Nonmyeloablative conditioning is sufficient to allow engraftment of EGFP-expressing bone marrow and subsequent acceptance of EGFP-transgenic skin grafts in mice.
ABSTRACT Immunologic reactions against gene therapy products may prove to be a frequent problem in clinical gene therapy protocols. Enhanced green fluorescence protein (EGFP) is commonly used as a marker in gene transfer protocols, and immune responses against EGFP-expressing cells have been documented. The present study was designed to investigate the effect of a pharmacologic, nonmyeloablative, conditioning regimen on the development of EGFP+ donor/recipient mixed bone marrow chimerism and ensuing tolerance to EGFP-expressing transplants. To this end, C57BL/6J (B6) mice were treated with soluble formulations of either busulfan (Busulfex) or the closely related compound treosulfan, followed by transplantation of bone marrow cells from EGFP-transgenic (B6-EGFP.Tg) donor mice. Such conditioning regimens resulted in long-term persistence of donor EGFP+ cells among various hematopoietic lineages from blood, bone marrow, and thymus. Stable hematopoietic chimeras transplanted at 10 to 17 weeks after bone marrow transplantation (BMT) with B6-EGFP.Tg skin grafts all accepted their transplants, whereas non-EGFP chimeric B6 control animals were able to mount rejection of the EGFP+ B6 skin grafts. Control third-party grafts from major histocompatibility complex (MHC)-mismatched mice were rejected within 20 days, indicating that acceptance of EGFP-expressing skin grafts was the result of specific immune tolerance induction by the transplantation of EGFP-transgenic bone marrow. Long-term tolerance to EGFP in chimeric recipients was confirmed by the absence of anti-EGFP-reactive T cells and antibodies. These results broaden the therapeutic potential for using hematopoietic molecular chimerism in nonmyeloablated recipients as a means of preventing rejection of genetically modified cells.
Article: Marker tolerant, immunocompetent animals as a new tool for regenerative medicine and long-term cell tracking.[show abstract] [hide abstract]
ABSTRACT: Immune-mediated rejection of labeled cells is a general problem in transplantation studies using cells labeled with any immunogenic marker, and also in gene therapy protocols. The aim of this study was to establish a syngeneic model for long-term histological cell tracking in the absence of immune-mediated rejection of labeled cells in immunocompetent animals. We used inbred transgenic Fischer 344 rats expressing human placental alkaline phosphatase (hPLAP) under the control of the ubiquitous R26 promoter for this study. hPLAP is an excellent marker enzyme, providing superb histological detection quality in paraffin and plastic sections. Transplantation of cells from hPLAP transgenic (hPLAP-tg) F344 rats into wild-type (WT) F344 recipients failed because of immune-mediated rejection. Here we show that this problem can be overcome by inducing tolerance to the marker gene by transplantation of bone marrow from hPLAP-tg F344 rats into WT F344 hosts after lethal irradiation, or by neonatal exposure of WT F344 rats to hPLAP-tg F344 cells. As proof-of-principle, we injected bone marrow cells (BMC) from hPLAP-tg rats into the knee joint of marker tolerant, bone marrow-transplanted WT rats, and found successful engraftment and differentiation of donor cells. In addition, hPLAP-tg BMC injected intravenously in neonatally tolerized WT F344 hosts could be traced in lymph nodes, 2 months post-injection. In combination with the excellent marker hPLAP, marker tolerant animals may open up new perspectives for all experiments requiring long-term histological tracking of genetically labeled cells.BMC Biotechnology 02/2007; 7:30. · 2.35 Impact Factor