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

Abstract

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 MGMT(P140K) 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 MGMT(P140K)-mediated chemoprotection of CD34(+) cells.

Efficiency and Safety of O6-Methylguanine DNA Methyltransferase (MGMTP140K)-MediatedIn VivoSelection in a Humanized Mouse Model

“…4). In accordance with previous studies employing lentiviral vectors (Mitchell et al, 2004;Laufs et al, 2006;Grund et al, 2010), in our experiments we found most of the integrations within gene-coding regions; 76.5% in pre-TX samples, 67.4% in treated samples, 60.5% in NT samples, and 74% in control BM samples. Furthermore, among these insertions, 65.4% -4% occurred in introns.…”

“…+ cells transduced with an MGMT lentiviral vector employing the SFFV promoter (Grund et al, 2010). Similarly, insertional analysis in a number of large animal models employing gammaretro and lentiviral vectors revealed polyclonal profiles without enrichment of specific clones after chemotherapy.…”

“…The top integrations were selected for further validation using locus-specific PCR (ls-PCR). Insertions in various genes from our study were also compared with integration site databases retrieved from different MGMT studies (Beard et al, , 2010Grund et al, 2010;Giordano et al, 2011;Adair et al, 2012) and cancer databases [D'Antonio et al (2012) and www.bushmanlab.org]. These post-HISAP processing steps were performed using in-house-developed scripts.…”

“…Thus, we mainly focused on the BM-derived insertions for further clonality assessment (Supplementary Table S2). We compared insertion sites retrieved from chemotherapy-treated, NT, and GFP control vector-transduced BM samples to the pre-TX samples using the retroviral insertion sites published in previous MGMT studies (Beard et al, , 2010Grund et al, 2010;Giordano et al, 2011;Adair et al, 2012). Moreover, we matched the genes closest to lentiviral integrations found in our model with the available cancer databases [D'Antonio et al (2012) and www.bushmanlab.org/links/genelists] summarized in Supplementary Table S3.…”

“…To screen for potential genotoxic side effects, the clonal repertoire of hematopoiesis after MGMT-mediated selection has been investigated for hematopoietic cells after exposure to alkylatying agents in vitro (Grund et al, 2010), in murine serial transplant (Giordano et al, 2011), canine and primate models (Neff et al, 2005;Beard et al, 2009Beard et al, , 2010Larochelle et al, 2009), as well as in the recent clinical phase I study (Adair et al, 2012). Reassuringly, these studies so far have failed to demonstrate any adverse effects of MGMT-mediated chemoselection on the clonal repertoire of MGMT-transduced hematopoietic cells.…”