| Co-Investigator(Kenkyū-buntansha) |
OKADA Hidechika Medical School, Nagoya City University Professor, 医学部, 教授 (30160683)
YOKOYAMA Itsuo Nagoya University School of Medicine M.D, 医学部, 講師 (60240206)
KITO Junzo Nagoya University School of Medicine Professor, 医学部, 教授 (60022802)
MURAMATSU Takashi Nagoya University School of Medicine Professor, 医学部, 教授 (00030891)
NAKASHIMA Izumi Nagoya University School of Medicine Professor, 医学部, 教授 (40022826)
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| Research Abstract |
The prospect of clinical xenotransplantation using pigs as donors holds out a potential solution to the shortage of human organs. The technical advance of genetic engineering has prompted the development of various strategies aimed at preventing xenograft rejection, including (i) expression of species-specific human complement regulatory proteins, (ii) modification of xenogeneic antigen expression and (iii) suppression of endothelial activation. The purpose of this study is to provide the useful information for clinical application of xenotransplantation.
As gene transfection of alpha1,2 fucosyl transferase (alpha1,2 FT), which was capable of competitive inhibition of alpha1,3 galactosyltransferase (alpha1,3 GT), caused the decreased expression of alphaGal antigens in pig cultured cells, we produced alpha1,2 FT transgenic pigs. However, this strain could not be established, since these pigs died of malignant tumor or incomplete development. We also showed the findings that such gene tra
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nsfer decreased not only alphaGal expression but also sialylation in cultured endothelial cells and inhibited the capacity for tube formation. We also demonstrated that alpha1,2 FT gene or alpha1,3 GT ribozyme transfer using adenoviral vectors decreased alphaGal expression in vitro. We are now exploring direct gene replacement of alpha1,3 GT gene with human alpha1,2 FTgene, namely knock-in method.
We analyzed the genomic structure of alpha1,3 GT in pigs and constructed a targeting vector with the aim of inducing alphaGal knockout pigs as soon as porcine embryonic stem cells or nuclear transplantation are available. Some alternative splicing variants without exon 8 or mutant cDNA (two mutations in the catalytic region ; exon 9) were also devoid of enzymatic activity. These observations provides a new strategy for reducing alphaGal antigen expression in a dominant-negative effect. With regard to non-alphaGal antigens, Hanganutziu-Deicher (HD) antigens were found to be expressed on the vascular endothelial cells of pig and baboon kidneys, but not in the human kidney, suggesting that HD antigens could elicit a strong humoral response in pig-to-human transplantation, even if HAR due to alphaGal epitopes is avoided. The pig-to-baboon transplantation model cannot demonstrate this possibility, because the baboon expresses HD antigens and does not appear to produce anti-HD antibodies.
We examined baboon sera to establish pig-to-baboon transplantation model. Some baboon sera showed the positivity for herpes B-virus using ELISA,however, this was ascribed to crossreactivity with human herpes simplex virus. Blood test revealed that all were within normal limits. All baboon sera contained anti-pig (anti-alphaGal) IgM antibodies as much as human sera, although anti-pig IgG and IgA in baboon sera were less than those in human sera. Usefulness and safety of pig-to-baboon transplantation model was demonstrated for clinical application. Less
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