- Email: [email protected]
Effectiveness of adenovirus-mediated gene transfer with CTLA4Ig in allo and xeno-transplantation
Effectiveness of Adenovirus-Mediated Gene Transfer With CTLA4Ig in Allo and Xeno-Transplantation S. Hayashi, L. Dai-Kaku, Y. Namii, T. Kato, T. Tokoro, T. Kobayashi, I. Yokoyama, H. Hamada, and A. Nakao
C
TLA4Ig is a recombinant fusion protein containing the extracellular domain of human CTLA4 fused to the human Ig1 region, which effectively binds the B7 molecule, interrupting the costimulatory pathway. It has been reported that CTLA4Ig can inhibit the rejection reaction both in allotransplantation and xenotransplantation. Adenovirus-mediated gene transfer is an excellent vehicle in the clinical setting of gene therapy due to the high efficiency of gene transduction to the organ. We examined the effect of adenovirus-mediated gene transfer with CTLA4Ig gene in organ transplantation of rats. MATERIALS AND METHODS In allotransplant experiments, DA and LEW rats were used as donor and recipient; whereas, in xenotransplant experiments, Syrian golden hamsters and Wister rats were used as donor and recipient, respectively. Adenoviral vectors with human CTLA4Ig gene (AdexhCTLA4Ig) and with beta galactosidase gene (AdexLacZ) were constructed by homologous recombination using cosmid casette and parental viruses. 1 ⫻ 109 PFU of adenoviral vectors were injected via the peripheral vein of the recipient rats to transduce hCTLA4Ig or LacZ gene 7 or 14 days before transplantation. The blood concentration of CTLA4Ig was analyzed by binding assay with NIH3T3 cells transduced with B7.1 cDNA (B7.1 transfectants). Heterotopic cardiac transplantation (Tx) was performed using the cuff technique in DA–to–LEW rats and hamster–to–Wister rats. FK506 was used for 7 days or 30 days, respectively.
RESULTS
In cardiac allotransplantation, the mean graft survival time (MST) in control rats administered with AdexLacZ was 6.0 days while the MST rats with AdexhCTLA4Ig was 80.8 days. Sixty percent of rats with AdexhCTLA4Ig survived longer than 100 days after Tx with a normally beating heart. The blood concentration of hCTLA4Ig in rats with AdexhCTLA4Ig was over 20 mg/mL before and 60 days after Tx. However, that showed almost zero 100 days after Tx. In cardiac xenotransplantation, MST in control rats administered with AdexLacZ was 3.4 days while in rats with AdexhCTLA4Ig the MST was 5.6 days. MST in rats with FK506 (1 mg/kg/day IM) was comparable to that in control rats. However, MST in rats with both AdexhCTLA4Ig and
FK506 (1 mg/kg/day IM for 30 days after Tx) showed over 30 days, the maximal survival of which was 55 days. In histological examination of the spleen, the size was prominently reduced without normal structure such as lymphocyte component after adenovirus-mediated CTLA4Ig gene transfer.
DISCUSSION
This study clearly showed that adenovirus-mediated gene transfer with CTLA4Ig gene is effective to inhibit rejection reaction in both allo- and xeno-transplantation. In xenotransplant experiments, the cardiac grafts in rats with AdexhCTLA4Ig significantly survived longer than those in control and FK506 –treated rats. It has been reported that antibody formation by splenic B cells has an important role in humoral immune response by T– cell independent mechanism, followed by graft destruction in hamster-to-rat xenotransplantation.1 After escaping antibody-mediated rejection in early stage, the grafts are exposed to T– cell mediated rejection. Considering that the spleen has an enormous alteration with the elimination of lymphocyte component after adenovirus-mediated CTLA4Ig gene transfer, antibody formation by T– cell independent mechanism will be influenced by CTLA4Ig gene transfer. It has been well known that adenoviral vector is effective for in vivo gene therapy due to high transduction efficiency, and gene therapy programs have already started in the fields such as genetic diseases, malignancies, and ischemic arterial diseases using this vector. In small animals like rodents, the gene transduction to the liver is susceptible by adenovirusmediated gene transfer; however, our investigations have demonstrated the transduction efficiency in large animals inferior to that in small ones (data not shown). Now we have examined the improved adenovirus–mediated gene
From the Department of Surgery II, Nagoya University School of Medicine and Cancer Chemotherapy Center, National Cancer Institute, Nagoya, Japan. Address reprint requests to Shuji Hayashi, Department of Surgery II, Nagoya University School of Medicine, 65 Tsurumaicho, Showa-ku, Nagoya 466, Japan.
