The development of lymphoid leukemia in two children with X-SCID who underwent gene therapy was partially due to activation of the LMO-2 proto-oncogene by the retroviral LTR of the vector which inserted nearby (Hacein-Bey-Abina et al., Science 2003), highlighting the importance of vector design on the potential to activate genes near vector integration sites. As gene therapy vectors for other blood disorders are evaluated, it seems prudent to assess the safety issues regarding insertion for each particular vector in appropriate pre-clinical models. We have focused on developing γ-globin lentiviral vectors for gene therapy of the hemoglobin disorders and have documented correction of a murine model of β-thalassemia in the absence of observable adverse events (Persons et al., Blood 2003; Hanawa et al., Blood 2004). To more thoroughly evaluate the potential for vector-induced genotoxicity, we have examined whether self-inactivating (SIN) γ-globin lentiviral vectors containing erythroid-specific, β-globin locus enhancer elements can alter the expression of genes nearby the vector insertion site, as the retroviral LTR did in the X-SCID trial. To ascertain whether an integrated globin vector could influence endogenous transcriptional activity in erythroid precursors, 15 clonal spleen colony erythroblast populations (≥ 95% erythroid) containing lentiviral globin vector insertions and 15 untransduced control clones were derived from bone marrow cells of β-thalassemic mice. The transcriptional profile of each clone was determined using the Affymetrix Mouse 430A microarray (representing ~15,000 genes). Expression of 4500–6000 genes was observed in all samples. Ligation-mediated PCR was used to obtain the vector-genomic DNA junction sequences, allowing identification of vector insertion locations in 13 of the clones using the NCBI database. Of these, 6 globin vector clones had 16 genes, including N-ras, which were located within 100kb of the vector insertion site and were represented on the array. Only one gene, D3Jfr1, encoding a “cold shock” DNA binding protein and which was disrupted by an intronic vector insertion, had a change in signal value relative to the mean signal value of the controls. Real time RT-PCR confirmed a 4-fold reduction in expression of this gene. Both microarray and real time RT-PCR demonstrated that expression of N-ras was unchanged. For comparison, 15 clones with insertions of a lentiviral vector containing the MSCV retroviral LTR, were also derived, along with 10 additional mock control clones. We are currently analyzing the expression of some 116 genes that lie within 300kb of the vector insertions, relative to the mean expression level in the 25 mock transduced clones. Additionally, we have expanded analysis of the globin vector clones to evaluate changes in expression of 107 genes located within 300kb of the vector insertions. These data should prove useful to assess whether integrated SIN globin lentiviral vectors containing erythroid-specific regulatory elements have a propensity to alter transcriptional activity in the progeny of genetically modified hematopoietic stem cells, relative to vectors containing viral LTR elements.