Introduction of a xenogeneic gene via hematopoietic stem cells leads to specific tolerance in a rhesus monkey model.

Host immune responses against foreign transgenes may be a major obstacle to successful gene therapy. To clarify the impact of an immune response to foreign transgene products on the survival of genetically modified cells, we studied the in vivo persistence of cells transduced with a vector expressing a foreign transgene compared to cells transduced with a nonexpressing vector in the clinically predictive rhesus macaque model. We constructed retroviral vectors containing the neomycin phosphotransferase gene (neo) sequences modified to prevent protein expression (nonexpressing vectors). Rhesus monkey lymphocytes or hematopoietic stem cells (HSCs) were transduced with nonexpressing and neo-expressing vectors followed by reinfusion, and their in vivo persistence was studied. While lymphocytes transduced with a nonexpressing vector could be detected for more than 1 year, lymphocytes transduced with a neo-expressing vector were no longer detectable within several weeks of infusion. However, five of six animals transplanted with HSCs transduced with nonexpression or neo-expression vectors, and progeny lymphocytes marked with either vector persisted for more than 2 years. Furthermore, in recipients of transduced HSCs, infusion of mature lymphocytes transduced with a second neo-expressing vector did not result in elimination of the transduced lymphocytes. Our data show that introduction of a xenogeneic gene via HSCs induces tolerance to the foreign gene products. HSC gene therapy is therefore suitable for clinical applications where long-term expression of a therapeutic or foreign gene is required.

High levels of lymphoid expression of enhanced green fluorescent protein in nonhuman primates transplanted with cytokine-mobilized peripheral blood CD34(+) cells.

We have used a murine retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-expressing cells in the peripheral blood (PB) of transplanted rhesus macaques. Cytokine mobilized CD34(+) cells were transduced with an amphotropic vector that expressed EGFP and a dihydrofolate reductase cDNA under control of the murine stem cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high concentrations of the flt-3 ligand and stem cell factor. Following transplantation of the transduced cells, up to 55% EGFP-expressing granulocytes were obtained in the peripheral circulation during the early posttransplant period. This level of myeloid marking, however, decreased to 0.1% or lower within 2 weeks. In contrast, EGFP expression in PB lymphocytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks, the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and Southern blot analysis, and EGFP expression was observed in CD4(+), CD8(+), CD20(+), and CD16/56(+) lymphocyte subsets. EGFP expression was only transiently detected in red blood cells and platelets soon after transplantation. Such sustained levels of lymphocyte marking may be therapeutic in a number of human gene therapy applications that require targeting of the lymphoid compartment. The transient appearance of EGFP(+) myeloid cells suggests that transduction of a lineage-restricted myeloid progenitor capable of short-term engraftment was obtained with this protocol. (Blood. 2000;95:445-452)