Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors.
Bidirectional lentiviral vectors mediate expression of two or more cDNAs from a single internal promoter. In this study, we examined mechanisms that control titer and expression properties of this vector system. To address whether the bidirectional design depends on lentiviral (LV) backbone components, especially the Rev/Rev responsive element (RRE) system, we constructed similar expression cassettes for LV and gammaretroviral (GV) vectors. Bidirectional expression levels could be adjusted by the use of different internal promoters. Furthermore, removal of the constitutive RNA transport element of Mason-Pfizer monkey virus, used in first generation bidirectional LV vectors, improved gene expression. Titers of bidirectional vectors were approximately 10-fold reduced in comparison to unidirectional vectors, independent of the Rev/RRE interaction. We reasoned that titer reductions were due to the formation of interfering double-stranded RNA in packaging cells. Indeed, cotransfection of Nodamuravirus B2 protein, an RNA interference suppressor, increased bidirectional vector titers at least fivefold. We validated the potential of high titer bidirectional vectors by coexpressing a fluorescent marker with O(6)-methylguanine-DNA methyltransferase from integrating, or with Cre recombinase from integrating and non-integrating GV and LV backbones. This allowed for the tracking of chemoprotected and recombined cells by fluorescence marker expression.
Department of Experimental Hematology, Hannover Medical School, Hannover, Germany.
T
T
Maetzig
M
M
Galla
M H
MH
Brugman
R
R
Loew
C
C
Baum
A
A
Schambach
D000818:Animals; D002460:Cell Line; D017988:Gammaretrovirus / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D015752:Genes, env; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D016093:Mason-Pfizer monkey virus / Q000235:genetics; D051379:Mice; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics; D011401:Promoter Regions, Genetic; D012330:RNA, Double-Stranded / Q000235:genetics; D019562:Viral Load / Q000235:genetics*
D012330:RNA, Double-Stranded; D019853:O(6)-Methylguanine-DNA Methyltransferase