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Comparison of viral gene-deleted adenoviral vectors with the E1-deleted adenoviral vector in islets
Comparison of Viral Gene-Deleted Adenoviral Vectors With the E1-Deleted Adenoviral Vector in Islets Z. Guo, J. Shen, D. Mital, Y. Hong, S.C. Jensik, R. Alemany, W.-W. Zhong, and J.W. Williams
A
LTHOUGH adenoviral vector-mediated gene transfer has significant potential for gene therapy, host immune response to virally expressed protein and small insert capacity may limit its clinical application.1 In order to overcome these disadvantages, a new adenoviral vector that lacks all viral genes and contains only the packing signal and ITRs of Ad5 has been developed.2,3 Using the green fluorescent (GFP) gene as a reporter gene, we here investigated the efficiency of this vector (miniAd-GFP) in islets in vitro and ex vivo, and compared it with the E1-deleted adenoviral vector (E1-GFP). MATERIALS AND METHODS Both the E1-GFP and the miniAd-GFP vectors contained the GFP expression cassette composed of the cytomegalovirus (CMV) immediate early enhancer/b-actin promoter and the SV40 polyadenylation signal. AdHb, a helper virus, was used to generate and propagate the miniAd-GFP vector in 293 cells. The islets were isolated according to our previously published isolation protocol.4 The E1-GFP or the mini-GFP was mixed with freshly isolated mouse islets at a multiplicity of infection (MOI) 100:1 for 1 hour. For in vivo study, islets were checked for GFP gene expression 1 day after E1-GFP or miniAd-GFP infection under a fluorescence microscope. For ex vivo study, 300 islets were transplanted into streptozocin (STZ)-induced diabetic mice under the left kidney capsule. The nonfasting blood glucose level of each recipient was measured to monitor the islet graft survival after transplantation. The islet grafts were collected at the 1, 2, and 20-week time point. Immunohistochemistry was performed to evaluate the virus-specific immune response in the islet grafts.
RESULTS AND DISCUSSION
One day after in vitro transfection, the GFP-positive single cells in the miniAd-GFP and the E1-GFP-transfected islets were 9.5 6 2.3% and 11.3 6 1.5%, respectively. Both
0041-1345/99/$–see front matter PII S0041-1345(98)01772-2
miniAd-GFP and E1-GFP and E1-GFP-transfected islet grafts reversed diabetes in syngeneic diabetic mice 3 days posttransplantation. Normal blood glucose level was maintained for over 20 weeks posttransplantation. Hematoxylin and eosin staining showed that no islet destruction was seen by light microscopy at 1, 2, and 20 weeks after transplantation. Immunohistochemically, mild CD4 and CD8-positive lymphocyte infiltration was found in all E1-GFP-transfected islet grafts at 1, 2, and 20 weeks. However, this was not seen in most miniAd-GFP-transfected islet grafts. A major disadvantage of the E1-deleted Ad vector is that it expresses other early and later viral genes and replicates viral DNA. Development of inflammation by the immune response to viral proteins and transgene products after AD-mediated gene transfer is the major reason for the loss of transgene expression. Our results indicate that gene transfer by an adenoviral vector that lacks all viral genes is as efficient as E1-deleted adenoviral vector-mediated gene transfer in islets, and this adenoviral vector is less immunogeneic than the E1-deleted adenoviral vector. REFERENCES 1. Yang Y, Nunes FA, Berencsi K, et al: Proc Natl Acad USA 91:44071, 1994 2. Fisher KJ, Choi H, Burda J, et al: Virology 217:11, 1996 3. Chen H-H, Mack LM, Kelly R, et al: Proc Natl Acad Sci USA 94:1645, 1997 4. Guo Z, Mital D, Shen J, et al: Transplantation 65:1310, 1998 From the Department of General Surgery, Rush-Presbyterian-St Luke’s Medical Center, and Baxter Healthcare Corporation, Chicago, Illinois. Address reprint requests to Z. Guo, Department of General Surgery, Rush-Presbyterian-St Luke’s Medical Center, Chicago IL 60612.
