Jeffrey Monahan

BACKGROUND: Inhibition of the T-cell-mediated immune response is a necessary component of preventing rejection following xenotransplantation with pig alpha1,3-galactosyltransferase gene-knockout (GTKO) organs. Cytotoxic T lymphocyte-associated antigen (CTLA4) is a co-stimulatory molecule that inhibits T-cell activity and may be useful in prolonging graft rejection. METHODS: An expression vector was built containing the extracellular coding region of porcine (p) CTLA4 fused to the hinge and CH2/CH3 regions of human IgG1 (pCTLA4-Ig). Pigs transgenic for pCTLA4-Ig, on either a GTKO or wild-type (WT) genetic background, were produced by nuclear transfer and characterized using Western blot analysis, immunofluorescence, ELISA, and necropsy. RESULTS: Fifteen pCTLA4-Ig-transgenic piglets resulted from five pregnancies produced by nuclear transfer. All transgenic pigs exhibited robust expression of the pCTLA4-Ig protein and most expressed the transgene in all organs analyzed, with significant levels in the blood as well. Despite initial good health, these pigs exhibited diminished humoral immunity, and were susceptible to infection, which could be managed for a limited time with antibiotics. CONCLUSIONS: Viable pigs exhibiting robust and ubiquitous expression of pCTLA4-Ig were produced on both a WT and GTKO background. Expression of pCTLA4-Ig resulted in acute susceptibility to opportunistic pathogens due at least in part to a significantly compromised humoral immune status. As this molecule is known to have immunosuppressive activity, high levels of pCTLA4-Ig expression in the blood, as well as defective development related to exposure to pCTLA4-Ig in utero, may contribute to this reduced immune status. Prophylactic treatment with antibiotics may promote survival of disease-free transgenic pigs to a size optimal for organ procurement for transplantation. Additional genetic modifications and/or tightly regulated expression of pCTLA4Ig may reduce the impact of this transgene on the humoral immune system.

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.

Introduction: Porcine cytomegalovirus (PCMV) is an upper respiratory tract infection, widespread among swine herds, that can be prevented by early weaning piglets at birth into high-health herd barns. Although PCMV infection has not been documented in humans, there is a concern that it could be transmitted through xenotransplantation and potentially lead to xenograft failure. Recently we reported the first experimental case of xeno-heart transplant to a human. The pig (A328.1) had tested negative for PCMV in routine nasal swab DNA tests before being used as a heart donor. However, PCMV was subsequently detected in the recipient’s blood by cell free DNA sequencing. This suggests that latent PCMV was present in donor pig tissues and that nasal swab DNA testing was inadequate for detecting latent PCMV. Here, we retrospectively screened for PCMV in the donor pig PBMCs, spleen and explanted heart biopsy by nested PCR, RT-PCR and ddPCR. Methods: DNA analysis for PCMV detection was performed by nested PCR, qRT-PCR and ddPCR, following standard protocols. ddPCR was performed using 3 different PCMV probes (PCMV DNA pol gene) for robust and redundant analysis. Results: In retrospective analyses, presence of PCMV was determined by qualitative, quantitative, and absolute DNA quantification analysis. PCMV was undetectable by nested PCR in the PBMC but was detected in spleen and postmortem explanted heart tissue. Low levels of PCMV DNA were detected by qRT-PCR in the pig’s PBMCs collected before heart procurement. Similar to qRT-PCR, ddPCR showed low copy number of PCMV in the pig’s PBMCs and spleen, but high copy number in the explanted heart. These results suggest that latent PCMV was present in the donor pig and that it subsequently replicated in the transplanted heart. Conclusions: Collectively, our results revealed the inadequacy of PCR-based PCMV assays and showed the need for more reliable and robust methods to detect latent PCMV. DNA tests on blood or nasal swab samples are more appropriate for viremic pigs with high viral loads. In pigs with latent or low levels of the virus, the PCR assays often produce hit-or-miss, ambiguous results, just above or below the threshold of detection, that could lead to false negatives, as in the case of this donor pig. Therefore, alternative approaches such as serological assays to screen donor serum for PCMV antibodies may be more appropriate before xenotransplantation.