62. Proteasome Activity Restricts Lentiviral Gene Transfer into Hematopoietic Stem Cells and Is Down-Regulated by Cytokines That Enhance Transduction
Authors: Francesca R. Santoni de Sio, Paolo Cascio, Anna Zingale, Mauro Gasparini, Luigi Naldini
Journal: Molecular Therapy (2006)

The therapeutic potential of hematopoietic stem cell (HSC) gene therapy can be fully exploited only by reaching efficient gene transfer into HSCs without compromising their biological properties. HIV- derived lentiviral vectors (LVs) are good candidates for this purpose. Although HSCs can be transduced by LVs in short ex vivo culture, they display low permissivity to the vector, requiring cytokine stimulation to reach high-frequency transduction. Using stringent assays of competitive xenograft repopulation, we show that early-acting cytokines (SCF, IL6, TPO, Flt3L) synergistically enhanced human HSC gene transfer by LV without impairing engraftment and repopulation capacity. Using S-phase suicide assays, we show that transduction enhancement by cytokines was not dependent on cell cycle progression and demonstrate that LVs can transduce quiescent HSCs. Cytokines enhancement of LV gene transfer was not dependent on the envelope pseudotype, indicating that cytokine stimulation did not affect a specific entry pathway. We then evaluated factors influencing the post-entry steps of viral infection. Pharmacological inhibition of the proteasome during transduction dramatically enhanced HSC gene transfer. HSCs transduced in the presence of proteasome inhibitors showed a dramatic increase in the levels of vector integration, without apparent adverse effects on engraftment capacity in NOD/SCID mice. This indicates that: i) a large excess of vector particles is uptaken by HSCs but does not become integrated; ii) LVs are effectively restricted at a post-entry step by proteasome activity; iii) proteasome inhibition may represent a new strategy to overcome the low HSC permissiveness to LV gene transfer. By measuring the proteasome activities in cytokine-stimulated and unstimulated hematopoietic progenitors, unexpectedly we found that they were significantly down-regulated by exposure to the cytokines. Importantly, this response was rapid, with a maximal effect at early time points corresponding to the period in which the first steps of LV transduction take place, and synergistic for SCF, TPO and Flt3L. These findings highlight one of the mechanisms by which cytokines may enhance permissiveness to LV gene transfer. Interestingly, we did not observe a similar extent of proteasome- mediated LV restriction in other primary cells, suggesting that it represents a specific feature of HSCs, both human and murine. Effective restriction correlated with high proteasome activity. We are currently investigating the molecular mechanism of restriction, whether the proteasome targets uncoating vector cores, directly or through the action of host restrictive factors, or rather acts by targeting factors required for the early steps of transduction. Our findings demonstrate that antiviral responses ultimately mediated by the proteasome strongly limit the efficiency of HSC transduction by LVs, and help to establish improved conditions for HSC-based gene therapy.