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 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 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 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 (10(6) 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.
Mautino
M R
MR
Clinical Gene Therapy Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-1851, USA.
Ramsey
W J
WJ
Reiser
J
J
Morgan
R A
RA
D001483:Base Sequence; D002460:Cell Line; D015694:Gene Products, rev / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005799:Genes, Dominant; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics*; D006678:HIV / Q000235:genetics*; D006801:Humans; D009154:Mutation; D011687:Purines / Q000737:chemistry; D012326:RNA Splicing; D015301:Retroviruses, Simian / Q000235:genetics; D019076:Transgenes; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D011687:Purines; D054321:rev Gene Products, Human Immunodeficiency Virus