Therapeutic windows in the genetic engineering of hematopoietic cells

Christopher Baum1,2, Zhixiong Li1,2, Bernd Schiedlmeier2, Hannes Klump2, Elke Will2, Jochen Düllmann3, Johann Meyer1, Martin Forster2, Carol Stocking2, Wolfram Ostertag2, Manfred Schmidt4, Christof von Kalle4, Axel R. Zander5, Boris Fehse5

1Department of Hematology and Oncology, Medical School, D-30625 Hannover. 2Deptartmentof Cell & Virus Genetics, Heinrich Pette Institute, D-20251 Hamburg 3Neuroanatomy, University Hospital Eppendorf, D-20251 Hamburg 4Department of Hematology and Oncology, Albert Ludwigs University, D-79106 Freiburg 5 Bone Marrow Transplantation, University Hospital Eppendorf, D-20251 Hamburg

AIM: Gene transfer into repopulating hematopoietic cells and lymphocytes offers new perspectives in the treatment of malignancies. Improvements in retroviral vector technology have resulted in a clinically relevant gene transfer efficiency. Promising selectable marker genes have been developed to enrich engineered cells prior to transplantation, or to control their survival and proliferation in vivo. However, elevating the efficiency may also increase the risk of genetic interventions, emphasizing the need to define therapeutic windows in somatic gene transfer. METHODS: We developed a set of retroviral vectors expressing different selectable marker genes to high levels in hematopoietic cells. The marker genes tested were all of human origin and encoded cell surface tags (tLNGFR, tCD34), regulatory proteins supporting stem cell self-renewal (HOXB4) or proteins conferring resistance to cytotoxic agents (MDR1). Primary hematopoietic stem cells were transduced with the different vectors of interest and transplanted into irradiated mice. Hematopoiesis was observed for up to 18 months to screen for anticipated and unexpected effects of the genetic manipulation. RESULTS: All marker genes tested mediated their anticipated function. However, side effects were also observed that could have originated either from the ectopic expression of the transgene or from genetic damage induced by transgene insertion. Retroviral vectors integrate without promoting genetic instability, but at random positions in the host genome. With respect to contributing to a malignant phenotype, the risk of mutagenesis of cellular sequences has been estimated to be in the order of 10-7 per insertion. As further genetic lesions are required for malignant transformation and tumor development, a single insertion of an otherwise innocuous retroviral transgene is not expected to produce severe side effects. Despite of this stochastic improbability, we observed leukemia induction in an animal model of retroviral gene marking of hematopoietic stem cells. Genetic and functional controls suggested that the leukemia arose by a combination of insertional activation of a proto-oncogene and signal interference induced by the expression of a truncated cell-surface receptor. Others have observed leukemia induction in mice when expressing MDR1, a gene proposed for chemoprotection of bone marrow cells (Bunting et al., Blood 1998; 92:2269). However, so far we have been unable to reproduce this observation using our vector and experimental conditions. Possibly, this discrepancy is due to differences in transgene copy numbers in single cells, or related to other aspects of vector design. CONCLUSIONS: Our observations suggest that the incidence of severe side effects in gene-modified hematopoietic cells depends on multiple factors, including the persistence, expansion and plasticity of engineered cell clones, the copy number and expression level of the transgene, and its interaction with cellular signaling networks. Genetic procedures proposed for the therapeutic manipulation of transplantable cells require a stringent search for preferred vector insertion sites and a careful analysis of transgene side effects. Systematic risk assessment involves long-term observations in well-designed animal models and multi-center efforts.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Molecular Genetics & Therapy - 2.