In vivo gene transfer of endo-β-galactosidase C removes αGal antigen on erythrocytes and endothelial cells of the organs

:  Background:  The presence of Galα1-3Galβ1-4GlcNAc (αGal) in pigs is a formidable barrier for pig-to-primate xenotransplantation. We have reported that administration of recombinant endo-β-galactosidase C (EndoGalC) removes αGal on porcine erythrocytes and kidneys. The present study examined the effects of EndoGalC gene therapy on αGal suppression. Methods:  Naked plasmid DNA encoding Igκ-EndoGalC was given to rats by rapid tail vein injection. The expression of αGal in the heart and kidney were studied by lectin staining followed by computer-assisted quantitative analyses. αGal 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 αGal epitopes. Elimination of αGal was further studied by injecting antibodies against αGal to rats 2 days after the gene transfer. Results:  Administration of 1 mg of Igκ-EndoGalC/pCAGGS plasmid eliminated αGal from the vascular endothelium of the heart and kidney on day 1 and day 2. Between days 4 and 7, αGal 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 αGal 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 αGal 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 αGal 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 α1,3galactosyltransferase.