221. Staurosporine Increases Lentiviral Transduction of Human CD34+ Cells

Lentiviral vector (LVV) mediated transduction of CD34+ hematopoietic stem and progenitor cells holds tremendous promise for the treatment of monogenic diseases of the blood. Critical to the success of this ex vivo gene therapy approach is the generation of a sufficient proportion of gene-modified cells. Here we investigated the potential of staurosporine, a protein kinase inhibitor, to enhance the transduction of LVVs in mobilized peripheral blood CD34+ cells both in vitro and in vivo. Staurosporine treatment has been previously demonstrated to increase HIV-1 integration in metaphase-arrested cells and shown to cause chromatin relaxation in metaphase cells (Manioui et al., Virology 2004). Additionally, in a separate study staurosporine treatment led to a 150% increase in transduction of CAp24+ CD4+ T cells (Permanyer et al., PLoS One 2013). Staurosporine increases vector copy number (VCN) approximately 2-fold when added to human mobilized peripheral blood (mPB) CD34+ enriched cells prior to transduction. The mechanism of this VCN improvement was investigated using the BLAM assay and a 1.5-fold improvement in viral entry was demonstrated. This effect was observed in at least six different mPB CD34+ cell lots that had various levels of transducibility. Interestingly, the effect of increased LVV transduction was most striking with low transducing cell lots and the effect diminished in higher transducing cell lots. Staurosporine treatment did not affect viability of cells post-transduction and there was no difference in in vitro colony formation in staurosporine-treated cells compared to vehicle-treated cells. NSG mice transplanted with cells transduced in the presence of 400 nM or 800 nM staurosporine demonstrated a statistically significant 3-fold and 4-fold increase in VCN in human CD45+ cells in bone marrow at 4 months post-transplantation compared to vehicle-treated cells. Importantly, there was no significant difference in engraftment nor any observed lineage-bias between groups. Based on these observations, the use of staurosporine to enhance LVV transduction of human CD34+ cells is a promising method to improve the potential therapeutic benefit of gene therapy drug products.