Clonal inventory screens uncover monoclonality following serial transplantation of MGMTP140K-transduced stem cells and dose-intense chemotherapy

Abstract Gene transfer of mutant O6-methylguanine-DNA-methyltransferase (MGMTP140K) into hematopoietic stem cells (HSCs) protects hematopoiesis from alkylating agents and allows efficient in vivo selection of transduced HSCs. However, insertional mutagenesis, high regenerative stress associated with selection, and the genotoxic potential of alkylating drugs represent considerable risk factors for clinical applications of this approach. Therefore, we investigated the long-term effect of MGMTP140K gene transfer followed by repetitive, dose-intensive treatment with alkylating agents in a murine serial bone marrow transplant model and assessed clonality of hematopoiesis up to tertiary recipients. The substantial selection pressure resulted in almost completely transduced hematopoiesis in all cohorts. Ligation-mediated PCR and next-generation sequencing identified several repopulating clones carrying vector insertions in distinct genomic regions that were ∼9 kb of size (common integration sites). Beside polycl...