Xenogeneic transplantation and tolerance in the era of CRISPR-Cas9

Purpose of Review: The use of genetically modified donor pigs has been integral to recent major advances in xenograft survival in preclinical nonhuman primate models. CRISPR-Cas9 gene editing technology has dramatically accelerated the development of multimodified pigs. This review examines the current and projected impact of CRISPR-Cas9-mediated donor modification on preventing rejection and potentially promoting tolerance of porcine xenografts. Recent Findings: CRISPR-Cas9 has been used to engineer several genetic modifications relevant to xenotransplantation into pigs, including glycosyltransferase knockouts (GGTA1, CMAH, β4GALNT2, A3GALT2 and combinations thereof), other knockouts (SLA-I, ULBP1, PERV and GHR), and one knock-in (anti-CD2 monoclonal antibody transgene knocked into GGTA1). Although the use of these pigs as donors in preclinical nonhuman primate models has been limited to a single study to date, in-vitro analysis of their cells has provided invaluable information. For example, deletion of three of the glycosyltransferases progressively decreased the binding and cytotoxicity of preexisting immunoglobulin G and immunoglobulin M in human sera, suggesting that this 'triple-KO' pig could be a platform for clinical xenotransplantation. Summary: CRISPR-Cas9 enables the rapid generation of gene-edited pigs containing multiple tailored genetic modifications that are anticipated to have a positive impact on the efficacy and safety of pig-to-human xenotransplantation.Peter J. Cowan, Wayne J. Hawthorne and Mark B. Nottl

Characterization of transgenic pigs expressing an anti-Cd2 monoclonal antibody. A continuing quest for the ideal islet donor

Background: We have restored long-term blood glucose control in diabetic immunosuppressed baboons by transplantation of GalT KO- human CD55-human CD59 neonatal porcine islets. Following with- drawal of immunosuppression, T cell infiltration, islet cell destruction, and loss of glucose control were observed. To combat this, we gener- ated transgenic pigs expressing diliximab, a chimeric anti-CD2 mono- clonal antibody that depletes and inhibits human and baboon T cells. CRISPR was used to integrate a single copy of the transgene into exon 10 of the GGTA1 gene, thus concurrently eliminating expression of the major xenoantigen αGal. Transgene expression was under the control of either (1) a mouse M HC class I (MHC-I) promoter for widespread expressionor (2) the pig insulin promoter (PIP) for islet-specific expres- sion. We hypothesize that local expression of diliximab will protect the islet xenograft from invading T cells, eliminating the need for contin- uous systemic immunosuppression and its associated risks and side effects.Aim: To evaluate the pattern and level of expression of diliximab in the transgenic pigs. Methods: Serum expression was detected by FACS of human T cells incubated with pig serum followed by anti-human IgG. Transgene mRNA expression was assessed by quantitative RT-PCR using TaqMan. Diliximab expression in spleen, kidney, and pancreas was assessed by immunohistochemistry and immunofluorescence. Results: MHC-I promoter transgenic pigs expressed diliximab in serum and tissues. Tissue expression was the strongest in a subset of spleen cells, with weaker expression throughout the kidney, and patchy pan- creatic staining, with minimal expression in islets as shown by IHC and IF. Transgene mRNA expression was detected in spleen and kidney. However, diliximab was undetectable in the PIP transgenic pigs Discussion/Conclusion: Our preliminary analysis suggests that the basal level of diliximab expression by native islets in the MHC-I promoter transgenic pigs is marginal at best. However, the MHC-I promoter is known to be induced by proinflammatory cytokines. Therefore, it is possible that transgenic islet xenografts will upregulate diliximab expression in response to cytokines produced by activated T cells recruited to the transplant site after withdrawal of immunosup pression, protecting them from rejection. To further investigate this possibility, we plan to isolate islets from the MHC-I promoter trans genic pigs and examine diliximab secretion following (1) in vitro culture in the presence and absence of proinflammatory cytokines, and (2) transplantation into diabetic immunodeficient mice. We will continue analysis of the PIP transgenic pigs as the lack of expression from the pig insulin promoter was surprising, given that this promoter had been reported to drive efficient islet-specific expression of another secreted immunomodulatory molecule (LEA29Y) in pigsEvelyn Salvaris, Adwin Thomas, Nicole Byrne, Nella Fisicaro, Mark B. Nottle, Wayne J. Hawthorne, Peter J. Cowa

FokI-dCas9 mediates high-fidelity genome editing in pigs

Friday, October 11, 2019 Oral Abstracts Session 200 on PERV. Abstract #326.5Nella Fisicaro, Evelyn J. Salvaris, Gayle K. Philip, Matthew J. Wakefield, Mark B. Nottle, Wayne J. Hawthorne, Peter Cowa

