Polyclonal fluctuation of lentiviral vector-transduced and expanded murine hematopoietic stem cells.
Gene therapy has proven its potential to cure diseases of the hematopoietic system. However, severe adverse events observed in clinical trials have demanded improved gene-transfer conditions. Whereas progress has been made to reduce the genotoxicity of integrating gene vectors, the role of pretransplantation cultivation is less well investigated. We observed that the STIF (stem cell factor [SCF], thrombopoietin [TPO], insulin-like growth factor-2 [IGF-2], and fibroblast growth factor-1 [FGF-1]) cytokine cocktail developed to effectively expand murine hematopoietic stem cells (HSCs) also supports the expansion of leukemia-initiating insertional mutants caused by gammaretroviral gene transfer. We compared 4 protocols to examine the impact of prestimulation and posttransduction culture in STIF in the context of lentiviral gene transfer. Observing 56 transplanted mice for up to 9.5 months, we found consistent engraftment and gene-marking rates after prolonged ex vivo expansion. Although a lentiviral vector with a validated insertional-mutagenic potential was used, longitudinal analysis identifying > 7000 integration sites revealed polyclonal fluctuations, especially in "expanded" groups, with de novo detection of clones even at late time points. Posttransduction expansion in STIF did not enrich clones with insertions in proto-oncogenes but rather increased clonal diversity. Our data indicate that lentiviral transduction in optimized media mediates intact polyclonal hematopoiesis without selection for growth-promoting hits by posttransduction expansion.
Department of Experimental Hematology, Hannover Medical School, Hannover, Germany.
Tobias
T
Maetzig
Martijn H
MH
Brugman
Stefan
S
Bartels
Niels
N
Heinz
Olga S
OS
Kustikova
Ute
U
Modlich
Zhixiong
Z
Li
Melanie
M
Galla
Bernhard
B
Schiedlmeier
Axel
A
Schambach
Christopher
C
Baum
D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D046528:Chimerism; D002999:Clone Cells; D003470:Culture Media / Q000494:pharmacology; D016207:Cytokines / Q000494:pharmacology; D018628:Gene Dosage / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D016086:Lentivirus / Q000187:drug effects / Q000235:genetics*; D007938:Leukemia / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D016254:Mutagenesis, Insertional / Q000187:drug effects; D009857:Oncogenes / Q000235:genetics; D010641:Phenotype; D016133:Polymerase Chain Reaction; D013997:Time Factors; D014161:Transduction, Genetic
D003470:Culture Media; D016207:Cytokines