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Nucleic Acids Research 2004 32(12):e106; doi:10.1093/nar/gnh104
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Published online 16 July 2004

Nucleic Acids Research, Vol. 32 No. 12 © Oxford University Press 2004; all rights reserved

Design and in vivo characterization of self-inactivating human and non-human lentiviral expression vectors engineered for streptogramin-adjustable transgene expression

Barbara Mitta, Cornelia C. Weber1, Markus Rimann and Martin Fussenegger*

Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Hoenggerberg, HPT D74, CH-8093 Zurich, Switzerland and 1 Institute of Biomedical Engineering, ETH Zurich and University of Zurich, CH-8044 Zurich, Switzerland

* To whom correspondence should be addressed. Tel: +41 1 633 34 48; Fax: +41 1 633 12 34; Email: fussenegger{at}biotech.biol.ethz.ch

Received January 26, 2004; Revised June 2, 2004; Accepted June 27, 2004

Adjustable transgene expression is considered key for next-generation molecular interventions in gene therapy scenarios, therapeutic reprogramming of clinical cell phenotypes for tissue engineering and sophisticated gene-function analyses in the post-genomic era. We have designed a portfolio of latest generation self-inactivating human (HIV-derived) and non-human (EIAV-based) lentiviral expression vectors engineered for streptogramin-adjustable expression of reporter (AmyS{Delta}S, EYFP, SAMY, SEAP), differentiation-modulating (human C/EBP-{alpha}) and therapeutic (human VEGF) transgenes in a variety of rodent (CHO-K1, C2C12) and human cell lines (HT-1080, K-562), human and mouse primary cells (NHDF, PBMC, CD4+) as well as chicken embryos. Lentiviral design concepts include (i) binary systems harboring constitutive streptogramin-dependent transactivator (PIT) and PIT-responsive transgene expression units on separate lentivectors; (ii) streptogramin-responsive promoters (PPIR8) placed 5' of desired transgenes; (iii) within modified enhancer-free 3'-long terminal repeats; and (iv) bidirectional autoregulated configurations providing streptogramin-responsive transgene expression in a lentiviral one-vector format. Rigorous quantitative analysis revealed HIV-based direct PPIR-transgene configurations to provide optimal regulation performance for (i) adjustable expression of intracellular and secreted product proteins, (ii) regulated differential differentiation of muscle precursor cell lines into adipocytes or osteoblasts and (iii) conditional vascularization fine-tuning in chicken embryos. Similar performance could be achieved by engineering streptogramin-responsive transgene expression into an autoregulated one-vector format. Powerful transduction systems equipped with adjustable transcription modulation options are expected to greatly advance sophisticated molecular interventions in clinically and/or biotechnologically relevant primary cells and cell lines.


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