Modified human immunodeficiency virus-based lentiviral vectors display decreased sensitivity to trans-dominant rev

As a first step toward the development of HIV-based conditionally replicati ng 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. Ve ctors with mutations in the splicing signals (SD-/RRE+) yielded high titers (5 x 10(6) CFU/ml) and showed higher levels of cytoplasmic unspliced mRNA than the corresponding SD+/RRE+ vectors either in the absence of Rev, in th e presence of TdRev, or in the presence of both TdRev and Rev. Proviral cop ies 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 R ev expression. Titers obtained with the SD-/CTE+ vectors (10(6) CFU/ml) wer e higher than the titers obtained with SD+/CTE+ vectors. We also tested the effect of other structural modifications such as the orientation of the ex pression cassette and the presence of the central polypurine tract (cPPT/CT S). We show that an expression cassette cloned in the reverse orientation w ith respect to the LTRs or elimination of the cPPT/CTS element severely aff ected vector titers. We also demonstrated that these vectors can be efficie ntly mobilized from their proviral state by HIV trans-complementing functio ns, and transduced into secondary target cells without suffering any genomi c rearrangement.