Modified Human Immunodeficiency Virus-Based Lentiviral Vectors Display Decreased Sensitivity to Trans -Dominant Rev

Abstract

As a first step toward the development of HIV-based conditionally replicating defective interfering particles expressing trans-dominant Rev (TdRev), we studied whether mutation of the splicing signals and replacement of the RRE by the SRV-1 CTE would render these vectors less sensitive to TdRev. Vectors with mutations in the splicing signals (SD-/RRE⁺) yielded high titers (5 X 10⁶ CFU/ml) and showed higher levels of cytoplasmic unspliced mRNA than the corresponding SD⁺/RRE⁺ vectors either in the absence of Rev, in the presence of TdRev, or in the presence of both TdRev and Rev. Proviral copies of SD^-/RRE⁺ vectors were rescued more efficiently than SD⁺/RRE⁺ vectors when TdRev was expressed. Vectors with the SRV-1 CTE(SD⁺/CTE⁺ and SD^-/CTE⁺) expressed high levels of cytoplasmic unspliced mRNA in the absence of Rev expression. Titers obtained with the SD^-/CTE⁺ vectors (10⁶ CFU/ml) were higher than the titers obtained with SD⁺/CTE⁺ vectors. We also tested the effect of other structural modifications such as the orientation of the expression cassette and the presence of the central polypurine tract (cPPT/CTS). We show that an expression cassette cloned in the reverse orientation with respect to the LTRs or elimination of the cPPT/CTS element severely affected vector titers. We also demonstrated that these vectors can be efficiently mobilized from their proviral state by HIV trans-complementing functions, and transduced into secondary target cells without suffering any genomic rearrangement.