Cell Cycle Status of CD34+ Hemopoietic Stem Cells Determines Lentiviral Integration in Actively Transcribed and Development-related Genes
2015
Gene therapy utilizing lentiviral-vectors (LVs) is postulated as a dynamic therapeutic alternative for
monogenicdiseases. However, retroviral gene transfer may cause
insertional mutagenesis. Although, such risks had been originally estimated as extremely low, several reports of leukemias or clonal dominance, have led to a re-evaluation of the mechanisms operating in
insertional mutagenesis. Therefore, unraveling the mechanism of retroviral integration is mandatory toward
safergene therapy applications. In the present study, we undertook an experimental approach which enabled direct correlation of the
cell cyclestage of the target cell with the integration profile of LVs. CD34+ cells arrested at different stages of
cell cycle, were transduced with a GFP-LV. LAM-PCR was employed for integration site detection, followed by microarray analysis to correlate transcribed genes with integration sites. The results indicate that ~10% of integration events occurred in actively transcribed genes and that the
cell cyclestage of target cells affects integration pattern. Specifically, use of thymine promoted a
saferprofile, since it significantly reduced integration within
cell cycle-related genes, while we observed increased possibility for integration into genes related to development, and decreased possibility for integration within
cell cycleand cancer-related genes, when transduction occurs during mitosis.
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