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Eliminating Xenoantigen Expression on Swine RBC

Wang, Zheng-Yu PhD; Martens, Gregory R. MD; Blankenship, Ross L. BS; Sidner, Richard A. PhD; Li, Ping PhD; Estrada, Jose L. DVM, PhD; Tector, Matthew PhD; Tector, A. Joseph MD, PhD

doi: 10.1097/TP.0000000000001302
Original Basic Science-General

Background: The rapidly improving tools of genetic engineering may make it possible to overcome the 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 were incubated with RBC isolated from various genetically engineered swine or from humans. The level of IgG and IgM binding to these cells were compared using either flow cytometry or a novel mass spectrometric assay.

Results: Mass spectroscopic quantitation of human antibody binding demonstrated that as few as 3 gene inactivations can reduce the levels human antibody binding to swine RBC that is as low as autologous human RBC. Flow cytometry showed that RBC from 2-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 IgM RBC xenoreactivity than alloreactivity.

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.

Mass spectroscopic quantification is used to show that the disruption of GGTA1, CMAH and A4GalNT2 genes results in the near complete elimination of glycans on swine erthrocytes that are recognized by IgM and IgG in human sera.

1 Department of Surgery, Indiana University School of Medicine, Indianapolis, IN.

2 Department of Surgery, Indiana University Health, Indianapolis, IN.

3 Indiana University Health Transplant Institute, Indianapolis, IN.

Received 18 November 2015. Revision received 14 April 2016.

Accepted 20 April 2016.

Indiana University and Indiana University Health Transplant Institute.

A. Joseph Tector has founded Xenobridge, LLC which has applied for patents related to xenotransplantation. R. Sidner receives consulting fees related to xenotransplantation. The other authors declare no conflicts of interest.

Experiments were designed by ZYW, GM, RLB, RAS, MT, and AJT. PL and JLE created the knockout animals. ZYW, GM, RLB, MT, and AJT wrote the manuscript. Data was discussed by all authors, and all authors approved of the final manuscript.

Zheng-Yu Wang, PhD and Gregory R Martens, MD contributed equally.

Abstract was accepted for poster presentation at the American Transplant Congress, 2016, Boston MA.

Correspondence: A. Joseph Tector, MD, PhD, University of Alabama at Birmingham, Department of Surgery-Transplantation, LHRB 752, 1720 2nd Ave S., Birmingham, AL 35294. (jtector@uab.edu).

Supplemental digital content (SDC) is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.transplantjournal.com).

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