Surgical organ perfusion method for somatic gene transfer : an experimental study on gene transfer into the kidney, spleen, lung and mammary gland
1University of Oulu, Faculty of Medicine, Department of Surgery
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|Persistent link:|| http://urn.fi/urn:isbn:9514255429
|Publish Date:|| 2000-02-04
|Thesis type:||Doctoral Dissertation
|Defence Note:||Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in Auditorium 1 of the University Hospital of Oulu, on February 4th, 2000, at 12 noon.
Docent Mikko Hippeläinen
Docent Matti Vapalahti
The progress in recombinant DNA technology has made possible the introduction of exogenous genetic material into cells. Gene therapy aims to correct a defective gene, introduce a therapeutic exogenous gene or a counteracting gene into somatic cells without modification of the germ-cell line. The most important technical interests in the field of gene therapy research have pertained to the development of safe and effective vectors and suitable methods for the delivery of the exogenous gene carrying vectors into the target cells. The aim of this study was to evaluate surgical methods used for gene delivery and to develop an effective gene transfer method for organ-specific gene transfer, primarily into the renal glomeruli.
There are genetic and acquired diseases that are candidates for gene therapy. Alport syndrome is an X-chromosome-linked disease caused by a mutation in the type IV collagen α5 chain gene, which causes a defect of the glomerular basement membrane in the kidney, leading to progressive renal failure in males. This manifestation could theoretically be prevented by the transfer of a normal α5 chain gene into the renal glomerular cells. Cystic fibrosis and α1-antitrypsin deficiency are examples of pulmonary diseases and genetic lysosomal storage diseases that are candidates for splenic gene transfer. The gene transfer strategies used so far have proved relatively ineffective. Recombinant adenovirus, retrovirus, adeno-associated virus and liposomes have been previously used as vectors. Direct injection, intra-arterial, intravenous and intratracheal delivery of vectors have been the most extensively studied methods.
This preclinical experimental work for marker gene transfer into the kidney, spleen, lung and mammary gland was done by using rabbits, pigs and goats as test animals. The adenoviral vector carrying a β-galactosidase reporter gene was first infused in the renal artery of rabbits and pigs in vivo with or without pharmacological agents. This did not result in any remarkable gene transfer into the kidney. Next, the incubation time between the vector and the target cells was prolonged by ex vivo perfusion of explanted kidneys for 12 hours. Perfusion at room temperature did not improve gene transfer. When the perfusion temperature was raised to 37°C, improved and mostly glomerular gene transfer was observed, with up to 80% of the glomeruli showing β-galactosidase expression in four ex vivo experiments.
A closed-circuit organ perfusion method for in vivo gene transfer was developed in this study. The surgical perfusion experiment was tested successfully in ten in vivo perfusions of the kidney, eight of the spleen and eight of the lung in a porcine model. This method led to effective, up to 75% gene transfer into the renal glomeruli as assessed after four days. In the spleen, the perfusion method resulted in relatively effective gene transfer into perifollicular splenic cells, mostly macrophages and endothelial cells. Lung perfusion yielded transgene expression in alveolar epithelial cells, bronchiolar epithelial cells and, to a lesser extent, arteriolar endothelial cells and alveolar macrophages. Perfusion of the goats mammary gland using a retroviral vector in three experiments resulted in growth hormone secretion into the milk.
The gene transfer operation was well tolerated by the animals, and no clinical signs of inflammation were observed. No remarkable humoral immunological response against adenovirus or β-galactosidase was elicited in the kidney experiments, but histological signs of inflammation as mononuclear cell clusters in the kidney and lung were seen four and seven days after the experiments. The spleen showed no macroscopic or microscopic pathologic alterations after the perfusion.
Acta Universitatis Ouluensis. D, Medica
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