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
794
Transplantation Proceedings, 31, 794 (1999)
A
LTHOUGH adenoviral vector-mediated gene transfer has significant potential for gene therapy, host immune response to virally expressed protein and small insert capacity may limit its clinical application.1 In order to overcome these disadvantages, a new adenoviral vector that lacks all viral genes and contains only the packing signal and ITRs of Ad5 has been developed.2,3 Using the green fluorescent (GFP) gene as a reporter gene, we here investigated the efficiency of this vector (miniAd-GFP) in islets in vitro and ex vivo, and compared it with the E1-deleted adenoviral vector (E1-GFP). MATERIALS AND METHODS Both the E1-GFP and the miniAd-GFP vectors contained the GFP expression cassette composed of the cytomegalovirus (CMV) immediate early enhancer/b-actin promoter and the SV40 polyadenylation signal. AdHb, a helper virus, was used to generate and propagate the miniAd-GFP vector in 293 cells. The islets were isolated according to our previously published isolation protocol.4 The E1-GFP or the mini-GFP was mixed with freshly isolated mouse islets at a multiplicity of infection (MOI) 100:1 for 1 hour. For in vivo study, islets were checked for GFP gene expression 1 day after E1-GFP or miniAd-GFP infection under a fluorescence microscope. For ex vivo study, 300 islets were transplanted into streptozocin (STZ)-induced diabetic mice under the left kidney capsule. The nonfasting blood glucose level of each recipient was measured to monitor the islet graft survival after transplantation. The islet grafts were collected at the 1, 2, and 20-week time point. Immunohistochemistry was performed to evaluate the virus-specific immune response in the islet grafts.
RESULTS AND DISCUSSION
One day after in vitro transfection, the GFP-positive single cells in the miniAd-GFP and the E1-GFP-transfected islets were 9.5 6 2.3% and 11.3 6 1.5%, respectively. Both
0041-1345/99/$–see front matter PII S0041-1345(98)01772-2
miniAd-GFP and E1-GFP and E1-GFP-transfected islet grafts reversed diabetes in syngeneic diabetic mice 3 days posttransplantation. Normal blood glucose level was maintained for over 20 weeks posttransplantation. Hematoxylin and eosin staining showed that no islet destruction was seen by light microscopy at 1, 2, and 20 weeks after transplantation. Immunohistochemically, mild CD4 and CD8-positive lymphocyte infiltration was found in all E1-GFP-transfected islet grafts at 1, 2, and 20 weeks. However, this was not seen in most miniAd-GFP-transfected islet grafts. A major disadvantage of the E1-deleted Ad vector is that it expresses other early and later viral genes and replicates viral DNA. Development of inflammation by the immune response to viral proteins and transgene products after AD-mediated gene transfer is the major reason for the loss of transgene expression. Our results indicate that gene transfer by an adenoviral vector that lacks all viral genes is as efficient as E1-deleted adenoviral vector-mediated gene transfer in islets, and this adenoviral vector is less immunogeneic than the E1-deleted adenoviral vector. REFERENCES 1. Yang Y, Nunes FA, Berencsi K, et al: Proc Natl Acad USA 91:44071, 1994 2. Fisher KJ, Choi H, Burda J, et al: Virology 217:11, 1996 3. Chen H-H, Mack LM, Kelly R, et al: Proc Natl Acad Sci USA 94:1645, 1997 4. Guo Z, Mital D, Shen J, et al: Transplantation 65:1310, 1998 From the Department of General Surgery, Rush-Presbyterian-St Luke’s Medical Center, and Baxter Healthcare Corporation, Chicago, Illinois. Address reprint requests to Z. Guo, Department of General Surgery, Rush-Presbyterian-St Luke’s Medical Center, Chicago IL 60612.
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
794
Transplantation Proceedings, 31, 794 (1999)