Comparison of single regulated lentiviral vectors with rtTA expression driven by an autoregulatory loop or a constitutive promoter.
Regulated expression of a therapeutic gene is crucial for safe and efficacious gene therapy. Many inducible regulatory systems use a constitutive promoter to express a regulatory protein, such as rtTA in the Tet-On system, which may restrict their use because of cytotoxicity and immunogenicity. Autoregulatory expression of rtTA provides extremely low levels of rtTA when transgene expression is off, with rapid transgene induction upon addition of doxycycline. Lentiviral vectors efficiently transfer genes to dividing and non-dividing cells with long-term gene expression both in vitro and in vivo. We compared regulatory function in a single lentiviral vector where rtTA was either expressed from a constitutive promoter or placed in an autoregulatory loop. Autoregulatory expression of rtTA was superior to constitutive promoter expression, resulting in higher viral titers, undetectable levels of both rtTA and transgene expression in the absence of doxycycline, improved induction kinetics and increased induction levels in all cells tested. We further expanded the utility of the autoregulatory vector by using an improved rtTA variant with an increased sensitivity to doxycycline. This lentiviral vector with doxycycline-regulated transgene expression may be useful for gene therapy applications and in experimental settings where strict temporal expression of a transgene is required.
Markusic
David
D
AMC Liver Center S1-172, Meibergdreef 69, 1105BK Amsterdam, The Netherlands. d.m.markusic@amc.uva.nl
Oude-Elferink
Ronald
R
Das
Atze T
AT
Berkhout
Ben
B
Seppen
Jurgen
J
D002460:Cell Line; D002478:Cells, Cultured; D004318:Doxycycline / Q000494:pharmacology; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006706:Homeostasis; D006801:Humans; D007700:Kinetics; D016086:Lentivirus / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics*; D015533:Transcriptional Activation; D014161:Transduction, Genetic
D012097:Repressor Proteins; D015534:Trans-Activators; C034811:tetracycline resistance-encoding transposon repressor protein; D004318:Doxycycline