title: Cell-intrinsic and vector-related properties cooperate to determine the incidence and consequences of insertional mutagenesis.
abstract: In gene therapeutic approaches targeting hematopoietic cells, insertional mutagenesis may provoke clonal dominance with potential progress to overt leukemia. To investigate the contribution of cell-intrinsic features and determine the frequency of insertional proto-oncogene activation, we sorted hematopoietic subpopulations before transduction with replication-deficient gamma-retroviral vectors and studied the clonal repertoire in transplanted C57BL/6J mice. Progressive clonal dominance only developed in the progeny of populations with intrinsic stem cell potential, where expanding clones with insertional upregulation of proto-oncogenes such as Evi1 were retrieved with a frequency of approximately 10(-4). Longitudinal studies by high-throughput sequencing and locus-specific quantitative PCR showed clones with >50-fold expansion between weeks 5 and 31 after transplantation. In contrast, insertional events in proto-oncogenes did not endow the progeny of multipotent or myeloid-restricted progenitors with the potential for clonal dominance (risk <10(-6)). Transducing sorted hematopoietic stem cells (HSCs) with self-inactivating (SIN) lentiviral vectors in short-term cultures improved chimerism, and although clonal dominance developed, there was no evidence for insertional events in the vicinity of proto-oncogenes as the underlying cause. We conclude that cell-intrinsic properties cooperate with vector-related features to determine the incidence and consequences of insertional mutagenesis. Furthermore, our study offers perspectives for refinement of animal experiments in the assessment of vector-related genotoxicity.
authors.affiliation: Department of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Olga S
authors.initials: OS
authors.lastname: Kustikova
authors.forename: Bernhard
authors.initials: B
authors.lastname: Schiedlmeier
authors.forename: Martijn H
authors.initials: MH
authors.lastname: Brugman
authors.forename: Maike
authors.initials: M
authors.lastname: Stahlhut
authors.forename: Stefan
authors.initials: S
authors.lastname: Bartels
authors.forename: Zhixiong
authors.initials: Z
authors.lastname: Li
authors.forename: Christopher
authors.initials: C
authors.lastname: Baum
mesh_terms: D000818:Animals; D005260:Female; D005822:Genetic Vectors / Q000235:genetics*; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D016254:Mutagenesis, Insertional / Q000235:genetics / Q000379:methods*; D000090063:Proto-Oncogene Mas; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic