Miniature Swine Expressing Human CD47 to Enhance Bone Marrow Engraftment in Non-Human Primates

Abstract

Background: Tolerance induction via bone marrow chimerism has been successfully applied to small and large allogeneic models. However, in xenogenic models (pig-to-primate) host macrophages participate in the clearance of porcine bone marrow cells when transplanted into baboons, hindering the ability to achieve mixed chimerism. CD47 interacts with SIRPa receptors on macrophages to inhibit their activation, but this interaction is species-specific. Recent experiments indicate that expression of human CD47 on porcine cells can inhibit phagocytosis by primate macrophages. We report the generation of hCD47 transgenic miniature swine as potential pig-to-primate bone marrow donors. Methods: We constructed a knock-in vector for expression of the human CD47 gene from EF1a promoter with concomitant disruption of the a-1,3-galactosyltransferase (GalT) locus. Selectable targeted cells, with loss of gal epitope and gain of hCD47 expression were generated by transfecting GalT heterozygous fibroblast cells. Somatic cell cloned embryos were generated using Chromatin Transfer technology and transferred into recipient sows at Minitube International Center for Biotechnology. Two early trimester pregnancies were terminated for fetal collection and analysis. Established secondary fetal fibroblasts were then used for a second embryo reconstruction and transfer. Results: RT-PCR and Fluorescent Activated Cell Scan (FACS) analysis of fetal fibroblast lines showed that all fetuses expressed hCD47 and were GalT null. Further, genomic PCR of fetal DNA confirmed proper transgenic locus structure in the cloned fetuses. Transfer of embryos reconstructed using transgenic fetal fibroblasts has resulted in the birth of two healthy transgenic piglets to date. The newborn transgenic piglets also had the desired phenotype; GalT null and hCD47 expression on all peripheral white blood cell lineages. Colony forming unit assay (CFU) analysis of transgenic piglets also confirmed the expression of hCD47 in bone marrow progenitor cells. Also, in preliminary transplant experiments of ckit+ bone marrow progenitors from transgenic piglets in NOD scid IL2 receptor gamma chain knockout mice (NSG) we could detect porcine cells that were hCD47 positive in the peripheral blood and bone marrow of the recipients. Conclusion: We have been able to successfully generate the first transgenic piglets with widespread expression of the human CD47 gene. The animals appear to be healthy and have normal development. Initial transplant experiments in NSG mice recipients show that hCD47 is advantageous for porcine cells to engraft in the bone marrow.