Adenovirus-mediated gene transfer in the transplant setting. I. Conditions for expression of transferred genes in cold-preserved hepatocytes.
Transplantation of genetically modified hepatocytes has been suggested as a therapeutic modality for impaired hepatocellular function. This study examined adenoviral-mediated gene transfer to isolated hepatocytes, under conditions mimicking clinical transplant preservation. Isolated rat hepatocytes were infected using replication-defective adenoviral vectors with an expression cassette containing the beta-galactosidase gene driven by a CMV promoter. Hepatocytes were infected in suspension immediately after isolation, then either cultured or transplanted immediately into a syngeneic host. Gene transfer efficiency was assessed by histochemical staining and FACS analysis for the gene product. The presence of viral DNA and mRNA, as well as viral-derived protein production, were assayed. Efficiency of gene transfer was examined as a function of several preservation conditions. Infection efficiency was best in cells preserved in UW solution, correlated directly with virus:hepatocyte ratio and with length of exposure to virus. Successful infection resulted in significant viral-derived protein production, persisting for at least two weeks in culture. These results demonstrate the versatility of adenoviral vectors in accomplishing rapid and efficient gene transfer into nondividing hepatocytes during cold preservation. Such genetically modified hepatocytes have potential use for immediate transplantation, without the need for further manipulation.
Csete
M E
ME
Department of Surgery, University of California at Los Angeles 90024-1778.
Drazan
K E
KE
van Bree
M
M
McIntee
D F
DF
McBride
W H
WH
Bett
A
A
Graham
F L
FL
Busuttil
R W
RW
Berk
A J
AJ
Shaked
A
A
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002470:Cell Survival; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000032:analysis; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D016031:Liver Transplantation / Q000379:methods*; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D011917:Rats, Inbred Lew
D017931:DNA Primers; D004279:DNA, Viral