Negative Control Region Is a Critical Element Of Insertional Oncogenesis After Gene Transfer Into Hematopoietic Progenitors With Moloney Murine Leukemia Viruses

Gene transfer into hematopoietic stem cells has been used successfully to treat a variety of human genetic diseases. Although protocols have shown positive clinical outcomes, the successes of clinical trials have been tempered by adverse events in which the use of gamma-retroviral vectors (GV) containing full-length long terminal repeats (LTRs) with strong enhancer activity increased transcription of cancer-related genes, and thereby contributed to development of leukemia. Assessing safety of integrating viral vectors for future clinical use is therefore of paramount importance. The negative control region (NCR) is a particularly well-conserved sequence among mammalian gamma-retroviruses with demonstrated regulating a transcription activity of GV in hematopoietic cells. This suggests that the NCR might play a crucial role of insertional oncogenesis after gene transfer into hematopoietic progenitors.

In a series of safety studies of viral gene transfer constructs, we used an in vitro assay of murine bone marrow (BM) cell immortalization and compared the consequences of hematopoietic stem cell transduction with three different kinds of viral vectors, including Moloney murine leukemia virus- (MMLV), lentivirus- (LV), and foamy virus (FV)-based constructs. To evaluate critical elements for cell immortalization by MMLV vectors, we also tested four different MMLV LTR variants deleted of either 1) most of the two 75-bp repeats associated with the viral enhancer (delE1), 2) all of the two 75-bp repeats and the NCR (delE2), 3) only the NCR (delNCR), or 4) carrying a deleterious mutation of the NCR NFAT motif (ΔNFAT). All vectors carried an internal expression cassette including the eGFP gene under the control of a UCOE (ubiquitously acting chromatin opening element) promoter. In this assay, BM cells are harvested from C57BL6 mice, exposed to retroviral supernatants and cultured long-term. Derived lines are considered immortalized based on their ability to continue to grow in vitro for more than six weeks in the presence of interleukin-3 and stem cell factor. Real-time PCR was performed to verify comparable transduction efficiency of bone marrow cells by different vectors.

In our analysis of MMLV LTR mutants, full-MMLV and delE1 transduction of 92 and 108 cultures, respectively, resulted in 37 and 37 immortalized lines (40% and 34% immortalization rate, respectively). The difference in immortalization rate between full-MMLV and delE1 was not statistically significant. Transductions using delE2-, delNCR- and ΔNFAT-carrying vectors of 60, 36 and 35 cultures resulted in 10, 3 and 10 immortalized lines (17%, 8.3% and 29% immortalization rate, respectively). The difference between the immortalization caused by delE1 and delE2 vectors was statistically significant (p<0.05). Moreover, the difference between the immortalization caused by full-MMLV and delNCR vectors was statistically significant (p<0.01), while there was no significant difference between the immortalization induced by full-MMLV and ΔNFAT vectors. Transduction of 57 and 34 cultures with LV and FV vectors, respectively, resulted in no immortalized lines. Transductions of 128 cultures with a LV construct carrying the U3 region from the murine stem cell virus LTR as an internal promoter (LV-U3) resulted in 2 immortalized lines which was not statistically different from the results obtained with LV vectors carrying the UCOE internal promoter.

These results confirm that GV are prone to causing immortalization of hematopoietic cells and indicate that deletion of the whole viral enhancer sequences may not be adequate to eliminate the insertional oncogenesis risk. Importantly, our data point to the NCR as a crucial element for immortalization and justify additional studies to evaluate its specific role in MMLV-mediated insertional oncogenesis. Finally, our results suggest that vectors based on LV and FV backbones are safer alternatives for clinical gene transfer into hematopoietic stem cells.