100. Inducible and Reversible Transgene Expression in Human Stem Cells Stably Transduced by Novel Lentiviral Vectors

It is ideal, and quite often necessary, to have regulated or inducible transgene expression in stem cell-based gene therapies or basic studies, in which stage-specific or lineage-specific gene expression is crucial in analyzing progeny cells after stable gene transfer to the ancestral stem cells. We have previously shown that self-inactivating (SIN) lentiviral vectors confer promoter-specific transgene expression. In this study, we used the tetracycline repressor (tetR) that is fused with the KRAB transcriptional suppression domain (tTS) to specifically suppress transgene expression from stably transduced human cells. The transgene expression is controlled by a regular promoter adjacent to a high affinity-binding site (tetO7) of tTS. After optimizing a lentiviral vector containing a strong ubiquitous promoter such as EF1a and UBC and the location of the tetO7 site in the vector, we achieved better inducible transgene expression than previous lentiviral system containing the |[ldquo]|tet-off|[rdquo]| or |[ldquo]|tet-on|[rdquo]| system. In the new system, the transgene transcription from a regular promoter such as EF1a or UBC is suppressed by the tTS bound to the nearby tetO7 site in the absence of a tet analog doxycycline (Dox). In the presence of Dox, however, the tTS binding is off from the vector DNA and the regular transcription is restored. Thus the new system (dubbed as tet-SUPER) simply adds an extra level of regulation on transgene transcription, regardless which type of promoter is used. We tested the tet-SUPER system in human CD34- positive multipotent hematopoietic progenitor cell line TF1 and human embryonic stem (hES) cell lines H1 and I-6, using a dual-gene vector containing the GFP reporter (titers remain at 10-20 million transducing units per ml). In both human cell systems, we observed a tight regulation (suppression) in the un-induced or |[ldquo]|off|[rdquo]| state when Dox is absent. However, the suppression is reversible even after long-term suppression in the absence of Dox. The specific suppression was abolished and transgene expression was restored at a very low level of Dox (5 ng/ml). The presence of the tet-SUPER system did not alter unique properties of hES cells. In addition, the tet-SUPER lentiviral vector also conferred inducible transgene expression of the human PIG-A gene in a mutated human cell line that is deficient in the PIG-A gene and thus GPI-anchored proteins on cell surface. Together with recent papers published by Dr. Trono and others, we show that it is now possible to achieve inducible and reversible transgene expression in essentially any mammalian cell types.