545. Lentiviral Vector Mediated Gene Transfer of the Anti HIV Gene revM10 into Primitive Hematopoietic Cells for Gene Therapy of HIV Infection

Abstract

Lentiviral vectors are well suited for the transduction of resting hematopoietic progenitor cells. Yet, delivery of an anti-HIV gene using such a vector poses a challenge because the anti-HIV gene might reduce the otherwise high titer of the lentiviral vector preparation needed for efficient transduction of resting hematopoietic progenitor cells. The revM10 anti-HIV gene might reduce the titer by preventing export of unspliced packaging or vector message. Therefore, we investigated packaging and vector constructs that either contained or lacked the 5' splice donor site, reasoning that the deletion of this splice site might reduce the dependence of the resulting transcripts on the rev protein. The presence of the RRE in such 5' splice site deleted vectors was also evaluated. Theoretically, the presence of the RRE could lead to competition with the packaging construct for rev binding during packaging, leading to a diminished titer. Alternatively, the presence of the RRE could lead to an increased amount of vector transcripts being exported into the cytoplasm, leading to an increased titer. Furthermore, the combination of the revM10 and the RRE in one vector construct might be additive, synergistic or even antagonistic during both the packaging process and in their anti-HIV activity in the transduced target cell. Therefore, the presence of the RRE was evaluated in two different locations in the lentiviral vector: located outside the internal transcript and thus active only during packaging or located inside the internal transcript and thus active during both packaging and in the transduced target cells. Analysis of the different combinations of modified vector and packaging constructs revealed that the combination of revM10 with RRE in a vector containing the 5' splice site resulted in high titer vector preparations independent of whether the packaging construct contained a 5' splice site or not. Analysis of the location of the RRE in the lentiviral vector containing the revM10 gene revealed that the presence of the RRE in the internal transcript increased the anti-HIV activity, indicating that the combination of revM10 and RRE is either additive or synergistic but not antagonistic.