Copy number determination of genetically-modified hematopoietic stem cells.
Human gene transfer with gammaretroviral, murine leukemia virus (MLV) based vectors has been shown to effectively insert and express transgene sequences at a level of therapeutic benefit. However, there are numerous reports of disruption of the normal cellular processes caused by the viral insertion, even of replication deficient gammaretroviral vectors. Current gammaretroviral and lentiviral vectors do not control the site of insertion into the genome, hence, the possibility of disruption of the target cell genome. Risk related to viral insertions is linked to the number of insertions of the transgene into the cellular DNA, as has been demonstrated for replication competent and replication deficient retroviruses in experiments. At high number of insertions per cell, cell transformation due to vector induced activation of proto-oncogenes is more likely to occur, in particular since more than one transforming event is needed for oncogenesis. Thus, determination of the vector copy number in bulk transduced populations, individual colony forming units, and tissue from the recipient of the transduced cells is an increasingly important safety assay and has become a standard, though not straightforward assay, since the inception of quantitative PCR.
Schuesler
Todd
T
Division of Experimental Hematology, Cincinnati Children's, Hospital Medical Center, Cincinnati, OH, USA.
Reeves
Lilith
L
Kalle
Christof von
Cv
Grassman
Elke
E
D001483:Base Sequence; D017931:DNA Primers; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D012015:Reference Standards; D015203:Reproducibility of Results
D017931:DNA Primers