Conditions of vector delivery improve efficiency of adenoviral-mediated gene transfer to the transplanted heart

Objectives: Conditions for ex vivo gene transfer to the transplanted heart were studied in a model of syngeneic abdominal heterotopic heart transplantation in the rat. Various methods of adenoviral-mediated gene transfer to the transplanted heart were compared. Methods: In the first experiment, a dose response study, an adenoviral vector encoding the P-galactosidase gene was infused into the donor heart with the pulmonary artery open and flushed out prior to performing the transplant. In the second experiment, the effects of clamping the pulmonary artery during vector infusion and not flushing out the viral solution, resulting in vector dwell during the warm ischemia, were examined. Results: In the first experiment, gene transfer was relatively inefficient; however, transgene expression improved with increases in the vector dose (range, 1 x 10(7)-1 x 10(9)). The efficiency of gene transfer was significantly greater when the conditions of the second experiment were applied. In all models studied, cardiomyocytes and not vascular endothelial cells were the predominant cell type transduced. Conclusions: This study indicates that the conditions of adenoviral vector delivery are critical for optimizing gene transfer in the transplant setting. In addition, intravascular administration of adenoviral vector to the donor heart results predominantly in cardiomyocyte transgene expression. (C) 2001 Elsevier Science B.V. All rights reserve

Systematic evaluation of distribution of transgene expression after adenovirus-mediated gene transfer to the transplanted heart.

In the transplantation setting, the study and potential treatment of acute and chronic rejection by means of gene therapy will require widespread transgene expression in the donor organ. The distribution of transgene expression after adenovirus-mediated gene transfer via the coronary vasculature in a model of abdominal heterotopic heart transplantation in syngeneic rats (n = 6) was evaluated at 1 week. Reporter gene expression was evenly distributed in the base, the midventricle, and the apex of the transplanted hearts. This study demonstrates that intracoronary administration of the adenoviral vector to the donor heart results in widespread transgene expression

Gene therapy in lung transplantation: Effective gene transfer via the airways

Objectives: Gene therapy may provide a means of modifying factors that contribute to the development of pathologic processes in transplanted lungs, Experiments were designed to study the feasibility of adenovirus-mediated gene transfer by way of the airways to the transplanted lung, Methods: Orthotopic left lung transplantation (Lewis to Lewis rats) was performed on four groups of animals, 300 mu l of adenovirus solution encoding for beta-galactosidase was infused into the left bronchus of donor rats at viral concentrations of 10(8) pfu/ml (n = 5), 10(9) pfu/ml (n = 6), and 10(10) pfu/ml (n = 6), and the lung was ventilated for 5 minutes, Controls (n = 6) received medium only, Seven days after transplantation, native and transduced, transplanted lungs were harvested, Sections of lung were fixed and stained with a solution of X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) and staining was evaluated for distribution by cell type and intensity, Results: beta-Galactosidase expression was absent in the control group and in the native lunes, Two of five lungs in the 10(8) group expressed beta-galactosidase, but in a limited distribution and intensity. All six lungs in the 10(9) group and five of sis lungs in the 10(10) group expressed beta-galactosidase, The distribution and intensity of beta-galactosidase expression ranged from only a few cells staining per slide to up to 75%, Pneumocytes were the most frequently stained cell type followed by alveolar macrophages. Conclusions: Gene transfer to the transplanted lung via the bronchial route is feasible and offers a novel technique to modify pathologic processes in the transplanted lung

Transbronchial gene transfer of endothelial nitric oxide synthase to transplanted lungs.

background. Experiments were designed to study the efficiency, distribution, and toxicity of transbronchial adenoviral-mediated transfer of endothelial constitutive nitric oxide synthase (ecNOS) gene to transplanted lungs. Methods. Syngeneic orthotopic single-lung transplantation in the rat was performed after airway administration (300 mu L, 1 x 10(9) pfu/mL) of either the ecNOS gene or the marker gene beta-Gal (control group) to donor lungs (n = 4 each). After 4 days, transgene expression, inflammation, and the presence of apoptosis in the transplanted lungs were assessed by molecular, immunohistochemical, and histologic techniques. Results. Gene transfer was confirmed by a positive polymerase chain reaction signal for the recombinant ecNOS gene, and recombinant messenger RNA by reverse transcription polymerase chain reaction. Positive immunohistochemical staining for ecNOS was present in more than 75% of pneumocytes only in ecNOS transduced lungs. Calcium-dependent nitric oxide synthase activity was increased in ecNOS- compared with beta Gal-transduced lungs (2,139 +/- 756 versus 47 +/- 28 pmol mg protein-l h-l; p < 0.05). Minimal to mild inflammation was observed in all transplanted lungs; fewer than 0.5% of cells in both groups were apoptotic. Conclusions. Transbronchial transfer of ecNOS gene to the transplanted lung results in transduction of pneumocytes with expression of a functionally active transgene product. (C) 1998 by The Society of Thoracic Surgeons

Distribution and function of recombinant endothelial nitric oxide synthase in transplanted hearts.

Introducing recombinant genes into donor hearts may offer a therapeutic intervention that could potentially attenuate the complications of heart transplantation, including rejection, infection and accelerated atherosclerosis. In the cardiovascular system, reduced bioactivity of endothelial nitric oxide is a feature of atherosclerosis and vascular injury. Nitric oxide is an arterial vasodilator that also inhibits proliferation of vascular smooth muscle cells and platelet aggregation. Experiments were designed to determine the distribution of adenoviral-mediated transfer of recombinant endothelial nitric oxide synthase gene (eNOS) and the effect of recombinant gene expression on the function of transplanted hearts. Adenoviral vectors for (a) bovine eNOS (AdeNOS) or (b) beta-galactosidase (AdLacZ; control) were infused into two groups (n=12, per group) of explanted rat hearts. The transduced hearts were then implanted heterotopically into the abdomen of syngeneic recipient rats. After four days, the hearts were excised and examined for distribution and function of the recombinant genes. Polymerase chain reaction (PCR) verified the presence of the recombinant eNOS gene in eNOS-transduced but not in beta-galactosidase-transduced hearts; reverse transcriptase-PCR identified mRNA for eNOS in AdeNOS-transduced hearts. NOS activity (conversion of tritiated L-arginine to citrulline) was greater in homogenates of AdeNOS- compared to AdLacZ-transduced hearts. Positive immunoreactivity for eNOS was present in cardiomyocytes predominantly in eNOS-transduced hearts. Myocardial contractility and coronary blood flow, as determined using a Langendorff preparation, were not different between hearts transduced with AdeNOS or AdLacZ. These results suggest that, up to four days post transplantation, adenoviral-mediated transfer of eNOS into transplanted hearts is possible. However, expression of the recombinant protein did not result in measurable changes in myocardial contractility or coronary perfusion. (C) 1999 Elsevier Science BN. All rights reserved