Download OPTIMISING GENE TRANSFER INTO EMBRYONIC KIDNEYS AS A

OPTIMISING GENE TRANSFER INTO EMBRYONIC KIDNEYS AS A
PRELUDE TO TREATING RENAL TRACT MALFORMATIONS.
INTRODUCTION. Renal tract malformations can be devastating, accounting for about
half of children, and a large subset of young adults, with end stage renal disease. Most
are identified at fetal ultrasound screening and definition of their genetic pathogenesis is
making it possible to conceive of novel biological therapies. We hypothesised that
viruses can be used to transfer genes into embryonic kidneys. Accordingly, our aim was
optimise efficient gene transfer without compromising normal nephron differentiation.
METHODS. We isolated wild type embryonic day 13 mouse kidneys, anatomically
equivalent to mid first trimester human kidneys. Each organ was comprised of a
branching ureteric tree and nephron precursors. Kidneys were exposed to replication
defective vesicular stomatitis virus glycoprotein (VSVg) pseudotyped lentiviruses
expressing green fluorescent protein (GFP) from either a cytomegalovirus (CMV) or a
3-phosphoglycerate kinase (PGK) promoter. Kidneys were grown in organ culture for
up to three days and transgene expression visualised as green fluorescent cells.
RESULTS. Intact kidneys, unexposed to virus, differentiated in culture to form Ecadherin+ collecting ducts and Wilms tumour 1+ glomeruli. When intact rudiments
were exposed to lentivirus, differentiation was also good but few if any cells expressed
GFP. Reasoning that the virus could not penetrate into the organ, we dissociated
embryonic kidneys into single cells, exposed them to virus, and then placed the cell
pellet into organ culture. Remarkably, even after being dissociated for one hour, the
pellet rapidly moulded itself into a kidney shape and then differentiated nearly as well
as intact controls. After dissociated cells were exposed to virus, efficient gene transfer
and strong GFP expression from each promoter occurred in the reconstituted organ. A
ratio of adding one to five viral particles per kidney cell provided the best balance
between strong transgene expression without compromising nephron differentiation.
CONCLUSION. Lentiviruses target embryonic kidney cells and the transduced
reporter gene is robustly expressed. Further studies are now needed to optimise ex vivo
and in vivo transfer of reporter, and then therapeutic, genes into embryonic kidneys
from mice with mutations of human renal tract malformation genes.