Core D: Animal Production Core

Massachusetts General Hospital, Boston MA

Investigators

Abstract

ABSTRACT Genetic manipulation of the pig genome through a combination of sophisticated gene editing tools and somatic cell nuclear transfer and offers the opportunity to provide an unlimited source of human-compatible donor organs, cells, and tissues for transplantation. As the Pig Core, Revivicor will apply their advanced transgenic pig production platform to provide the Genetically Engineered (GE) source pig organs (lung, kidney, heart) necessary to meet the goals of the specific aims in these projects. Elimination of immunogenic Galα1,3Gal (Gal) sugars through inactivation of the α1,3- galactosyl transferase gene (GTKO) was a critical first step, resulting in the elimination of hyperacute rejection, and prolonged survival of xenografts in non-human primates, compared to wild-type controls. To provide further protection from innate immunity and nonGal- mediated humoral rejection, next-generation pigs were produced with further knockout of the beta-4- GalNT2 gene (double knockout or DKO pigs), and thus lacked the α-Gal antigen and were null for the Sda antigen. Pigs with a third sugar gene knocked out (triple knockout or TKO), with added CMAH-knockout (null for Neu5Gc), as well as, pigs with knockout of the human growth hormone receptor gene (GHRKO) will be included to create smaller donor pigs, to prevent rapid organ growth, which is valuable for both preclinical studies and for eventual clinical application. Constitutive high-level expression of the human complement- regulatory genes, hCD46 and hCD55, will also be included in the donor pigs for added protection from antibody directed at any other innate or adaptive immune targets. In addition, for inhibition of coagulation and thrombosis, human anticoagulant genes, endothelial protein C receptor (EPCR) and thrombomodulin (TBM), under endothelial cell-specific promoter systems, were added, as well as human hemeoxygenase-1 (HO1) as an anti-inflammatory agent, and human CD47 to further inhibit activation of monocytes, resulting in pigs 10-GE genetic modifications. In addition, HLA-E/beta-2 microglobulin has been added to the 9GE and 10-GE pig platform, and porcine vWF will be ‘humanized’ through use of Crispr/CAS9 technology on the 9GE and 10-GE backgrounds as these are modifications that are highly likely to be clinically useful for islet and whole organ transplants. Further modification of the source pig could in the future include the addition of the programmed death ligand 1 (PDL-1) transgene to inhibit T cell activation.