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

Highly efficient ex vivo gene transfer to the transplanted heart by means of hypothermic perfusion with a low dose of adenoviral vector.

Background: Hypothermic conditions required for donor heart preservation may reduce gene-transfer efficiency Experiments were designed to determine whether a perfusion technique could improve the efficiency of gene transfer to donor hearts. Methods: An adenoviral vector encoding beta-galactosidase (3.5 x 10(8) plaque-forming units) was infused into explanted rat hearts under 4 conditions teach n = 6): (1) the virus was diluted in 350 mu L of University of Wisconsin solution and infused as a high-pressure bolus into the coronary arteries of donor hearts through the aortic root; (2) the virus was diluted in 5 mL of University of Wisconsin solution and circulated by means of a peristaltic pump (flow, 0.75 mL/min) through the vasculature of the donor heart for 30 minutes; (3) 5 mi of viral solution was circulated as for group 2 for 15 minutes; and (4) 5 mt of viral solution was circulated for 5 minutes at a flow rate of 2.4 mL/min, Transduced hearts were transplanted into the abdomen of syngeneic rats, and transgene expression was assessed by means of immunoassay 4 days later. Results: The median beta-galactosidase content was (1) 45.0 ng/mg protein (25th-75th percentile, 33-73 ng/mg), (2) 6 10 ng/mg protein (25th-75th percentile, 613-878 ng/mg), (3) 493.8 ng/mg protein (25th-75th percentile, 456-527 ng/mg), and (4) 503.3 ng/mg protein (25th-75th percentile, 475-562 ng/mg; P <.01 for group 2 vs group 1, and P <.05 for groups 3 and 4 vs group 1), Transgene expression was predominantly in myocytes and favored the subepicardial region of the right ventricle, Conclusion: Hypothermic perfusion of the donor heart with an adenoviral vector resulted in efficient transgene expression compared with that induced by a single bolus injection

Gene transfer of endothelial nitric oxide synthase to pulmonary allografts: impact on acute rejection

Experiments were designed to study whether overexpression of nitric oxide (NO) from endothelial nitric oxide synthase (eNOS) affects acute rejection. Allogenic, orthotopic single-lung transplantation was performed after transbronchial adenoviral-mediated gene transfer (3 x 10(8) pfu) of either of eNOS or beta-galactosidase to donor lungs of rats (n = 6 each). No immunosuppression was used. After 4 days, transplanted lungs were prepared for enzyme activity, cGMP and histology. Calcium-dependent NOS activity, reflecting eNOS, was greater in eNOS-transduced lungs (587 +/- 97 vs 2.1 +/- 1.4 pmol/mg protein per h, P <0.001). In contrast, calcium-independent NOS activity, reflecting iNOS, was comparable. Concentrations of cGMP were higher in eNOS-transduced lungs (13.2 +/- 2.3 vs 4.9 +/- 0.5 pmol/mg protein). Positive immunostaining for eNOS was present in pneumocytes only in eNOS-transduced lungs. No difference in histological grade of rejection was observed. eNOS gene transfer to pulmonary allografts results in a functionally active transgene product and increased NO production. Increasing NO from eNOS does not affect histogically identified acute rejection

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