Evaluation of engraftment and immunological tolerance after reduced intensity conditioning in a rhesus hematopoietic stem cell gene therapy model.

Reduced intensity conditioning (RIC) is desirable for hematopoietic stem cell (HSC) targeted gene therapy; however, RIC may be insufficient for efficient engraftment and inducing immunological tolerance to transgenes. We previously established long-term gene marking in our rhesus macaque autologous HSC transplantation model following 10 Gy total body irradiation (TBI). In this study, we evaluated RIC transplantation with 4 Gy TBI in two rhesus macaques that received equal parts of CD34(+) cells transduced with green fluorescent protein (GFP)-expressing lentiviral vector and empty vector not expressing transgenes. In both animals, equivalently low gene marking between GFP and empty vectors was observed 6 months post-transplantation, even with efficient transduction of CD34(+) cells in vitro. Autologous lymphocyte infusion with GFP marking resulted in an increase of gene marking in lymphocytes in a control animal with GFP tolerance, but not in the two RIC-transplanted animals. In vitro assays revealed strong cellular and humoral immune responses to GFP protein in the two RIC-transplanted animals, but this was not observed in controls. In summary, 4 Gy TBI is insufficient to permit engraftment of genetically modified HSCs and induce immunological tolerance to transgenes. Our findings should help in the design of conditioning regimens in gene therapy trials.