Profiling Human CD55 Transgene Performance Assist in Selecting Best Suited Specimens and Tissues for Swine Organ Xenotransplantation

SIMPLE SUMMARY: The unbalance between availability and needs of human organs has drawn researchers’ attention to xenotransplantation as an option to cope with this shortage. Pig organs have received substantial attention for being comparable to human’s; nevertheless, compatibility constrains still block clinical applications. Transgenesis of human complement regulatory proteins, including the CD55 gene and its product the decay-accelerating factor (DAF), has been proposed to overcome xenorejection. This line of research has obtained interesting results along the years; however, most works assessing the impact of this strategy for xenotransplantation are limited to analyzing gene expression and assessing resistance to conventional serum challenge hemolysis assays, which provide somewhat reduced information prior to surgery. In this work, we tried to expand the analysis of the hCD55 transgene performance beyond common practice and into a better molecular understanding of its impact in xenotransplantation. We determined hCD55 gene expression, as well as hDAF protein presence, in different organs from five transgenic pigs, comparing readings from organs worthy for transplantation and other non-valuable organs and tissues. We also assessed the ability of transgenic cells, compared to non-transgenic, to withstand hemolysis and cytolysis. Finally, we made an effort to establish potential correlations between the hCD55 mRNA and hDAF protein levels detected. ABSTRACT: Xenotransplantation of pig organs receives substantial attention for being comparable to human’s. However, compatibility constraints involving hyper-acute rejection (HAR) still block clinical applications. Transgenesis of human complement regulatory proteins has been proposed to overcome xenorejection. Pigs expressing human-CD55 have been widely tested in experimental surgery. Still, no standardized method has been developed to determine tissue expression of human decay-accelerating factor (DAF), hCD55’s product, or to predict the ability to overpass HAR. Here we describe objective procedures addressing this need. Organs and tissues from five hCD55 transgenic pigs were collected and classified according to their xenotransplantation value. The ability to overcome HAR was assessed by classical complement pathway hemolysis assays. Quantitative PCR mRNA expression and Western blot protein level studies were performed. Real-time cytotoxicity assays (RTCA) on fibroblast cultures exposed to baboon and human sera informed on longer-term rejection dynamics. While greater hCD55/DAF expression correlated with better performance, the results obtained varied among specimens. Interestingly, the individual with highest mRNA and protein levels showed positive feedback for hCD55 transcript after challenge with human and baboon sera. Moreover, hCD55 expression correlated to DAF levels in the liver, lung and intestine, but not in the heart. Moreover, we found significant correlations among valuable and non-valuable tissues. In sum, the methodology proposed allows us to characterize the hCD55 transgene functioning and performance. Moreover, the correlations found could allow us to predict hCD55/DAF expression in surrogate tissues, thus eliminating the need for direct biopsies, resulting in preservation of organ integrity before xenotransplantation.