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Recent reports linking insertional activation of LMO2following gene therapy for X-linked severe combined immunodeficiency (X-SCID) have led to a re-evaluation of risks following gene therapy with retroviral vectors. In our analysis of 702 integration sites in rhesus macaques that underwent transplantation up to 7 years earlier with autologous CD34+cells transduced with amphotropic murine leukemia virus (MLV)-derived retroviral vectors containing marker genes, we detected insertion into one locus, the Mds1/Evi1region, a total of 14 times in 9 animals. Mds1/Evi1integrations were observed stably long term, primarily in myeloid cells. We hypothesize that this over-representation likely results from an impact on the self-renewal and engraftment potential of CD34+progenitor cells via insertional mutagenesis at this specific locus. There is no evidence of ongoing in vivo clonal expansion of the Mds1/Evi1populations, and all animals are hematologically normal without evidence for leukemia. Characterization of integration sites in this relevant preclinical model provides critical information for gene therapy risk assessment as well as identification of genes controlling hematopoiesis. (Blood. 2005;106:2530-2533)

Gene transfer experiments in nonhuman primates have been shown to be predictive of success in human clinical gene therapy trials. In most nonhuman primate studies, hematopoietic stem cells (HSCs) collected from the peripheral blood or bone marrow after administration of granulocyte colony-stimulating factor (G-CSF) + stem cell factor (SCF) have been used as targets, but this cytokine combination is not generally available for clinical use, and the optimum target cell population has not been systematically studied. In our current study we tested the retroviral transduction efficiency of rhesus macaque peripheral blood CD34+cells collected after administration of different cytokine mobilization regimens, directly comparing G-CSF+SCF versus G-CSF alone or G-CSF+Flt3-L in competitive repopulation assays. Vector supernatant was added daily for 96 hours in the presence of stimulatory cytokines. The transduction efficiency of HSCs as assessed by in vitro colony-forming assays was equivalent in all 5 animals tested, but the in vivo levels of mononuclear cell and granulocyte marking was higher at all time points derived from target CD34+cells collected after G-CSF+SCF mobilization compared with target cells collected after G-CSF (n = 3) or G-CSF+Flt3-L (n = 2) mobilization. In 3 of the animals long-term marking levels of 5% to 25% were achieved, but originating only from the G-CSF+SCF–mobilized target cells. Transduction efficiency of HSCs collected by different mobilization regimens can vary significantly and is superior with G-CSF+SCF administration. The difference in transduction efficiency of HSCs collected from different sources should be considered whenever planning clinical gene therapy trials and should preferably be tested directly in comparative studies.

To determine whether the multidrug resistance gene MDR1could act as a selectable marker in human subjects, we studied engraftment of peripheral blood progenitor cells (PBPCs) transduced with either MDR1or the bacterial NeoR gene in six breast cancer patients. This study differed from previous MDR1gene therapy studies in that patients received only PBPCs incubated in retroviral supernatants (no nonmanipulated PBPCs were infused), transduction of PBPCs was supported with autologous bone marrow stroma without additional cytokines, and a control gene (NeoR) was used for comparison with MDR1. Transduced PBPCs were infused after high-dose alkylating agent therapy and before chemotherapy with MDR-substrate drugs. We found that hematopoietic reconstitution can occur using only PBPCs incubated ex vivo, that theMDR1gene product may play a role in engraftment, and that chemotherapy may selectively expand MDR1gene-transduced hematopoietic cells relative to NeoR transduced cells in some patients.