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

Abstract

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.

Current Barriers to Clinical Liver Xenotransplantation

“…Additionally, further optimization of transgenic hTM expression in organ source pig livers would provide valuable information regarding its utility. Indeed, novel approaches in genetic engineering of pig cells and testing capabilities of their phenotypes without making those genetically modified pigs each time for each genetic modification will reduce the time and effort to understand which genetic modification combinations would be optimal for preclinical/clinical trials ( 56 , 57 ). With the strong data in preclinical trials in pig-to-NHP xenotransplantation trials as well as recent progress in ‘first-in-man’ genetically-engineered pig heart xenotransplantation into a patient, we would also strongly suggest that complement regulatory proteins, such as CD55 and CD46 as well as hTM and human EPCR expression would be helpful in extending the xenograft survival ( 13 , 42 , 58 ).…”

Strategies to induce natural killer cell tolerance in xenotransplantation

“…Hein et al reported that decreased SLA-I expression (SLA-I low) did not decrease NK cell activation (61). A recent study demonstrated that SLA-I is not an inhibitory ligand for human NK cells using an immortalized pEC with SLA-I gene disruption (5). The absence of inhibitory ligands on pEC triggers human NK cell destruction.…”

“…Xenotransplantation represents one of the most promising approaches. Elimination of major xenoantigens on xenografts by gene-editing tools has proven to be an effective approach to preventing hyperacute xenograft rejection (HXR) (3)(4)(5)(6). Earlier this year, the first pigto-human heart transplantation was performed and supported the patient's life for two months.…”

Advances in CRISPR-Based Functional Genomics and Nucleic Acid Detection in Pigs

“…Pigs carrying multi-fold xenoprotective transgenes and knockouts of xenoreactive antigens have been generated ( Fischer et al, 2016 ; Zhang et al, 2018 ; Fischer et al, 2020 ; Fu et al, 2020 ; Shim et al, 2021 ; Yue et al, 2021 ), with great potential to completely eliminate immunological barriers. It remains a challenge, however, to effectively assess the human immune response induced by various genetic modifications and to identify the ideal gene combinations ( Li P. et al, 2021 ). Recently, the world’s first porcine-to-human transplantation was performed at the University of Maryland Medical Center, successfully transplanting a genetically modified porcine heart into a 57-year-old man with end-stage heart disease, and the patient lived for two months after the transplant ( Shah and Han, 2022 ).…”