Indiana University-Purdue University Indianapolis (IUPUI)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
