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Miniature swine expressing human CD47 to enhance bone marrow engraftment in non-human primates

Aseda Tena¹, Nicole Turcotte¹, Angelo A. Leto Barone¹, Scott Arn¹, Steven L. Terlouw², John R. Dobrinsky², David H. Sachs¹, Robert J. Hawley¹

¹Transplantation Biology Research Center, Massachusetts General Hospital, Boston, MA; ²Minitube of America, Genetics, Verona, WI; United States

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 our progress in generating 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. GalT locus was chosen for ubiquitous expression of hCD47 gene because loss of GalT function is also advantageous in pig-to-primate transplantation. 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 and transferred at Minitube International Center for Biotechnology. Of three established pregnancies, two early trimester pregnancies were terminated for fetal collection and the third continued to natural term. The early second trimester cloned fetuses were obtained following hysterectomy and analyzed for hCD47 and gal epitope expression.

Results: RT-PCR analysis and Fluorescent Activated Cell Sorter (FACS) of fetal fibroblast lines showed that all three fetuses expressed hCD47 and were GalT null. Further, expression of hCD47 expression was examined in a cell relevant to bone-marrow transplantation; c-kit+ cells were isolated from the liver of the CT fetuses and placed in culture for colony forming unit assay (CFU). RT-PCR analysis of erythroid and myeloid colonies confirmed expression of hCD47 in bone marrow progenitor cells. Also, genomic PCRof fetal DNAconfirmed proper transgenic locus structure in the cloned fetuses.

Conclusion: We have been able to generate early genetically modified fetuses which have the expected gene expression patterns. The late transgenic fetuses which were lost 14 days before natural birth were also GalT null and hCD47 positive. The codominant expression of hCD47 gene in the GalT locus does not appear to have deleterious effects on fetal development.