Dose finding with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population.
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.
Kustikova
Olga S
OS
Department of Bone Marrow Transplantation, University Hospital Hamburg-Eppendorf, D-20251 Hamburg, Germany.
Wahlers
Anke
A
Kuhlcke
Klaus
K
Stahle
Birgit
B
Zander
Axel R
AR
Baum
Christopher
C
Fehse
Boris
B
D018929:Cell Culture Techniques; D002999:Clone Cells; D005434:Flow Cytometry; D018628:Gene Dosage; D015870:Gene Expression; D005822:Genetic Vectors / Q000008:administration & dosage*; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic / Q000592:standards*
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins