Lentiviral vector gene transfer is limited by the proteasome at post-entry steps in various types of stem cells

The isolation of human embryonic and somatic stem cells of different types has made it possible to design novel gene and cell replacement therapies. Vectors derived from retro/lentiviruses are used to stably introduce genes into stem cells and their progeny. Yet, the permissivity to retroviral infection varies among cell types. We previously showed that hematopoietic stem cells are poorly permissive to HIV-derived vectors and that pharmacological inhibition of the proteasome strongly enhances gene transfer. Here we report that the proteasome limits lentiviral gene transfer in all stem cell types tested, including embryonic, mesenchymal and neural, both of human and mouse origin. Remarkably, this inhibitory activity was sharply reduced upon differentiation of the stem cells, suggesting that it represents a novel feature of the stem cell/immature progenitor phenotype. Proteasomemediated inhibition was specific for lentiviral vectors and occurred at a post-entry infection step. It was not mediated by activation of NF-kB, a major signaling pathway modulated by the proteasome, and did not correlate with high proteasome activity. Interaction of the virion core with Cyclophilin A was required to maximize the effect of proteasome inhibitor on the infection pathway. These findings are relevant to uncover new mediators of HIV gene transfer and help designing more effective protocols for the genetic modification of stem cells.