A novel mammalian expression system derived from components coordinating nicotine degradation in arthrobacter nicotinovorans pAO1.
We describe the design and detailed characterization of 6-hydroxy-nicotine (6HNic)-adjustable transgene expression (NICE) systems engineered for lentiviral transduction and in vivo modulation of angiogenic responses. Arthrobacter nicotinovorans pAO1 encodes a unique catabolic machinery on its plasmid pAO1, which enables this Gram-positive soil bacterium to use the tobacco alkaloid nicotine as the exclusive carbon source. The 6HNic-responsive repressor-operator (HdnoR-O(NIC)) interaction, controlling 6HNic oxidase production in A.nicotinovorans pAO1, was engineered for generic 6HNic-adjustable transgene expression in mammalian cells. HdnoR fused to different transactivation domains retained its O(NIC)-binding capacity in mammalian cells and reversibly adjusted transgene transcription from chimeric O(NIC)-containing promoters (P(NIC); O(NIC) fused to a minimal eukaryotic promoter [P(min)]) in a 6HNic-responsive manner. The combination of transactivators containing various transactivation domains with promoters differing in the number of operator modules as well as in their relative inter-O(NIC) and/or O(NIC)-P(min) spacing revealed steric constraints influencing overall NICE regulation performance in mammalian cells. Mice implanted with microencapsulated cells engineered for NICE-controlled expression of the human glycoprotein secreted placental alkaline phosphatase (SEAP) showed high SEAP serum levels in the absence of regulating 6HNic. 6HNic was unable to modulate SEAP expression, suggesting that this nicotine derivative exhibits control-incompatible pharmacokinetics in mice. However, chicken embryos transduced with HIV-1-derived self-inactivating lentiviral particles transgenic for NICE-adjustable expression of the human vascular endothelial growth factor 121 (VEGF121) showed graded 6HNic response following administration of different 6HNic concentrations. Owing to the clinically inert and highly water-soluble compound 6HNic, NICE-adjustable transgene control systems may become a welcome alternative to available drug-responsive homologs in basic research, therapeutic cell engineering and biopharmaceutical manufacturing.
Institute for Chemical and Bio-Engineering (ICB), Swiss Federal Institute of Technology, ETH Hoenggerberg, HCI F115 Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland.
Laetitia
L
Malphettes
Cornelia C
CC
Weber
Marie Daoud
MD
El-Baba
Ronald G
RG
Schoenmakers
Dominique
D
Aubel
Wilfried
W
Weber
Martin
M
Fussenegger
D000818:Animals; D001173:Arthrobacter / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002642:Chick Embryo; D006224:Cricetinae; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D051379:Mice; D018919:Neovascularization, Physiologic; D009538:Nicotine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology*; D009875:Operator Regions, Genetic; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics; D014161:Transduction, Genetic; D019076:Transgenes; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
C502317:6-hydroxynicotine; D012097:Repressor Proteins; D015534:Trans-Activators; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D009538:Nicotine