The integration characteristics of retroviral (RV) vectors increase the probability of interfering with the regulation of cellular genes, and account for a tangible risk of insertional mutagenesis in treated patients. To assess the potential genotoxic risk of conventional or self-inactivating (SIN) gamma-RV and lentiviral (LV) vectors independently from the biological consequences of the insertion event, we developed a quantitative assay based on real-time reverse transcriptase--PCR on low-density arrays to evaluate alterations of gene expression in individual primary T-cell clones. We show that the Moloney leukemia virus long terminal repeat (LTR) enhancer has the strongest activity in both a gamma-RV and a LV vector context, while an internal cellular promoter induces deregulation of gene expression less frequently, at a shorter range and to a lower extent in both vector types. Downregulation of gene expression was observed only in the context of LV vectors. This study indicates that insertional gene activation is determined by the characteristics of the transcriptional regulatory elements carried by the vector, and is largely independent from the vector type or design.
Transplantation of genetically modified hematopoietic stem cells (HSCs) has therapeutic potential for a variety of blood genetic disorders. Engraftment of HSCs, however, requires toxic myeloablative treatments, which render this approach questionable for non-life-threatening disorders. A potential alternative is the use of transgenes, which allows positive selection of HSCs in vivo. We used retroviral vectors to express a truncated derivative of the erythropoietin receptor (tEpoR) in murine and human hematopoietic cells. Murine HSCs expressing tEpoR at different levels (1500 to 13,000 receptors/cell) acquire a competitive repopulation capacity in vivo upon transplantation into fully or partially myeloablated co-isogenic mouse recipients. Long-term analysis of transplanted mice showed that expression of tEpoR at paraphysiological levels (approximately 1500 receptors/cell) has no effect on steady-state hematopoiesis and induces no further expansion of transduced cells after the engraftment period. Human cord blood-derived CD34+ stem/progenitor cells transduced with a lentiviral vector expressing tEpoR expand their clonogenic capacity in vitro, and significantly increase their marrow repopulation capacity upon xenotransplantation into sublethally irradiated NOD-SCID mice, with no alteration in their phenotype, survival, and differentiation properties. These data indicate that expression of tEpoR is an effective strategy to promote selective engraftment of genetically modified HSCs upon transplantation in both myeloablative and nonmyeloablative conditions, without the use of toxic drugs for selection.
A number of reports indicate that peroxisome proliferator-activated receptor (PPAR) delta is involved in the molecular control of monocyte-macrophage differentiation. In this regard, the recent demonstration that PPARdelta is a primary response gene of 1alpha,25-dihydroxyvitamin D3 (VD), i.e. a powerful inducer of such process, allowed us to hypothesize the existence of a cross talk between PPARdelta and VD receptor pathways. To address this issue, we analyzed the effects promoted by stimulation with PPARdelta ligands and by overexpression of this nuclear receptor in monoblastic cell lines undergoing exposure to VD. The results obtained evidenced that, although promoting a weak differentiation effect by themselves, PPARdelta ligands efficiently co-operated with VD treatment. In spite of this, PPARdelta overexpression exerted a remarkable inhibitory effect on monocyte-macrophage differentiation induced by VD that was, at least partly, reverted by stimulation with a highly specific PPARdelta ligand. These data indicate that, although acting through a ligand-dependent modality, PPARdelta is a negative regulator of VD-mediated monocyte differentiation, allowing us to hypothesize a role of the investigated nuclear receptor in the differentiation block of M5 type (monoblastic) acute myeloid leukemias (AMLs). Bioinformatic analysis of a microarray database, containing the expression profiles of 285 AML cases, further supported this hypothesis demonstrating the existence of a subset of M5 type (monoblastic) AMLs that overexpress PPARdelta gene.
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