Genetic engineering of porcine endothelial cell lines for evaluation of human-to-pig xenoreactive immune responses

• Published in: Scientific reports Vol. 11; no. 1; p. 13131
• Main Authors: Li, Ping, Walsh, Julia R., Lopez, Kevin, Isidan, Abdulkadir, Zhang, Wenjun, Chen, Angela M., Goggins, William C., Higgins, Nancy G., Liu, Jianyun, Brutkiewicz, Randy R., Smith, Lester J., Hara, Hidetaka, Cooper, David K. C., Ekser, Burcin
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 23-06-2021; Nature Publishing Group; Nature Portfolio
• Subjects: 631/1647/1511; 631/250/1854; 631/250/2504; Antigenicity; Cell activation; Cell lines; Clinical trials; CRISPR; Endothelial cells; Genetic engineering; Humanities and Social Sciences; Immune response; multidisciplinary; Natural killer cells; Science; Science (multidisciplinary); Swine; Transplantation; Xenoantigens; Xenografts; Xenotransplantation; β2 Microglobulin
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-021-92543-y

Xenotransplantation (cross-species transplantation) using genetically-engineered pig organs offers a potential solution to address persistent organ shortage. Current evaluation of porcine genetic modifications is to monitor the nonhuman primate immune response and survival after pig organ xenotransplantation. This measure is an essential step before clinical xenotransplantation trials, but it is time-consuming, costly, and inefficient with many variables. We developed an efficient approach to quickly examine human-to-pig xeno-immune responses in vitro. A porcine endothelial cell was characterized and immortalized for genetic modification. Five genes including
GGTA1
,
CMAH
,
β4galNT2
,
SLA-I α chain,
and
β2-microglobulin
that are responsible for the production of major xenoantigens (αGal, Neu5Gc, Sda, and SLA-I) were sequentially disrupted in immortalized porcine endothelial cells using CRISPR/Cas9 technology. The elimination of αGal, Neu5Gc, Sda, and SLA-I dramatically reduced the antigenicity of the porcine cells, though the cells still retained their ability to provoke human natural killer cell activation. In summary, evaluation of human immune responses to genetically modified porcine cells in vitro provides an efficient method to identify ideal combinations of genetic modifications for improving pig-to-human compatibility, which should accelerate the application of xenotransplantation to humans.