Analysis of Self-Inactivating Lentiviral Vector Integration Sites and Flanking Gene Expression in Human Peripheral Blood Progenitor Cells After Alkylator Chemotherapy

A major side effect of chemotherapy is hematoxicity. Gene transfer of drug resistance genes, such as methylguanine-DNA-methyltransferase (MGMT), to primitive hematopoietic progenitor cells has been examined as a possible solution for circumventing this problem. This study by Grund et al. examines the effect of alkylator chemotherapy treatment on the integration pattern of lentiviral self-inactivating (SIN) vector mediated delivery of MGMT in human peripheral blood progenitor cells. Hematotoxicity is a major and frequently dose-limiting side effect of chemotherapy. Retroviral methylguanine-DNA-methyltransferase (MGMT; EC 2.1.1.63) gene transfer to primitive hematopoietic progenitor cells (CD34+ cells) might allow the application of high-dose alkylator chemotherapy with almost mild to absent myelosuppression. Because gammaretroviral vector integration was found in association with malignant or increased proliferation, novel lentiviral vectors with self-inactivating (SIN) capacity might display a safer option for future gene transfer studies. We assessed the influence of chemoselection on integration patterns in 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-treated and untreated human CD34+ cells transduced with an SIN lentiviral vector carrying the MGMTP140K transgene, using ligation-mediated PCR (LM-PCR) and next-generation sequencing. In addition, for the first time, the local influence of the lentiviral provirus on the expression of hit and flanking genes in human CD34+ cells was analyzed at a clonal level. For each colony, the integration site was detected (LM-PCR) and analyzed (QuickMap), and the expression of hit and flanking genes was measured (quantitative RT-PCR). Analyses of both treated and untreated CD34+ cells revealed preferential integration into genes. Integration patterns in BCNU-treated cells showed mild, but not significant, differences compared with those found in untreated CD34+ cells. Most importantly, when analyzing the local influence of the provirus, we saw no significant deregulation of the integration-flanking genes. These findings demonstrate that SIN vector-mediated gene transfer might display a feasible and possibly safe option for MGMTP140K-mediated chemoprotection of CD34+ cells.