Cell-intrinsic and vector-related properties cooperate to determine the incidence and consequences of insertional mutagenesis.
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.
Kustikova
Olga S
OS
Department of Experimental Hematology, Hannover Medical School, Hannover, Germany.
Schiedlmeier
Bernhard
B
Brugman
Martijn H
MH
Stahlhut
Maike
M
Bartels
Stefan
S
Li
Zhixiong
Z
Baum
Christopher
C
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