1038. Lentiviral Gene Transfer into HSC Is Enhanced by Early-Acting Cytokines without Impairing Stem Cell Properties and Involves Cellular Responses Distinct from Cell Cycle Control

Abstract

Hematopoietic stem cells (HSC) are attractive targets for gene therapy because they can be transduced ex vivo and infused in the donor to generate gene-corrected progeny potentially for a lifespan. To broaden the scope of HSC-based gene therapy, transduction of the majority of HSC in a transplant sample would be required, without affecting stem cell features. HIV-derived lentiviral vectors (LV) are good candidates for this purpose, because they integrate in non-proliferating cells, thus bypassing the requirement of retroviral vectors for ex vivo cell cycle induction. We and others have demonstrated that LV transduce efficiently cord blood-derived SCID/NOD mouse repopulating cells (SRC), assumed to be closely related to HSC, by a short ex vivo incubation. Contrary to the expectations, however, hematopoietic progenitors treatment with the early-acting cytokines IL6, SCF, TPO and Flt3L, strongly enhanced SRC transduction. To determine if cytokine treatment affected SRC repopulating ability, we performed a competitive repopulation assay, injecting half CD34+ cells transduced with and half transduced without cytokines into the same NOD/SCID mouse. We observed that SRC transduced with and without stimulation contributed equally to host repopulation, indicating that cytokine stimulation did not impair engraftment and repopulating ability of HSC in this stringent model. We then asked if cytokines enhanced transduction triggering the cells through the S-phase of the cell cycle, a condition that could lead to the loss of some SRC features. To address this issue, we performed an S-phase suicide assay, transducing CD34+ cells with or without cytokines and exposing them or not to an agent that kills all the cells that pass through the S-phase, before injecting them into NOD/SCID mice. We found higher transduction efficiency in stimulated SRC, and this higher level was also observed for cells exposed to the killing agent, indicating that cytokine enhancement of SRC transduction was not due to progression into S-phase. We then aimed to determine at what stage of transduction cytokines enhanced LV gene transfer. We evaluated viral entry, transducing CD34+ cells with LV vectors pseudotyped with different envelopes, such as A-MLV and RD114/TR. In all cases, we observed higher transduction efficiency in stimulated cells, indicating that the cytokine effect was not linked to a specific entry pathway. Performing transduction with different vector doses, we showed a similar one slope-trend in the dose-response curve both for stimulated and not stimulated CD34+ cells, suggesting that the different transduction efficiency was not due to the expression of saturable restrictive factor by not stimulated cells. Preliminary results rather indicate that, in hematopoietic progenitors, susceptibility to LV transduction is differentially regulated by the activity of a specific enzymatic complex in the cytosol, and that interference with this system strongly up-regulates transduction efficiency. Overall, our results will help to establish experimental conditions for safe and efficient HSC gene therapy, and shed new light on the mechanism of HSC transduction by LV.