Introduction: The first transplant of a pig heart into a human patient supported life for 60 days. We present here the genotype and phenotype of the donor pig and post-transplant heart. Nine genetic edits were introduced to mitigate rejection and one to limit post-transplant organ growth. The 3 major xenoantigen genes (GGTA1, b4GalNT2, CMAH were knocked out (KO) to reduce hyperacute rejection (HAR), which was further reduced by transgenes for human complement regulatory proteins (CD46, DAF). Dysregulated coagulation was controlled by transgenes for human thrombomodulin (TBM) and human endothelial protein C receptor (EPCR). Human heme oxygenase-1 (HO1) was added to reduce inflammation, and human CD47 was expressed to block human macrophage attack. These 6 transgenes were co-expressed using a multicistronic vector platform.

Methods: GGTA1 was KO’d by insertional mutagenesis while b4GalNT2, CMAH and GHR were KO’d via gene editing. The multicistronic vector was composed of 3 bicistrons, with each transgene pair linked by a 2A sequence to permit expression of both from a single promoter. The TBM-2A-EPCR bicistron was controlled by the porcine thrombomodulin promoter (pTBMpr) for endothelial-specific expression, while the chicken β actin (CAG) promoter was used to drive constitutive expression of CD46-2A-DAF and CD47-2A-HO1.  The single multicistronic vector was flanked by homology arms for targeting to the CMAH locus. Single cell colonies (SCC) were screened for targeted insertion of the vector by PCR and copy number by digital droplet PCR. KO edits were confirmed by NextGen sequencing. Correctly modified SCC were used in somatic cell nuclear transfer to generate pigs. Pig genotype was evaluated in genomic DNA by Southern blots, sequencing, and PCR. For phenotype, transgene expression was assessed by quantitative Western blot, flow cytometry (FC), and immunohistochemistry (IHC). Xenoantigen KOs were evaluated by FC and GHRKO by reduced serum IGF-1. Immunological reactivity between the donor pig (A328.1) and the patient was assessed by antibody binding and complement-dependent cytotoxicity (CDC) assays.

Results: We confirmed single-copy integration of the multicistronic vector on one CMAH allele, and KO of GGTA1, CMAH, b4GalNT2, and GHR genes. Expression of all 6 transgenes was confirmed by Western blot before transplant. Patient IgM and IgG binding to donor aortic endothelial cells was low and moderate, respectively, and cytotoxicity in the CDC assay was minimal. Expression of all 6 transgenes was confirmed by IHC in cardiac biopsy and postmortem samples at Days 30 and 60 post-transplant, respectively. TBM and EPCR expression was upregulated in postmortem heart vs. Day 30 heart biopsy, a possible indicator of stress, inflammation, or coagulation.

Conclusions: The gene edits described permitted a pig heart to avoid rejection and support life in a human patient for 60 days.