5. Evaluation of the Genotoxic Potential of a Self-Inactivating γ-Globin Lentiviral Vector

Abstract

The development of lymphoid leukemia in two children with X-SCID who underwent gene therapy appears to be partially due to activation of the LMO-2 proto-oncogene by the retroviral vector LTR (Hacein-Bey-Abina et al., Science 302:415, 2003), underscoring the importance of vector design on the potential to activate genes near integration sites. In order to determine whether integrated, self-inactivating (SIN) globin lentiviral vectors which lack a viral LTR also have the potential to cause transcriptional activation, we initially compared the mobilization rate of an integrated SIN globin lentiviral vector containing β-globin locus enhancer elements with that of a SIN lentiviral vector containing the MSCV U3 LTR region. 293T cells were transduced to a high vector copy number with the SIN globin vector or a SIN lentiviral vector containing the LTR element, followed by transfection of viral packaging genes to rescue aberrantly transcribed vector genomes. Although both vectors had a comparable transducing titer (globin 2 × 107 ± 4 × 106 TU/ml, n=4 vs. MSCV 4 × 107 ± 2 × 106 TU/ml, n=9), the globin vector had a “mobilized” titer 60-fold less than that of the MSCV LTR vector (p<0.0001). We estimate that 1 in 300,000 globin vector integration events were mobilized, compared to a frequency of 1 in 12,000 with the MSCV vector, consistent with the possibility of less transcriptional activation attributable to the globin vector. To ascertain whether an integrated globin vector could influence endogenous transcriptional activity in red cell presursors, clonal spleen colony erythroblasts containing a single globin vector insertion were derived from transduced bone marrow cells of β-thalassemic mice. The transcriptional profile of 21 single-copy globin vector clones and 15 untransduced, control clones was determined using the Affymetrix Mouse 430A microarray (representing ~15,000 genes). Expression of 4500–6000 genes was observed in all samples. Using a 2-fold difference in the level of expression as a cutoff, only 0.02% of all the genes among the 21 clones had potentially different expression compared to the control clones, well below the expected 2% rate of random differences (Genomics 83:321, 2004). Ligation-mediated PCR was then used to clone the sequences of the vector-genomic DNA junctions, allowing identification of vector insertion locations using the NCBI database. Six clones had 16 genes, including N-ras, that were located within 100kb of the vector insertion site and were represented on the array. Of those genes that were expressed, only 2 had a 2.0–2.8-fold difference in signal value between the clone and the controls. These differences were unlikely to be real due to a high false discovery rate in those clones. Evaluation of these potential changes by real time RT-PCR is underway. Both microarray and real time RT-PCR demonstrated that expression of N-ras was unchanged. These data suggest that integrated SIN globin lentiviral vectors have a low rate of altering transcriptional activity in target cells and therefore may be inherently less genotoxic than vectors containing a viral LTR.