Selective germline genome edited pigs and their long immune tolerance in Non Human Primates

Abstract

Organ transplantation is the only curative treatment for patients with terminal organ failure, however, there is a worldwide organ shortage. Genetically modified pig organs and tissues have become an attractive and practical alternative solution for the severe organ shortage, which has been made possible by significant progress in xenotransplantation in recent years.The past several decades witnessed an expanding list of genetically engineered pigs due to technology advancements, however, the necessary combination of genetic modifications in pig for human organ xenotransplantation has not been determined. In the current study, we created a selective germline genome edited pig (SGGEP). The first triple xenoantigens (GGTA, B4GAL, and CAMH) knockout somatic cells were generated to serve as a prototype cells and then human proteins were expressed in the xenoantigen knockout cells, which include human complement system negative regulatory proteins (CD46, CD55, and CD59); human coagulation system negative regulatory proteins thrombomodulin (THBD); tissue factor pathway inhibitor (TFPI); CD39; macrophage negative regulatory proteins (human CD47); and natural killer cell negative regulatory human HLA-E. After the successful establishment of SGGEP by the nuclear tranfer, we engrafted SGGEP skin to NHP, up to 25 days graft survival without immunosuppressive drugs was observed. Because a pig skin graft does not impact the success of a subsequent allograft or autograft or vice versa, thus our SGGEP could have a great potential for clinical value to save severe and large area burn patients and the other human organ failure. Therefore, this combination of specific gene modifications is a major milestone and provides proof of concept to initiate investigator-initiated clinical trials (IITs) in severe burn patients with defined processes and governance measures in place and the other clinical application.

Cardiac xenotransplantation: from concept to clinic

“…A summary of GMs proposed for xeno-organ donor pigs is provided in Table 1 . Progress in gene editing technologies facilitated the generation of pigs carrying several of these GMs (reviewed previously 93 ), in some cases up to 11 94 or 12. 95 We focus here on the combinations that have been tested in heterotopic or orthotopic HTx experiments in baboons (Section 3).…”

Pathways to Clinical Cardiac Xenotransplantation

“…Recently, a genetically engineered pig with 11 modifications was reported. 40 The modifications were introduced in 2 steps: first, GGTA1 , CMAH , and B4GALNT2 were simultaneously knocked out using CRISPR/Cas9. Second, the triple knockout cells were cotransfected with 3 PiggyBac vectors harboring multigene expression cassettes (hCD46/hCD55/hCD59, hTHBD/hTFPI/hCD39, and HLA-E/hCD47) and PiggyBac transposase.…”

Select Porcine Elongation Factor 1α Sequences Mediate Stable High-Level and Upregulated Expression of Heterologous Genes in Porcine Cells in Response to Primate Serum

“…Promoters for ubiquitous expression seem to be suitable for expressing human complement regulating genes and immune-cell response factor genes, such as CD46, CD55, human HLAs, and CD47. The ICAM2 promoter has been frequently used for specific expression of anti-coagulation and anti-inflammatory proteins, including tissue factor pathway inhibitor (TFPI), thrombomodulin (THBD), C1 inhibitor, and A20 [ 3 , 19 , 20 ]. In previous studies, we reported the production of GTKO pigs carrying human CD46 (GTKO/CD46) or THBD (GTKO/CD46/THBD) driven by the CMV promoter [ 21 , 22 ], CAG promoters for ubiquitous expression, and the pig ICAM2 promoter for endothelium-specific expression of THBD and CD73 [ 23 ].…”