In vivo gene transfer of endo-beta-galactosidase C removes alphaGal antigen on erythrocytes and endothelial cells of the organs.
BACKGROUND
The presence of Galalpha1-3Galbeta1-4GlcNAc (alphaGal) in pigs is a formidable barrier for pig-to-primate xenotransplantation. We have reported that administration of recombinant endo-beta-galactosidase C (EndoGalC) removes alphaGal on porcine erythrocytes and kidneys. The present study examined the effects of EndoGalC gene therapy on alphaGal suppression.


METHODS
Naked plasmid DNA encoding Igkappa-EndoGalC was given to rats by rapid tail vein injection. The expression of alphaGal in the heart and kidney were studied by lectin staining followed by computer-assisted quantitative analyses. alphaGal expression on erythrocytes was determined by flow cytometric analyses. Enzymatic activity of EndoGalC in the serum was also determined by evaluating the capacity of EndoGalC in removing alphaGal epitopes. Elimination of alphaGal was further studied by injecting antibodies against alphaGal to rats 2 days after the gene transfer.


RESULTS
Administration of 1 mg of Igkappa-EndoGalC/pCAGGS plasmid eliminated alphaGal from the vascular endothelium of the heart and kidney on day 1 and day 2. Between days 4 and 7, alphaGal started to reappear, but remained suppressed. No serious adverse effect was observed in rats treated with EndoGalC. Flow cytometric analyses showed that EndoGalC digested 97% of alphaGal on erythrocytes when measured 4 days after the gene transfer. Enzymatic activity of EndoGalC in the serum peaked on day 1, and significant levels were still observed on day 7. When antibodies against alphaGal were given, rats treated with EndoGalC showed no change, while all control rats died within 40 min.


CONCLUSIONS
The present study demonstrates the potential of an EndoGalC gene transfer, using a hydrodynamics-based delivery system, in eliminating alphaGal from endothelial cells in vivo. The results also ensured that EndoGalC is not harmful suggesting that the production of pigs overexpressing EndoGalC would be a reasonable alternative to pigs deficient in alpha1,3galactosyltransferase.
Department of Clinical Immunology, Nagoya University Graduate School of Medicine, Tsurumaicho, Nagoya, Japan.
Yusuke
Y
Miki
Shoichi
S
Maruyama
DaGe
D
Liu
Takaaki
T
Kobayashi
Fumihiko
F
Sato
Hideaki
H
Shimizu
Sawako
S
Kato
Waichi
W
Sato
Yoshiki
Y
Morita
Yukio
Y
Yuzawa
Takashi
T
Muramatsu
Seiichi
S
Matsuo
D000818:Animals; D003001:Cloning, Molecular; D004730:Endothelium, Vascular / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D004912:Erythrocytes / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D008297:Male; D009206:Myocardium / Q000378:metabolism; D009844:Oligosaccharides / Q000276:immunology*; D051381:Rats; D011917:Rats, Inbred Lew; D013552:Swine; D013997:Time Factors; D014183:Transplantation, Heterologous / Q000276:immunology
D000939:Epitopes; D007145:Immunoglobulin kappa-Chains; D009844:Oligosaccharides; C404741:galactopyranosyl-1-3-galactopyranosyl-1-3(4)-N-acetylglucosamine; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase