KONDO Akihiro TAKARA SYUZOU CO., LTD Biotechnology Research Laboratories staff, バイオ研究所, 主任研究員
OKABE Masaru OSAKA Univ.Med.schl.professor, 遺伝情報実験施設, 教授 (30089875)
SHIRAKURA Ryouta OSAKA Univ.Med.schl.professor, 医学部, 教授 (00116047)
井原 義人 大阪大学, 医学部, 助手 (70263241)
|Budget Amount *help
¥10,800,000 (Direct Cost : ¥10,800,000)
Fiscal Year 1998 : ¥2,800,000 (Direct Cost : ¥2,800,000)
Fiscal Year 1997 : ¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1996 : ¥5,000,000 (Direct Cost : ¥5,000,000)
Xenotransplantation ofers a potential oto the world-wide shortage of organs for transplantation. In this study, in addition to attempting to reduce the alpha-galactosyl epitope by competitive enzymes such as alpha 1,2fucosyltransferase (alpha
1,2FT), alpha 2,3sialyltransferase(alpha 2,3ST)and a 2, 6sialyltransferase(alpha 2, 6ST), we focused of N-acetylucosaminyltaransferase III(GnT-III). Our strategy to diminish the antigenicity of swine cells is directed, not only at the alpha-galactosyl epitope, but to other unknown epiopes, as well.
Introductio of GnT-III gene into swine endothelial cells (SEC) reduced the susceptibility to normal human natural antibodies as revealed by flow cytometric analysis as well as 1B4 lectin binding to the alpha -galactosyl epitope. Westeun blot analysis indicated that proteins smaller than 66 kDa and diminished reactivity to NHS and 1B4 lectin. GnT-III.
alpha 1,2 FT.alpha 2,3 ST and alpha 2,6 ST, putative enzymes which reduce the alpha-galactosyl epitope by i
ntracellular competition with the alpha 1,3 galactosyltransferase for a common acceptor substrate. The data suggest that the alpha 2,3 ST and alpha 2,6 ST effectively reduced the alpha -falactosyl epitope as well or better than the alpha 1,2 FT.and that the reductin of the alpha -galactosyl epitope is due, not only to substrate competition but also to an overall reduction of endogenous alpha 1,3 galactosyltransferase enzyme activity.
Additionally, SEC were co-transfected with cDNAs encoding two different types of glycosyltransferase involving GnT-III and alpha 2,3 ST.As expected, the combined transfer appeared to be quite effective as judged by flow cytometric analysis and a cytotoxicity assay. In vivo study, we have demonstrated the remodeling of xenoantigen by making transgenic mice with GnT-III.
Both western and IB4 lectin blotting revaled that whole pwoteins, especially those under 66 kDa had diminished reactivity to NHS and IB4 lectin, respectively, in many organs in which the endogenious enzyme activity is low. FAGS analysis of the spleen cells revealed marked reduction of antigenicity to human natural antibodies. These results imply the importance of this approach for clinical xenotransplantatio. Less