New Aspects on the Safety of Multidrug-Resistance 1 Gene Transfers: No Indication for Clonal Dominance after Long-Term Follow-Up in a Primate Transplantation Model

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 multidrug-resistance 1 gene transfer may be associated with a myeloproliferative disorder. In human or primate cells this could not be reproduced in bulk cell populations. Analysis on the clonal level were lacking. One method to study the in vivo behaviour of repopulating progenitor and stem cells is marking the cells with replication-incompetent retroviral vectors that integrate into identifiable host DNA sequences, thus allowing the tracking of cell progeny based on unique proviral insertion sites. In this study CD34-enriched peripheral blood stem cells from 2 rhesus macaque monkeys were split into two aliquots and transduced either with a multidrug-resistance 1 gene-retroviral vector based on the Harvey murine sarcoma virus (HaMDR1-vector) or a NeoR-retroviral vector based on the Moloney murine leukaemia virus (G1Na-vector). After autologous retransplantation, DNA from blood and bone marrow was collected at different time points in a period of 4 years and the animals are still alive. By using a highly sensitive and specific ligation-mediated polymerase chain reaction (LM-PCR) followed by sequencing of vector integration sites, we found in animal M120 32 different contributing hematopoietic clones 8 weeks and 50 weeks after transplantation and in animal M038 17 clones 58 weeks after transplantation. Based on the difference between the sequences of the HaMDR1-LTR and the G1Na-LTR, the clones can be allocated definitely to one of the two vectors. Remarkably, 36 clones descend from the G1Na-vector, whereas only 13 clones descend from the HaMDR1-vector. 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.