Nuclease-based editing in the porcine genome : a strategy to facilitate porcine-to human xenotransplantation
| dc.contributor.advisor | Tector, A. Joseph | |
| dc.contributor.advisor | White, Kenneth E. | |
| dc.contributor.author | Butler, James R. | |
| dc.contributor.other | Schmidt, C. Max | |
| dc.contributor.other | Radovich, Milan | |
| dc.date.accessioned | 2018-01-10T14:34:19Z | |
| dc.date.available | 2018-01-10T14:34:19Z | |
| dc.date.issued | 2017-04-18 | |
| dc.degree.date | 2017 | en_US |
| dc.degree.discipline | Department of Medical & Molecular Genetics | |
| dc.degree.grantor | Indiana University | en_US |
| dc.degree.level | Ph.D. | en_US |
| dc.description | Indiana University-Purdue University Indianapolis (IUPUI) | en_US |
| dc.description.abstract | Solid organ transplantation is severely limited by a shortage of available donor allografts. Pig-to-human xenotransplantation offers a potential solution to this growing problem. For xenotransplantation to achieve clinical relevance, both immunologic and physiologic barriers must be understood. Genetic modification of pigs has proven to be a valuable means of both studying and eliminating these barriers. The present body of work describes a method for greatly increasing the efficiency and precision of genome editing within the porcine genome. By combining non-integrating selection and homologous recombination of exogenous oligonucleotides, a method for rapidly creating genetic modification without reliance on phenotypic sorting was achieved. Furthermore this work employs the technique of CRISPR/Cas9-directed mutagenesis to create and analyze several new animal models of porcine-to-human xenotransplantation with respect to both immunologic and physiologic parameters. First, Isoglobotrihexosylceramide -a controversial glycan to the field of xenotransplantation- was studied in a knockout model and found not to affect human-anti-porcine humoral reactions. Second, a new combination of glycan modifications is described that significantly lowers the human anti-porcine humoral immune response. This model animal suggests that glycan-deletion alone will be sufficient to promote clinical application, and that conventional immunosuppression will be successful in mediating the human cellular response. Finally, two potential physiologic barriers to xenotransplantation are studied in genetically modified model animals. Xenogenic consumption of human platelets was studied across hepatic and renal organ systems; xenogenic platelet consumption was reduced by glycan modifications to the porcine liver while human platelet sequestration was not identified in the study of renal endothelium. Porcine FcRN –an essential receptor expressed in kidneys to maintain serum proteostasis- was studied as a final potential barrier to pig-to human renal transplantation. Because albumin is the primary driver of serum oncotic pressure, the protein-protein interaction of endogenous porcine FcRN and human albumin was studied. Porcine FcRN was found capable of binding human albumin under physiologic parameters. In summary, the results of the present work suggest that the salient barriers to clinical xenotransplantation have been removed and that porcine-to human renal transplantation may soon offer an answer to the current organ shortage. | en_US |
| dc.identifier.doi | 10.7912/C2F341 | |
| dc.identifier.uri | https://hdl.handle.net/1805/14967 | |
| dc.identifier.uri | http://dx.doi.org/10.7912/C2/1976 | |
| dc.language.iso | en_US | en_US |
| dc.subject | CRISPR | en_US |
| dc.subject | Genetics | en_US |
| dc.subject | Nuclease | en_US |
| dc.subject | Transplantation | en_US |
| dc.subject | Xenotransplantation | en_US |
| dc.title | Nuclease-based editing in the porcine genome : a strategy to facilitate porcine-to human xenotransplantation | en_US |
| dc.type | Dissertation |