FokI-dCas9 mediates high-fidelity genome editing in pigs

Brief communicationGene editing using clustered regularly interspaced short palindromic repeats/Cas9 has great potential for improving the compatibility of porcine organs with human recipients. However, the risk of detrimental off-target mutations in gene-edited pigs remains largely undefined. We have previously generated GGTA1 knock-in pigs for xenotransplantation using FokI-dCas9, a variant of Cas9 that is reported to reduce the frequency of off-target mutagenesis. In this study, we used whole genome sequencing (WGS) and optimized bioinformatic analysis to assess the fidelity of FokI-dCas9 editing in the generation of these pigs. Genomic DNA was isolated from porcine cells before and after gene editing and sequenced by WGS. The genomic sequences were analyzed using GRIDSS variant-calling software to detect putative structural variations (SVs), which were validated by PCR of DNA from knock-in and wild-type pigs. Platypus variant-calling software was used to detect single-nucleotide variations (SNVs) and small insertions/deletions (indels). GRIDSS analysis confirmed the precise integration of one copy of the knock-in construct in the gene-edited cells. Three additional SVs were detected by GRIDSS: deletions in intergenic regions in chromosome 6 and the X chromosome and a duplication of part of the CALD1 gene on chromosome 18. These mutations were not associated with plausible off-target sites, and were not detected in a second line of knock-in pigs generated using the same pair of guide RNAs, suggesting that they were the result of background mutation rather than off-target activity. Platypus identified 1375 SNVs/indels after quality filtering, but none of these were located in proximity to potential off-target sites, indicating that they were probably also spontaneous mutations. This is the first WGS analysis of pigs generated from FokI-dCas9-edited cells. Our results demonstrate that FokI-dCas9 is capable of high-fidelity gene editing with negligible off-target or undesired on-target mutagenesis.Nella Fisicaro, Evelyn J. Salvaris, Gayle K. Philip, Matthew J. Wakefield, Mark B. Nottle, Wayne J. Hawthorne, Peter J. Cowa

GGTA1 knock-in pigs expressing the anti-CD2 monoclonal antibody diliximab from the porcine insulin promoter

Oral Abstracts Session 200 on PERVEvelyn Salvaris, Nella Fisicaro, Stephen McIlfatrick, Ivan Vassiliev, Andrew M. Lew, Wayne J. Hawthorne, Mark B. Nottle, Peter J. Cowa

Hypoxia-inducible factor-1alpha (HIF-1alpha) potentiates beta-cell survival after islet transplantation of human and mouse islets

A high proportion of β-cells die within days of islet transplantation. Reports suggest that induction of hypoxia-inducible factor-1α (HIF-1α) predicts adverse transplant outcomes. We hypothesized that this was a compensatory response and that HIF-1α protects β-cells during transplantation. Transplants were performed using human islets or murine β-cell-specific HIF-1α-null (β-HIF-1α-null) islets with or without treatment with deferoxamine (DFO) to increase HIF-1α. β-HIF-1α-null transplants had poor outcomes, demonstrating that lack of HIF-1α impaired transplant efficiency. Increasing HIF-1α improved outcomes for mouse and human islets. No effect was seen in β-HIF-1α-null islets. The mechanism was decreased apoptosis, resulting in increased β-cell mass posttransplantation. These findings show that HIF-1α is a protective factor and is required for successful islet transplant outcomes. Iron chelation with DFO markedly improved transplant success in a HIF-1α-dependent manner, thus demonstrating the mechanism of action. DFO, approved for human use, may have a therapeutic role in the setting of human islet transplantation.Rebecca A. Stokes, Kim Cheng, Natasha Deters, Sue Mei Lau, Wayne J. Hawthorne ... Shane Grey ... et al

Human islets express a marked proinflammatory molecular signature prior to transplantation

In the context of islet transplantation, experimental models show that induction of islet intrinsic NF-κB-dependent proinflammatory genes can contribute to islet graft rejection. Isolation of human islets triggers activation of the NF-κB and mitogen-activated kinase (MAPK) stress response pathways. However, the downstream NF-κB target genes induced in human islets during the isolation process are poorly described. Therefore, in this study, using microarray, bioinformatic, and RTqPCR approaches, we determined the pattern of genes expressed by a set of 14 human islet preparations. We found that isolated human islets express a panel of genes reminiscent of cells undergoing a marked NF-κB-dependent proinflammatory response. Expressed genes included matrix metallopeptidase 1 (MMP1) and fibronectin 1 (FN1), factors involved in tissue remodeling, adhesion, and cell migration; inflammatory cytokines IL-1β and IL-8; genes regulating cell survival including A20 and ATF3; and notably high expression of a set of chemokines that would favor neutrophil and monocyte recruitment including CXCL2, CCL2, CXCL12, CXCL1, CXCL6, and CCL28. Of note, the inflammatory profile of isolated human islets was maintained after transplantation into RAG(-/-) recipients. Thus, human islets can provide a reservoir of NF-κB-dependent inflammatory factors that have the potential to contribute to the anti-islet-graft immune response. To test this hypothesis, we extracted rodent islets under optimal conditions, forced activation of NF-κB, and transplanted them into allogenic recipients. These NF-κB activated islets not only expressed the same chemokine profile observed in human islets but also struggled to maintain normoglycemia posttransplantation. Further, NF-κB-activated islets were rejected with a faster tempo as compared to non-NF-κB-activated rodent islets. Thus, isolated human islets can make cell autonomous contributions to the ensuing allograft response by elaborating inflammatory factors that contribute to their own demise. These data highlight the potential importance of islet intrinsic proinflammatory responses as targets for therapeutic intervention.Mark J. Cowley, Anita Weinberg, Nathan W. Zammit, Stacey N. Walters, Wayne J. Hawthorne ... Shane T. Grey ... et al

Anticoagulation practices in total pancreatectomy with autologous islet cell transplant patients: an international survey of clinical programs

Diabetes mellitus: pathophysiological changes and therap