Polyclonal fluctuation of lentiviral vector-transduced and expanded murine hematopoietic stem cells

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 -