Quantitative Real Time PCR Analysis of MDR1 Transduced Rhesus Macaque Hematopoietic Stem Cells Shows No Indication for Proliferative Advantage after Long-Term Follow-Up.

Introduction: It is of concern whether the introduction of a transgene into hematopoietic stem cells by retroviral vectors will lead to an alteration of the growth and engraftment characteristics. Earlier studies in mice indicated that retroviral MDR1 gene transfer may be associated with a myeloproliferative disorder. In human or primate cells this could not be reproduced in bulk cell populations. Analyses on the clonal level and a long-term follow-up in a model with more direct relevance to human HSC biology were lacking.

Methods: In this study CD34-enriched peripheral blood stem cells from 2 rhesus macaque monkeys were transduced either with a MDR1 gene-retroviral vector (HaMDR1-vector) or a NeoR-retroviral vector (G1Na-vector). After autologous transplantation granulocytes and mononuclear cells of each animal were obtained at different time points and analysed by using a highly sensitive and specific ligation-mediated PCR (LM-PCR) and by quantitative real time PCR.

Results: In monkey M120 73 different cell populations (clones) were detected between 8 weeks and 4 years after transplantation; in monkey M038 49 clones could be identified between 16 weeks and 4 years after transplantation. Remarkably, 99 clones descend from the G1Na-vector, whereas only 23 clones descend from the HaMDR1-vector. Quantitative real time PCR analyses support these data: in both animals, the mean proportion of the G1Na-vector and the HaMDR1-vector, respectively, is 2,46%±0,014% and 0,455%±0,003%. Furthermore, single G1Na clones identified with LM-PCR 4 years after transplantation were quantified, showing a copy number between 1.43±0,52 and 7.1±0.34, according to 0.001%±0.0003% and 0.004%±0.0003% of the granulocytes fraction. In the MNC fraction, the G1Na clones were not detectable. At the same time point, 2 HaMDR1 clones were quantified, one in the granulocyte and one in the MNC fraction, respectively. Here, the copy numbers were 2.87±0.56 and 3.52±1.11, according to 0.002%±0.0003% and 0.002%±0.0008% of the granulocyte and MNC fraction, respectively.

Conclusions: We conclude that hematopoiesis in these monkeys is polyclonal for prolonged periods after transplantation and that MDR1 gene transfer does not confer a proliferative advantage over vector-control-transduced hematopoietic stem cells.