4. β-Deliverin: A Small Molecule for Improving Gene Transfer to Hematopoietic Stem Cells and Probing Mechanisms of Lentiviral Vector Restriction
Authors: Nina Timberlake, Stosh Ozog, Saritha D'souza, Igor Slukvin, Sergio D. Catz, Scott Snyder, Bruce E. Torbett
Journal: Molecular Therapy (2016)

A major obstacle to the success of gene therapy for hematologic disorders is the inefficiency of lentiviral vector (LV) gene transfer to hematopoietic stem cells (HSCs). Achieving clinically relevant gene delivery levels requires prolonged ex vivo culture of HSCs with cytokines and large LV doses. Rapamycin, PGE2, and other small molecules, have been reported to increase LV transduction of HSCs, however the mechanism of action of these drugs and the basis for LV restriction in HSCs are poorly understood. Here, we report a novel small molecule, β-deliverin, which improves LV gene transfer to HSCs up to 3 fold over DMSO control in vitro. This effect is most pronounced in human adult peripheral blood-derived HSCs, but also observed in human cord-derived HSCs, and in non-human primate bone marrow-derived HSCs. Importantly, treatment with β-deliverin does not significantly affect the viability or expansion of HSCs in culture. Furthermore, for cord-derived HSCs, there was no reduction in the number or type of colony-forming cells. In vivo, β-deliverin treated HSCs engraft in the peripheral blood of NSG mice at levels comparable to control at 16 weeks post-engraftment. Despite an only modest increase in transduction efficiency in β-deliverin-treated cord-derived HSCs transduced in vitro (26% versus 20% for control), the marking frequency for control cells dropped to <5% in the peripheral blood of NSG mice 16 weeks post-engraftment, while mice engrafted with β-deliverin treated HSCs retained on average 20% marking. Our data suggest that β-deliverin treatment increases gene marking in long-term repopulating cells without reducing proliferation, differentiation or engraftment potential. Mechanistically, β-deliverin acts at the stage of viral fusion; the rate of fusion is increased more than 2-fold in the presence of β-deliverin. We show, by confocal microscopy, that the proportion and intensity of virus signal localized to EEA1-labelled early endosomes is unchanged by drug treatment, suggesting that viral entry is not affected. In further support of this, LDLR surface expression and receptor density is unchanged following β-deliverin treatment. In β-deliverin treated cells, 2 hours post-transduction, we observed reduced accumulation of virus in late endosomes, a 3-fold reduction in the total number of late endosomes, as measured by LAMP1 puncta, and a nearly 2-fold increase in the proportion of early endosomes when compared to control cells. Together, our data imply that β-deliverin facilitates more efficient exit of virus from the endosome, possibly by inhibiting endosomal maturation. Further mechanistic studies are ongoing. Notably, a monomer of β-deliverin has no effect on HSC transduction, suggesting an important structure-function relationship. β-deliverin is a promising tool not only for the development of novel gene delivery applications, but also to further elucidate LV-HSC interactions. We are investigating whether the mechanisms we observed here hold true for other small molecule transduction enhancers, and using these drugs to probe the biology of LV restriction in HSCs.