SummaryThe development of safe methods for inducing donor-specific tolerance across xenogeneic barriers could potentially relieve the critical shortage of allograft donors that currently limits the applicability of organ transplantation . We report here that such tolerance can be induced in a xenogeneic combination (rat -mouse) using a nonmyeloablative and nonlethal preparative regimen . Successful induction of chimerism and donor-specific transplantation tolerance required pretreatment of recipients with monoclonal antibodies (mAbs) against NK1 .1, Thy-1.2, CD4 and CD8, followed by administration of 3 Gy whole body radiation (WBI), 7 Gy thymic irradiation, and infusion of T cell-depleted rat bone marrow cells (BMC). Rat cells appeared among peripheral blood lymphocytes (PBL) of such recipients by 2-3 wk, and rat T cells by 2-5 wk following bone marrow transplantation (BMT) . Donor-type rat skin grafts placed 4 mo after BMT were accepted, while simultaneously placed non-donor-type rat skin grafts were promptly rejected. In addition to its clinical potential, the ability to induce donor-specific tolerance across xenogeneic barriers using such a nonlethal preparative regimen provides a valuable model for the study of mechanisms of xenogeneic transplantation tolerance .A n inadequate supply of allogeneic donors has become the major limitation to the rapidly advancing field of clinical organ transplantation . Transplantation oforgans across species barriers could potentially provide a solution to this problem . Although the use of lethal irradiation and bone marrow transplantation (BMT)' has been shown to induce transplantation tolerance across xenogeneic barriers in animal models (1-5), the clinical applicability of such an approach is limited by the toxicity of the preparative regimen . It will therefore be essential to develop less toxic methods for inducing xenogeneic chimerism ifthis approach is to be useful clinically. Previous work from this laboratory has demonstrated that chimerism and transplantation tolerance can be induced across complete allogeneic MHC barriers in mice using a nonlethal preparative regimen, in which anti-CD4 and anti-CD8 mAbs are used instead of lethal irradiation to eliminate mature T cells from the host. Following this mAb treatment, relatively nontoxic whole body irradiation (WBI) at 3 Gy plus 7 Gy thymic irradiation were sufficient to permit 'Abbreviations used in this paper: BMC, bone marrow cells ; BMT, bone marrow transplantation ; FCM, flow cytometry; LCA, leukocyte common antigen ; TCD, T cell-depleted ; TI, thymic irradiation; TRA, Texas red streptavidin ; WBI, whole-body irradiation .195 engraftment of allogeneic bone marrow (6) . Attempts to extend this model to a xenogeneic rat/mouse strain combination were not successful using the same nonlethal preparative regimen that was effective in allogeneic combinations. However, addition of antibodies against host NK cells and Thy-1+ cells to the preparative regimen led to successful induction ofxenogeneic chimerism and specific tr...

Human alpha-galactosidase A (alpha-Gal A; EC.3.2.1.22) is a lysosomal exoglycosidase encoded by a gene on Xq22. Deficiencies of this enzyme result in Fabry disease, an X-chromosome-linked recessive disorder that leads to premature death in affected males. For treatment of genetic diseases, we have developed a retroviral vector system, pSXLC/pHa, that enables coexpression of drug-selectable markers with a second nonselectable gene as part of a bicistronic message using the promoter from the Harvey murine sarcoma virus and an internal ribosomal entry site (IRES) from encephalomyocarditis virus. Retroviral vectors based on this system that carry the human alpha-Gal A cDNA either upstream (pHa-alpha Gal-IRES-MDR) or downstream (pHa-MDR-IRES-alpha Gal) from the IRES relative to the drug-selectable MDR1 (P-glycoprotein) cDNA were constructed. Each of eight independent vincristine-resistant, pHa-alpha Gal-IRES-MDR-transfected clones and all four vincristine-resistant, pHa-alpha Gal-IRES-MDR retrovirus-transduced clones showed significantly higher activity of alpha-Gal A than the parental cells. More than 50% of the vincristine-resistant, pHa-MDR-IRES-alpha Gal-transfected clones and all four vincristine-resistant, pHa-MDR-IRES-alpha Gal retrovirus-transduced clones showed significantly higher activity of alpha-Gal A than the parental cells. In these bicistronic vectors, the cDNA whose translation was cap-dependent (upstream) was expressed at higher levels than when the same cDNA was translated in an IRES-dependent manner (downstream). These vectors may prove useful in the gene therapy of Fabry disease.

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We describe a new retroviral vector system pSXLC/pHa that utilizes a putative internal ribosome entry site (IRES) from encephalomyocarditis virus downstream from a multicloning site to co-express drug-selectable markers with a second non-selectable cDNA in a eukaryotic expression vector. The positive drug-selectable marker, MDR1, and the positive-negative marker, herpes simplex virus thymidine kinase (HSV-TK), were successfully introduced and expressed in the pSXLC/pHa system. The pSXLC-MDR and pSXLC-TK vectors contain the drug-selectable genes under translational control of the IRES and multiple cloning sites upstream for insertion of second cDNAs which can be co-expressed in this system. The inserts of these pSXLC plasmids were designed for easy transfer to the pHa retrovirus vector which has a strong promoter from Harvey murine sarcoma virus. The IRES-MDR-carrying retroviral vector, pHa-MCS-IRES-MDR, conferred resistance to vincristine and adriamycin. The IRES-TK-containing vector, pHa-MCS-IRES-TK conferred HAT-resistance in TK-deficient cells and the transfectants showed hypersensitivity to ganciclovir. These "flexible" vectors should be useful for co-expression of genes for selectable gene transfer and for positive-negative (suicide) selections in vitro and in vivo.