Establishment of gene‐edited pigs expressing human blood‐coagulation factor VII and albumin for bioartificial liver use

Abstract

Background and Aim Bioartificial livers (BALs) are considered as a solution to bridge patients with acute liver failure to liver transplantation or to assist in spontaneous recovery for patients with end‐stage liver disease. Pig is the best donor of hepatocytes for BALs in clinical trials, because metabolic and detoxification function of its liver are close to human. However, using pig hepatocytes for BALs remains controversial for safety concern owing to nonhuman proteins secretion. Herein, we attempt to establish modified pigs expressing humanized liver proteins, blood‐coagulation factor VII (F7), and albumin (ALB). These pigs should also be porcine endogenous retrovirus subtype C (PERV‐C) free so that their ability of transmitting PERV to human could be diminished seriously. Methods We devised both homology‐dependent and independent knock‐in approaches to insert a fusion of hF7 and hALB gene downstream the site of pig endogenous F7 promoter in pig fetal fibroblasts negative for PERV‐C. The modified pigs were then generated through somatic cell nuclear transfer. Results We obtained 14 and 10 cloned pigs by homology‐dependent and independent approaches, respectively. Among them, 19 cloned pigs were with expected gene modification and 13 are alive to date. These modified pigs can successfully express hF7 and hALB in the liver and serum, and the expressed hF7 exhibits normal coagulation activity. Conclusions The gene‐edited pigs expressing hF7 and hALB in the liver were generated successfully. We anticipate that our pigs could provide an alternative cell source for BALs as a promising treatment for patients with acute liver failure.

Pig Coat Color Manipulation by MC1R Gene Editing

“…This result indicates HR remains a low-efficiency event in primary fibroblast, even with the aid of CRISPR. Previous reports showed that CRISPR-mediated HR occurred in an efficiency range from 0 to 41.2% in pig fetal fibroblast with a very low rate in most cases, far less than the CRISPR-mediated knockout efficiency [ 14 , 15 , 16 ].…”

Current status of the application of gene editing in pigs

“…to reduce complement activity in xenotransplantation [ 41 ]. Although most knock-in pigs have been generated by SCNT using gene-edited somatic cells carrying transgenes as donor cells [ 31 , 43 , 44 , 104 , 105 , 106 , 107 , 108 , 109 ], cytoplasmic microinjection of gene editors can also be used to successfully generate knock-in pigs [ 49 , 110 ]. However, knock-in pigs have not been successfully generated by electroporation-mediated methods, because the introduction of large transgenes for knock-in is difficult using electroporation alone in pigs, as described above.…”

Stem cell therapies and benefaction of somatic cell nuclear transfer cloning in COVID-19 era

“…Targeted changes in the animal genome are likely to initiate a new era of bio-pharming [ 195 ]. Healthcare applications include production of target-specific stem cells and therapeutic proteins [ 196 ], mAbs [ 190 ], released into the milk of animals, to the use of genetically modified (GM) animals to produce organs for xenotransplantation are envisaged.…”