Polyclonal fluctuation of lentiviral vector-transduced and expanded murine hematopoietic stem cells
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Polyclonal fluctuation of lentiviral vector-transduced and expanded murine hematopoietic stem cells. / Maetzig, Tobias; Brugman, Martijn H; Bartels, Stefan; Heinz, Niels; Kustikova, Olga S; Modlich, Ute; Li, Zhixiong; Galla, Melanie; Schiedlmeier, Bernhard; Schambach, Axel; Baum, Christopher.
In: BLOOD, Vol. 117, No. 11, 17.03.2011, p. 3053-64.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Polyclonal fluctuation of lentiviral vector-transduced and expanded murine hematopoietic stem cells
AU - Maetzig, Tobias
AU - Brugman, Martijn H
AU - Bartels, Stefan
AU - Heinz, Niels
AU - Kustikova, Olga S
AU - Modlich, Ute
AU - Li, Zhixiong
AU - Galla, Melanie
AU - Schiedlmeier, Bernhard
AU - Schambach, Axel
AU - Baum, Christopher
PY - 2011/3/17
Y1 - 2011/3/17
N2 - 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.
AB - 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.
KW - Animals
KW - Cell Proliferation/drug effects
KW - Cell Survival/drug effects
KW - Cells, Cultured
KW - Chimerism
KW - Clone Cells
KW - Culture Media/pharmacology
KW - Cytokines/pharmacology
KW - Gene Dosage/genetics
KW - Genetic Vectors/genetics
KW - Hematopoietic Stem Cells/cytology
KW - High-Throughput Nucleotide Sequencing
KW - Lentivirus/drug effects
KW - Leukemia/pathology
KW - Mice
KW - Mice, Inbred C57BL
KW - Mutagenesis, Insertional/drug effects
KW - Oncogenes/genetics
KW - Phenotype
KW - Polymerase Chain Reaction
KW - Time Factors
KW - Transduction, Genetic
U2 - 10.1182/blood-2010-08-303222
DO - 10.1182/blood-2010-08-303222
M3 - SCORING: Journal article
C2 - 21248062
VL - 117
SP - 3053
EP - 3064
JO - BLOOD
JF - BLOOD
SN - 0006-4971
IS - 11
ER -