10. The Effect of Globin Locus Control Region (LCR) Elements or the MSCV-LTR on Promoter Trapping by Integrated Lentiviral Vector Genomes

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Integrating vectors are desirable for gene therapy of inherited blood disorders to achieve permanent genetic modification and stable expression of transgenes in hematopoietic cells. However, three patients with severe combined immunodeficiency enrolled in a gene therapy trial of a retroviral vector developed leukemia and activation of the LMO2 proto-oncogene by vector integration has been demonstrated in two. Retroviral vectors prefer the transcriptional start sites for integration while lentiviral vector integration sites are distributed throughout the active genes. Thus, the safety profile of integrating vectors is a critical issue. As a strategy to evaluate interaction of regulatory elements in the vectors with genes into which they have integrated, a series of lentiviral promoter trap vectors were designed to compare the effect of regulatory elements within the vector on the potential for promoter trapping. The trapping cassette consists of a splice acceptor followed by the GFP coding sequences and a polyadenylation site. The cassette was inserted immediately upstream of the 3|[prime]| LTR in a reverse orientation in vectors containing either a globin LCR element or the oncoretroviral MSCV-LTR also in a reverse orientation to the lentiviral LTR. As a control, a vector containing the cassette without regulatory elements was also constructed. HeLa or erythroid K562 cells were transduced with these vectors and analyzed for GFP expression by flow cytometry. Transduction frequency was estimated by Southern hybridization and the efficiency of promoter trapping was then calculated as a percentage of GFP expressing cells in transduced cells. Our results showed 5% promoter trapping in HeLa cells transduced with the control vector. This efficiency is similar to that recently reported by others (De Palma et al, Blood 2004 Nov 12). Vectors containing either the globin LCR or the MSCV-LTR gave 15 to 20% trapping efficiency. Similarly, in K562 cells, the percentage of promoter trapping of vectors containing either the globin or MSCV regulatory elements was significantly higher (up to 6-fold) of that of the control vector. To further investigate the effect of the globin LCR or the MSCV-LTR on promoter trapping, we isolated single cell clones from GFP-positive HeLa and K562 cells that had been transduced with a trapping vector. Many clones had lost GFP expression when reanalyzed, but the proportion (43%) in which more than 60% of the cells were positive was higher with vectors containing the globin LCR compared to those containing the MSCV-LTR (27%). In addition, the MFI of globin LCR clones was, on average, slightly higher than that of MSCV-LTR clones (20 and 17, respectively). Eighteen clones having a single integrated proviral genome were identified and 15 integration sites have been mapped to date. Ten integration sites were in genes and 8 of these were in the first intron with the promoter trap in tandem with the natural transcript. Our results to date indicate that the presence and nature of regulatory elements within a lentiviral vector influences the probability of vector genome transcription either by trapping of a natural promoter or by activation of cryptic promoters.