Globin Vectors Research Articles

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

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.

Globin gene transfer: a paradigm for transgene regulation and vector safety

The β-thalassemias and sickle cell disease are severe congenital anemias that are caused by the defective synthesis of the β chain of hemoglobin. Allogeneic hematopoietic stem cell (HSC) transplantation is curative, but this therapeutic option is not available to the majority of patients. The transfer of a regulated β-globin gene in autologous HCSs thus represents a highly attractive alternative treatment. This strategy, simple in principle, raises major challenges in terms of controlling transgene expression, which ideally should be erythroid-specific, differentiation stage-restricted, elevated, position-independent, and sustained over time. Using lentiviral vectors, we recently demonstrated that an optimized combination of proximal and distal transcriptional control elements permits lineage-specific and elevated expression of the β-globin gene, resulting in therapeutic hemoglobin production and correction of anemia in β-thalassemic mice. Several groups have now confirmed and extended these findings in various mouse models of severe hemoglobinopathies, thus generating enthusiasm for a genetic treatment based on globin gene transfer. Furthermore, globin vectors provide a paradigm for improving vector safety by restricting transgene expression to the differentiated progeny within a single lineage, thereby reducing the risk of activating oncogenes in hematopoietic progenitors. Here we review the principles underlying the genesis of regulated vectors for stem cell therapy.

Development of virus vectors for gene therapy of β chain hemoglobinopathies: flanking with a chromatin insulator reduces γ-globin gene silencing in vivo

AbstractWe have previously described the development of oncoretrovirus vectors for human γ-globin using a truncated β-globin promoter, modified γ-globin cassette, and α-globin enhancer. However, one of these vectors is genetically unstable, and both vectors exhibit variable expression patterns in cultured cells, common characteristics of oncoretrovirus vectors for globin genes. To address these problems, we identified and removed the vector sequences responsible for genetic instability and flanked the resultant vector with the chicken β-globin HS4 chromatin insulator to protect expression from chromosomal position effects. After determining that flanking with the cHS4 element allowed higher, more uniform levels of γ-globin expression in MEL cell lines, we tested these vectors using a mouse bone marrow transduction and transplantation model. When present, the γ-globin cassettes from the uninsulated vectors were expressed in only 2% to 5% of red blood cells (RBCs) long term, indicating they are highly sensitive to epigenetic silencing. In contrast, when present the γ-globin cassette from the insulated vector was expressed in 49% ± 20% of RBCs long term. RNase protection analysis indicated that the insulated γ-globin cassette was expressed at 23% ± 16% per copy of mouse α-globin in transduced RBCs. These results demonstrate that flanking a globin vector with the cHS4 insulator increases the likelihood of expression nearly 10-fold, which in turn allows for γ-globin expression approaching the therapeutic range for sickle cell anemia and β thalassemia.

Globin vectors: control without the locus

Hereditary hemoglobinopathies have long been favorites of gene therapists, and a genetic cure would be a wonderful extension of the decades of research spent understanding the molecular biology of hemoglobin gene expression. The most realistic approaches rely on retroviral vectors, which have the