Adenovirus-mediated gene therapy to liver grafts: successful gene transfer by donor pretreatment.
BACKGROUND
We have previously shown excellent adenoviral (Ad) gene transfection to transplanted liver grafts with the clamp technique (CT) where viral vector was delivered ex vivo and trapped in cold preserved liver grafts. In this study, we adopted a new gene therapy approach to achieve early transgene expression by donor pretreatment with viral vector and compared the efficacy of these two methods by using Ad vector encoding enhanced green fluorescent protein (AdEGFP) marker gene.


METHODS
AdEGFP (1 x 10(9)plaque forming units) was delivered to the liver grafts by: (1) single intravenous injection to donor Lewis rats 48 hours before harvesting, (2) ex vivo cold infusion into the harvested liver with CT, or (3) a combination of both methods. Liver grafts were stored in University of Wisconsin solution at 4 degrees C for 18 hours and then orthotopically transplanted into syngeneic recipients, and the expression of EGFP was studied.


RESULTS
With intravenous pretreatment of donor liver grafts, EGFP-expressing cells were detected as early as 3 hours after transplant, and moderate expression was seen by 12 hours. In contrast, EGFP was not detected until 12 to 24 hours after transplant with CT. High levels of EGFP-producing cells were seen with each technique at 7 days ( approximately 30% transfection efficiency). A combination of both methods did not enhance infectivity. Liver preservation injury was comparable between groups.


CONCLUSIONS
Gene transfer by donor pretreatment with AdEGFP induces early and efficient gene transduction to liver grafts compared with back-table delivery with CT. This method is simple and provides early transgene expression in liver grafts that potentially could be used to deliver genes to decrease preservation injury or rejection.
Thomas E. Starzl Transplantation Institute, Department of Surgery and Center for Biologic Imaging, University of Pittsburgh Medical Center, PA 15213, USA.
Y
Y
Takahashi
D A
DA
Geller
A
A
Gambotto
S C
SC
Watkins
J J
JJ
Fung
N
N
Murase
D000241:Adenosine; D000256:Adenoviridae; D000493:Allopurinol; D000818:Animals; D002460:Cell Line; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D005978:Glutathione; D049452:Green Fluorescent Proteins; D006801:Humans; D007328:Insulin; D008099:Liver; D016031:Liver Transplantation / Q000379:methods / Q000502:physiology*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008297:Male; D019188:Organ Preservation Solutions; D009928:Organ Specificity; D011887:Raffinose; D051381:Rats; D011917:Rats, Inbred Lew; D014162:Transfection; D014185:Transplantation, Isogeneic
D007328:Insulin; D008164:Luminescent Proteins; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D049452:Green Fluorescent Proteins; D000493:Allopurinol; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose