Dose finding with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population
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Dose finding with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population. / Kustikova, Olga S; Wahlers, Anke; Kuhlcke, Klaus; Stahle, Birgit; Zander, Axel R; Baum, Christopher; Fehse, Boris.
in: BLOOD, Jahrgang 102, Nr. 12, 01.12.2003, S. 3934-7.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › Kurzpublikation › Forschung › Begutachtung
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TY - JOUR
T1 - Dose finding with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population
AU - Kustikova, Olga S
AU - Wahlers, Anke
AU - Kuhlcke, Klaus
AU - Stahle, Birgit
AU - Zander, Axel R
AU - Baum, Christopher
AU - Fehse, Boris
PY - 2003/12/1
Y1 - 2003/12/1
N2 - Retroviral vectors are commonly used in clinical gene therapy, but recent observations of insertional oncogene activation in preclinical and clinical settings have forced a discussion of their safety. Here we investigated the relationship between retroviral transduction efficiency in mass cultures and the actual number of integrated vector copies in single cells using K562 leukemia and primary CD34+ cells. We found an exponential increase of integration numbers correlated to gene transfer rates and a linear increase of expression levels with insertion frequency. On average we detected one vector insertion per transduced cell for a gene transfer of less than 30%, 3 for 60%, and approximately 9 for 90% (in K562). Clonal analysis revealed strikingly increased variations of both transgene copy numbers (more than 20-fold in primary cells) and expression levels associated with higher transduction. Therefore, limiting retroviral gene transfer to approximately 30% may be suggested to avoid generating clones containing multiple insertions.
AB - Retroviral vectors are commonly used in clinical gene therapy, but recent observations of insertional oncogene activation in preclinical and clinical settings have forced a discussion of their safety. Here we investigated the relationship between retroviral transduction efficiency in mass cultures and the actual number of integrated vector copies in single cells using K562 leukemia and primary CD34+ cells. We found an exponential increase of integration numbers correlated to gene transfer rates and a linear increase of expression levels with insertion frequency. On average we detected one vector insertion per transduced cell for a gene transfer of less than 30%, 3 for 60%, and approximately 9 for 90% (in K562). Clonal analysis revealed strikingly increased variations of both transgene copy numbers (more than 20-fold in primary cells) and expression levels associated with higher transduction. Therefore, limiting retroviral gene transfer to approximately 30% may be suggested to avoid generating clones containing multiple insertions.
KW - Cell Culture Techniques
KW - Clone Cells
KW - Flow Cytometry
KW - Gene Dosage
KW - Gene Expression
KW - Genetic Vectors/administration & dosage
KW - Green Fluorescent Proteins
KW - Hematopoietic Stem Cells/metabolism
KW - Humans
KW - K562 Cells
KW - Luminescent Proteins/analysis
KW - Retroviridae/genetics
KW - Transduction, Genetic/standards
U2 - 10.1182/blood-2003-05-1424
DO - 10.1182/blood-2003-05-1424
M3 - Short publication
C2 - 12881303
VL - 102
SP - 3934
EP - 3937
JO - BLOOD
JF - BLOOD
SN - 0006-4971
IS - 12
ER -