MULTI-TRANSGENIC PIGS FOR XENOTRANSPLANTATION

Abstract

Successful development of somatic cell nuclear transfer (cloning) technology in pigs has allowed for precise genetic manipulation of the pig genome. For xenotransplantation applications, pigs have been produced in which both copies of the α1,3-galactosyl transferase (GT) gene were inactivated (GTKO pigs). Analysis of tissues from GTKO pigs demonstrated a complete lack of immunogenic Galα1,3Gal (Gal) sugars, while in vivo pre-clinical studies in nonhuman primates, using cells (i.e. pancreatic islets) or whole organs (heart, kidney, liver, lung), demonstrated the elimination of hyperacute rejection, and prolonged survival compared to wild-type controls. While survival of GTKO xenografts was extended, challenges including induced antibody responses to non-gal antigens, thrombosis, inflammation, and cell-mediated rejection remained, pointing to the need for further genetic modification of the source pig. Towards this goal, through a combination of cloning and breeding, in combination with GTKO, we have produced multi-transgenic pigs (some with 5 different transgenes) with controlled expression of genes for (1) complement regulation to address the humoral response to anti-non-gal targets (DAF, CD46); (2) inhibition of inflammation and thrombosis (TFPI, CD39, thrombomodulin, EPCR); and (3) local protection against the human cellular response (CTLA4Ig, CIITA-DN). For some transgenes a constitutive promoter system can be used for expression in all tissues, such that one animal can be used for multiple transplant applications, however, our results have shown that for certain transgenes, tissue-specific gene expression is preferred. Since inhibition of thrombosis, complement modulation, and suppression of T-cell responses are important to delayed xenograft rejection of both whole organs and islet cell xenografts, pigs have been produced with tissue-specific transgene expression in either the vascular endothelium or endocrine pancreas compartments, or constitutively in all tissues. In vivo results in nonhuman primates have demonstrated complete normalization of blood glucose for up to 1 year in diabetic monkeys, and 8-month survival of multigenic pig hearts in baboons, evidence for the promise of this technology for human clinical applications.