Expression of swine MHC class II genes in a cynomolgus monkey: Retrovirus-mediated gene therapy in a preclinical transplantation model

Immune reactivity against products of the major histocompatibility complex (MHC) is the major barrier to allotransplantation, Conversely, sharing of MHC class II antigens appears to be of overwhelming importance in permitting the induction of immune tolerance to vascularized organ allografts, as demonstrated previously in miniature swine. Class II antigen has also been shown to be recognized predominantly in human anti-pig xenoreactions in vitro. To achieve tolerance in the xenogeneic pig-to-primate model, we are therefore investigating an approach involving retrovirus-mediated gene therapy to transfer swine MHC (SLA) class II genes into primate primitive hematopoietic stem cells, so that swine MHC class II antigens may participate in the education of the recipient's newly developing T cell repertoire. We report here the in vitro and in vivo use of a recombinant retrovirus containing a polycistronic retroviral vector which carries swine MHC class II DRA and DRB cDNA sequences to transduce CD34(+) bone marrow cells (BMC) from a cynomolgus monkey. Transduction efficiency was assessed by reverse transcriptase-polymerase chain reaction analysis of the colony-forming unit progenitor colonies grown in the absence of gentamycin; 55% and 24% of the progenitor colonies were determined to express the retroviral transcript at 1 week and 3 weeks post-transduction, respectively. These in vitro studies have been extended to the transplantation of retrovirally transduced autologous stem cells into a cynomolgus monkey prepared with a non-myeloablative conditioning regimen. Prolonged expression of SLA-DR transcripts in monkey peripheral blood mononuclear cells (PBMC) has been documented over a 56-week period after transplantation of retrovirus-transduced bone marrow cells, However, we could not detect any protein expression by FAGS analysis on the surface of primate PBMC or bone marrow, using a porcine SLA-DR-specific antibody. Engraftment of hematopoietic cells with the transduced genes was further detected in the progenitor colonies grown from the bone marrow cells harvested at 4 weeks and 25 weeks after bone marrow transplantation. Our results thus document that long-term engraftment of retrovirally transduced hematopoietic cells can be achieved in a primate model using a non-myeloablative preparative regimen.