The average levels of gene marking in bone marrow CD34 phospholipases cells from months 4, 5, and 6 post transplant for all 18 recipients are shown in Figure 6. While many of the monkeys had essentially no gene marked CD34 cells in their bone marrow duringmonths 4 6 post transplant, marking from 0.0001 to 0.01 vector copies/cell was achieved in several of the animals. In general, the levels of marking by both vectors were similar, independently of whether they had been treated with busulfan and fludarabine or busulfan alone. There was no evidence of preferential loss of cells expressing GFP compared to those with the NoN gene suggesting the absence of a cytolytic immunologic response to cells expressing GFP. The levels of gene marking of bone marrow CD34 cells in the recipients custom peptide synthesis during months 4 6 were grouped, based on the busulfan levels that had been achieved during conditioning. There were positive correlations between the busulfan AUC achieved and the levels of marking with both the NoN and GFP genes. For the third series of transplants, observations were extended over 1 year from transplantation.
The levels of marking in bone marrow CD34 cells averaged for months 7 12 post transplant were consistent with those measured over months 4 6, with 3B continuing to show clopidogrel relatively high levels of marking with both the GFP and the NoN genes, 3D with GFP, and 3E and 3F with the NoN genes. Evaluation of immune responses to the GFP transgene product In Group 3 recipients, humoral and cellular immune responses to the GFP transgene product were monitored monthly for 1 year post transplant. Serum antibodies to recombinant GFP were quantified by enzyme linked immunosorbent assay and the frequencies of PBMCs with antigen specific responses to GFP by interferon production were measured by enzyme paclitaxel linked immunosorbent spot. As a control to test the effects of the pretransplant conditioning regimen on immunity, all six monkeys were preimmunized with clinical grade tetanus toxoid at 1 and 2 months postnatal age and titers of serum antibodies to tetanus were measured by ELISA and IFN responses were measured by ELISPOT.
To test the ability of the monkeys to respond to a neo antigen post transplant, they were immunized with clinical grade hepatitis B surface antigen vaccine at 6 months after transplant and evaluated by ELISA and ELISPOT. The anti tetanus antibody levels were stable in all six recipients over the entire year, with no decline seen immediately after conditioning. All six recipients had a three to tenfold increase in serum antibodies to hepatitis B vaccine after immunization at 6 months, indicating immune competence. During the first six months after transplant, only recipient 3B with the highest level of GFP marked blood cells developed a serological response to GFP. There was no change in the levels of GFP marked cells in this recipient following the increased levels of anti GFP antibodies at 2 months post transplant. Upon immunization with rGFP at 6 months post transplant, the remaining five monkeys seroconverted with development of serum antibodies to GFP. ELISA for GFP antibodies were also performed for the first 12 recipients. In the first 6 months, positive ELISA measurements of antibodies to GFP were observed.