Adenovirus-mediated gene transfer into kidney glomeruli using an ex vivo and in vivo kidney perfusion system - first steps towards gene therapy of Alport syndrome.
In order to develop gene therapy for Alport syndrome, we have examined the efficiency of adenovirus-mediated transfer of the beta-galactosidase gene into cultured cells and intact glomeruli in vitro, and developed an organ perfusion system for gene transfer into kidney ex vivo and in vivo. Human endothelial and mesangial cells, as well as isolated human glomeruli, were readily infected and exhibited expression of the reporter gene. Single or multiple injections of the virus solution into the renal artery of pig in vivo did not lead to significant gene transfer and expression of the reporter gene in kidney cells. To increase the exposure time of kidney cells to the virus we perfused kidneys ex vivo and in vivo for up to 12 and 2 h, respectively. The perfusion system consisted of a perfusate container, a peristaltic pump and an artificial membrane lung gassed with carbogen. Using this system, intense expression of the reporter gene could be achieved in up to about 85% of the glomeruli after perfusion of an explanted kidney ex vivo and about the same efficiency of gene transfer could be obtained in glomerular cells after 2-h perfusion in vivo. Some expression was observed in other vascular endothelial cells following the perfusion, but no expression was observed in cells of the Bowman's capsule or epithelial cells of the tubuli. The X chromosome-linked form of Alport syndrome is caused by defects in the gene for the type IV collagen of alpha5 chain, which is primarily expressed in the kidney in glomerular cells. The present results demonstrated that efficient gene transfer can be achieved into glomerular cells, a prerequisite for gene therapy of this disease. The organ perfusion method developed in this study might also be applicable for gene therapy of other diseases.
Heikkila
P
P
Biocenter Oulu and Department of Biochemistry, University of Oulu, Finland.
Parpala
T
T
Lukkarinen
O
O
Weber
M
M
Tryggvason
K
K
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007261:Infusions, Intra-Arterial; D007668:Kidney / Q000378:metabolism; D007678:Kidney Glomerulus / Q000166:cytology / Q000378:metabolism; D009394:Nephritis, Hereditary / Q000628:therapy; D010477:Perfusion / Q000379:methods*; D013552:Swine; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics
D001616:beta-Galactosidase