P37. Epigenetic changes induced in human hematopoietic progenitor/stem cells following ex vivo lentiviral transduction

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Gene therapy is a promising strategy to treat certain inherited disorders. In that context, lentiviral vectors (LV) have been used in ex vivo protocols for the stable gene-modification of hematopoietic stem cells. There are concerns about the potential genotoxicity of integrative vectors such as LV. So far, these concerns have only been envisioned as resulting from unwanted genomic insertion leading to insertional mutagenesis. At present, there has been no investigation of potential epigenetic modification induced by the procedure of gene transfer into stem cells. Epigenetic changes are known to be induced in cells following culture and stress. This prompted us to investigate if the procedure used for ex vivo lentiviral transduction which involves a period of cytokine pre-activation prior to LV infection, could induce epigenetic alterations in human CD34+ haematopoietic/progenitor cells. The expression of a panel of known epigenetic proteins was explored by immunohistochemical analysis at initiation of the treatment, post-stimulation and post-infection of umbilical cord blood CD34+ cells. To measure the patterns and levels of expression of these proteins in the cell population and to account for the heterogeneity of the CD34+ cell population, we used high throughput image analysis with the Cell Profiler Software. As expected, the cytokine pre-activation of the cells led to a heterogeneous up regulation of several proteins known to be involved in metabolic changes and stress response. Importantly, the systematic analysis of up to 30 morphological features per cell indicated that lentiviral infection itself had a distinct effect than those of cytokines. Cells infected with the LV displayed significantly different and more heterogenous phenotypic profiles compared to non-transduced cells or pre-activated only cells. These constitute the first observations to our knowledge that ex vivo lentiviral transduction of hematopoietic cells may induce epigenetic changes. Our data suggest that lentiviral transduction may represent a stress for the cell leading to an “epigenetic storm” which could induce variable deregulation of certain genes in the cells. We show that these effects could be monitored and further experiments are ongoing to evaluate the impact of different experimental conditions on such epigenetic changes.