Background:

The rapidly improving tools of genetic engineering may make it possible to overcome humoral immune barrier that prevents xenotransplantation. We hypothesize that levels of human antibody binding to donor tissues from swine must approximate the antibody binding occurring in allotransplantation. It is uncertain if this is an attainable goal. Here we perform an initial analysis of this issue by comparing human antibody binding to red blood cells (RBC) isolated from knockout swine and to allogeneic or autologous human RBC.

Methods:

Human sera (83) were incubated with RBC isolated from humans, wild type pigs and various genetically engineered pigs including: GGTA1 knockout, GGTA1/CMAH knockout and GGTA1/CMAH/β4GalNT2 knockout. The level of IgG and IgM binding to these cells were analyzed using flow cytometry, or SDS-PAGE gel, or a novel mass spectrometric assay.

Results:

Flow cytometry showed that RBC from two-gene knockout swine exhibited less human antibody binding than human blood group O allogeneic RBC in 22% of tested sera. Deletion of a third gene from pigs resulted in 30% of human samples having less IgG and IgMRBC xenoreactivity than alloreactivity. Mass spectrometric quantitation of human antibody binding confirmed that as few as three gene inactivations can reduce the levels human antibody binding to swine RBC that is as low as or lower than to autologous human RBC.

Conclusions:

Xenoantigenicity of swine RBC can be eliminated via gene disruption. These results suggest that the gene knockout approach may be able reduce antigenicity in other pig tissues to levels that enable the xenotransplantation humoral barrier to be overcome.

CITATION INFORMATION: Wang Z.-Y, Martens G, Blankenship R, Sidner R, Li P, Estrada J, Tector M, Tector A. Eliminating Xenoantigen Expression on Swine Red Blood Cells. Am J Transplant. 2016;16 (suppl 3).

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