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.