short duration and the inability of repeated administration remain limitations. The aims of this study were to evaluate whether adenoviral vector encoding soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase cotransfection prolongs gene expression and facilitates repeated vector administration to investigate the feasibility of a cotransfection strategy. METHODS F344 rats received intratracheal administration of 1 x 10(9) plaque-forming units of adenoviral vector encoding beta-galactosidase or both adenoviral vector encoding beta-galactosidase and adenoviral vector encoding soluble tumor necrosis factor alpha receptor immunoglobulin. In the expression study beta-galactosidase gene expression in the lung was examined by means of enzyme-linked immunosorbent assay on days 2, 7, 14, 28, and 56 (n = 4/day). In the repeated transfection study, soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase were readministered once (7 days after the first adenovirus administration) or twice (on days 7 and 14; n = 4/day). A 2-way factorial analysis of variance was used for statistical analysis. RESULTS Soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase cotransfection prolonged the duration of beta-galactosidase expression. However, antiadenovirus antibody production was significantly increased in the cotransfection group. In addition, there was no increase in beta-galactosidase expression after readministration of soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase. CONCLUSION Adenoviral vector encoding soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase cotransfection prolongs beta-galactosidase expression but does not increase beta-galactosidase expression after repeated administration. These results suggest that tumor necrosis factor alpha is one of the most important factors in regulating the duration of gene expression. The cotransfection approach is feasible, but the increase of antiadenovirus antibodies might make repeated cotransfection unfeasible.

Potential for gene therapy in lung transplantation

Lung transplantation has become a well-established management for many end-stage pulmonary diseases; however, the same success has not been achieved as with other solid organ transplants. Despite substantial improvements in organ preservation and the perioperative management of lung transplant recipients, ischemia–reperfusion injury, acute rejection, and chronic rejection persist. Severe ischemia–reperfusion injury affects as many as 20% of lung transplant recipients, prolongs mechanical ventilation, and may result in hemodynamic compromise. Acute rejection is the most common risk factor for bronchiolitis obliterans, a manifestation of chronic graft rejection, which ultimately progresses to graft failure. Gene therapy is a promising therapeutic option for these transplant-related injuries. Potential targets include donor and recipient, with a host of available delivery options and gene constructs. This review addresses the challenges of gene therapy and reviews its experimental progress in lung transplantation.

Vascular immunotargeting to lung graft endothelium

versus cooling on the non-perfused pulmonary graft. Methods: Domestic pigs (30,8 0,35 Kg) were anesthetized and divided in 3 groups. In group I lungs were flushed with cold Perfadex , explanted and stored in saline solution (4°C) for 4 hours (Control group HBD, n 5). In the two study groups pigs were sacrificed by myocardial fibrillation and left untouched for 1 hour after death. Lungs in group II were topically cooled (NHBD-TC, n 5) in situ for three hours with saline solution (6°C). Lungs in group III were ventilated (NHBD-V, n 5) in situ for three hours. The left lung was then prepared for evaluation in an isolated circuit. The temperature of the perfusate and the pulmonary artery pressure were gradually increased up to 37,5 °C and 20 mmHg respectively. Functional parameters were assessed at this point. Results: Results are listed in table. Data are presented as mean SEM, ° p 0,05 group NHBD-V vs NHBD-TC, ^ p 0,05 NHBD-V vs HBD, not significant NHBD-TC vs HBD.