0041-1345/00/$–see front matter PII S0041-1345(00)01775-9
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2520
Transplantation Proceedings, 32, 2520–2521 (2000)
ADENOVIRUS-MEDIATED GENE
transfer to transduce genes selectively using the strategy of viral tropism in transplanted organs. In this study, we demonstrated the effect of in vivo gene therapy with Adex CTLA4Ig to the recipient of allo and xeno-transplant model; whereas, recent investigations have reported the adenovirus–mediated CTLA4Ig gene transfer to the donor with an excellent graft survival.2,3 The continuous injection of CTLA4Ig protein can induce the suppression of allo and xeno–immune response, but the adenovirus–mediated CTLA4Ig gene transfer can follow the long–term immunosuppressive status by single use. The adverse effect by
2521
adenovirus–mediated CTLA4Ig gene transfer remains to be determined; however, it is a promising immunosuppressive strategy for the clinical setting of gene therapy in the future. REFERENCES 1. Yasutomi M, Ito M, Hayashi S, et al: J Heart Lung Transplant 17:452, 1998 2. Olthoff KM, Da Chen X, Gelman A, et al: Transplant Proc 29:1030, 1997 3. Namii Y, Hayashi S, Yokoyama I, et al: Transplant Proc 29:1738, 1997
C
TLA4Ig is a recombinant fusion protein containing the extracellular domain of human CTLA4 fused to the human Ig1 region, which effectively binds the B7 molecule, interrupting the costimulatory pathway. It has been reported that CTLA4Ig can inhibit the rejection reaction both in allotransplantation and xenotransplantation. Adenovirus-mediated gene transfer is an excellent vehicle in the clinical setting of gene therapy due to the high efficiency of gene transduction to the organ. We examined the effect of adenovirus-mediated gene transfer with CTLA4Ig gene in organ transplantation of rats. MATERIALS AND METHODS In allotransplant experiments, DA and LEW rats were used as donor and recipient; whereas, in xenotransplant experiments, Syrian golden hamsters and Wister rats were used as donor and recipient, respectively. Adenoviral vectors with human CTLA4Ig gene (AdexhCTLA4Ig) and with beta galactosidase gene (AdexLacZ) were constructed by homologous recombination using cosmid casette and parental viruses. 1 ⫻ 109 PFU of adenoviral vectors were injected via the peripheral vein of the recipient rats to transduce hCTLA4Ig or LacZ gene 7 or 14 days before transplantation. The blood concentration of CTLA4Ig was analyzed by binding assay with NIH3T3 cells transduced with B7.1 cDNA (B7.1 transfectants). Heterotopic cardiac transplantation (Tx) was performed using the cuff technique in DA–to–LEW rats and hamster–to–Wister rats. FK506 was used for 7 days or 30 days, respectively.
RESULTS
In cardiac allotransplantation, the mean graft survival time (MST) in control rats administered with AdexLacZ was 6.0 days while the MST rats with AdexhCTLA4Ig was 80.8 days. Sixty percent of rats with AdexhCTLA4Ig survived longer than 100 days after Tx with a normally beating heart. The blood concentration of hCTLA4Ig in rats with AdexhCTLA4Ig was over 20 mg/mL before and 60 days after Tx. However, that showed almost zero 100 days after Tx. In cardiac xenotransplantation, MST in control rats administered with AdexLacZ was 3.4 days while in rats with AdexhCTLA4Ig the MST was 5.6 days. MST in rats with FK506 (1 mg/kg/day IM) was comparable to that in control rats. However, MST in rats with both AdexhCTLA4Ig and
FK506 (1 mg/kg/day IM for 30 days after Tx) showed over 30 days, the maximal survival of which was 55 days. In histological examination of the spleen, the size was prominently reduced without normal structure such as lymphocyte component after adenovirus-mediated CTLA4Ig gene transfer.
DISCUSSION
This study clearly showed that adenovirus-mediated gene transfer with CTLA4Ig gene is effective to inhibit rejection reaction in both allo- and xeno-transplantation. In xenotransplant experiments, the cardiac grafts in rats with AdexhCTLA4Ig significantly survived longer than those in control and FK506 –treated rats. It has been reported that antibody formation by splenic B cells has an important role in humoral immune response by T– cell independent mechanism, followed by graft destruction in hamster-to-rat xenotransplantation.1 After escaping antibody-mediated rejection in early stage, the grafts are exposed to T– cell mediated rejection. Considering that the spleen has an enormous alteration with the elimination of lymphocyte component after adenovirus-mediated CTLA4Ig gene transfer, antibody formation by T– cell independent mechanism will be influenced by CTLA4Ig gene transfer. It has been well known that adenoviral vector is effective for in vivo gene therapy due to high transduction efficiency, and gene therapy programs have already started in the fields such as genetic diseases, malignancies, and ischemic arterial diseases using this vector. In small animals like rodents, the gene transduction to the liver is susceptible by adenovirusmediated gene transfer; however, our investigations have demonstrated the transduction efficiency in large animals inferior to that in small ones (data not shown). Now we have examined the improved adenovirus–mediated gene
From the Department of Surgery II, Nagoya University School of Medicine and Cancer Chemotherapy Center, National Cancer Institute, Nagoya, Japan. Address reprint requests to Shuji Hayashi, Department of Surgery II, Nagoya University School of Medicine, 65 Tsurumaicho, Showa-ku, Nagoya 466, Japan.
0041-1345/00/$–see front matter PII S0041-1345(00)01775-9
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2520
Transplantation Proceedings, 32, 2520–2521 (2000)
ADENOVIRUS-MEDIATED GENE
transfer to transduce genes selectively using the strategy of viral tropism in transplanted organs. In this study, we demonstrated the effect of in vivo gene therapy with Adex CTLA4Ig to the recipient of allo and xeno-transplant model; whereas, recent investigations have reported the adenovirus–mediated CTLA4Ig gene transfer to the donor with an excellent graft survival.2,3 The continuous injection of CTLA4Ig protein can induce the suppression of allo and xeno–immune response, but the adenovirus–mediated CTLA4Ig gene transfer can follow the long–term immunosuppressive status by single use. The adverse effect by
2521
adenovirus–mediated CTLA4Ig gene transfer remains to be determined; however, it is a promising immunosuppressive strategy for the clinical setting of gene therapy in the future. REFERENCES 1. Yasutomi M, Ito M, Hayashi S, et al: J Heart Lung Transplant 17:452, 1998 2. Olthoff KM, Da Chen X, Gelman A, et al: Transplant Proc 29:1030, 1997 3. Namii Y, Hayashi S, Yokoyama I, et al: Transplant Proc 29:1738, 1997