Efficiency and Safety of O 6 -Methylguanine DNA Methyltransferase (MGMT P140K )-Mediated In Vivo Selection in a Humanized Mouse Model

Abstract

Efficient O⁶-methylguanine DNA methyltransferase (MGMT^P140K)-mediated myeloprotection and in vivo selection have been demonstrated in numerous animal models and most recently in a phase I clinical study in glioblastoma patients. However, this strategy may augment the genotoxic risk of integrating vectors because of chemotherapy-induced DNA damage and the proliferative stress exerted during the in vivo selection. Thus, to improve the safety of the procedure, we evaluated a self-inactivating lentiviral MGMT^P140K vector for transduction of human cord blood-derived CD34⁺ cells followed by transplantation of the cells into NOD/LtSz-scid/Il2rγ^−/− mice. These experiments demonstrated significant and stable enrichment of MGMT^P140K transgenic human cells in the murine peripheral blood and bone marrow. Clonal inventory analysis utilizing linear amplification-mediated polymerase chain reaction and high-throughput sequencing revealed a characteristic lentiviral integration profile. Among the bone marrow insertions retrieved, we observed considerable overlap to previous MGMT^P140K preclinical models or the clinical study. However, no significant differences between our chemotherapy-treated and nontreated cohorts were observed. This also hold true when specific cancer gene databases and a functional annotation of hit genes by the Panther Database with respect to molecular function, biological process, or cellular component were assessed. Thus, in summary, our data demonstrate efficient and long-term in vivo selection without overt hematological abnormalities using the lentiviral MGMT^P140K vector. Furthermore, the study introduces humanized mouse models as a novel tool for the pre-clinical assessment of human gene therapy related toxicity.