Adenovirus-mediated gene transfer using ex vivo perfusion of the heart graft.
A replication-deficient adenovirus was used for ex vivo gene transfer into rat heart grafts under conditions simulating clinical transplantation. The adenoviral vector, AdHCMVsp1LacZ, containing an expression cassette of Escherichiae coli lacZ, was used to perfuse heart grafts during cold ischemia before transplantation. Heart grafts were perfused with University of Wisconsin (UW) solution containing either 0 pfu, 5 x 10(10) pfu, or 1 x 10(11) pfu of viral vector, and were preserved for either 2 or 4 h and then transplanted into syngeneic recipients. The animals were killed at 1, 7, and 14 days after transplantation. The infection rate was assessed by histochemical staining for beta-galactosidase. Using polymerase chain reaction (PCR), viral DNA presence was confirmed in every graft perfused with viral vectors. The protein production from the transfected gene was confirmed by a functional protein assay. An efficient gene transfer was achieved with an infection rate of 1%-1.5% for all cardiac myocytes, as assessed by 5-bromo-4-chloro-indolyl-beta-D-galactopyranoside (X-gal) staining. All studies were negative in the control grafts. Gene expression persisted for at least 10 days after transplantation. We thus conclude that an efficient adenovirus-mediated gene transfection and expression of gene products can be achieved in ex vivo perfusion of the heart graft during cold preservation.
Shiraishi
M
M
First Department of Surgery, University of Ryukyu, School of Medicine, Okinawa, Japan.
Kusano
T
T
Hara
J
J
Hiroyasu
S
S
Shao-Ping
M
M
Makino
Y
Y
Muto
Y
Y
D000005:Abdomen; D000241:Adenosine; D000256:Adenoviridae / Q000235:genetics*; D000493:Allopurinol; D000818:Animals; D002314:Cardioplegic Solutions; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005978:Glutathione; D006085:Graft Survival; D016027:Heart Transplantation / Q000379:methods*; D007328:Insulin; D019188:Organ Preservation Solutions; D010477:Perfusion; D016133:Polymerase Chain Reaction; D011887:Raffinose; D051381:Rats; D011917:Rats, Inbred Lew; D016042:Transplantation, Heterotopic
D002314:Cardioplegic Solutions; D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose