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 com plex (MHC) is the major barrier to allotransplantation, Conversely, sh aring of MHC class II antigens appears to be of overwhelming importanc e in permitting the induction of immune tolerance to vascularized orga n allografts, as demonstrated previously in miniature swine. Class II antigen has also been shown to be recognized predominantly in human an ti-pig xenoreactions in vitro. To achieve tolerance in the xenogeneic pig-to-primate model, we are therefore investigating an approach invol ving retrovirus-mediated gene therapy to transfer swine MHC (SLA) clas s II genes into primate primitive hematopoietic stem cells, so that sw ine MHC class II antigens may participate in the education of the reci pient's newly developing T cell repertoire. We report here the in vitr o and in vivo use of a recombinant retrovirus containing a polycistron ic retroviral vector which carries swine MHC class II DRA and DRB cDNA sequences to transduce CD34(+) bone marrow cells (BMC) from a cynomol gus monkey. Transduction efficiency was assessed by reverse transcript ase-polymerase chain reaction analysis of the colony-forming unit prog enitor colonies grown in the absence of gentamycin; 55% and 24% of the progenitor colonies were determined to express the retroviral transcr ipt at 1 week and 3 weeks post-transduction, respectively. These in vi tro studies have been extended to the transplantation of retrovirally transduced autologous stem cells into a cynomolgus monkey prepared wit h a non-myeloablative conditioning regimen. Prolonged expression of SL A-DR transcripts in monkey peripheral blood mononuclear cells (PBMC) h as been documented over a 56-week period after transplantation of retr ovirus-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 th us document that long-term engraftment of retrovirally transduced hema topoietic cells can be achieved in a primate model using a non-myeloab lative preparative regimen.