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Nucleic Acids Research, 2002, Vol. 30, No. 21 e113
© 2002 Oxford University Press

Advanced modular self-inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction

Barbara Mitta, Markus Rimann, Markus U. Ehrengruber1, Martin Ehrbar2, Valentin Djonov3, Jens Kelm and Martin Fussenegger*

Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Zurich, ETH Hoenggerberg, HPT, CH-8093 Zurich, Switzerland, 1 Brain Research Institute, University of Zurich, CH-8057 Zurich, Switzerland, 2 Department of Materials and Institute for Biomedical Research, ETH Zurich and University of Zurich, CH-8044 Zurich, Switzerland and 3 Institute of Anatomy, University of Bern, CH-3009 Bern, Switzerland

*To whom correspondence should be addressed. Tel: +41 1 633 34 48; Fax: +41 1 633 10 51; Email: fussenegger{at}biotech.biol.ethz.ch

In recent years, lentiviral expression systems have gained an unmatched reputation among the gene therapy community for their ability to deliver therapeutic transgenes into a wide variety of difficult-to-transfect/transduce target tissues (brain, hematopoietic system, liver, lung, retina) without eliciting significant humoral immune responses. We have cloned a construction kit-like self-inactivating lentiviral expression vector family which is compatible to state-of-the-art packaging and pseudotyping technologies and contains, besides essential cis-acting lentiviral sequences, (i) unparalleled polylinkers with up to 29 unique sites for restriction endonucleases, many of which recognize 8 bp motifs, (ii) strong promoters derived from the human cytomegalovirus immediate-early promoter (PhCMV) or the human elongation factor 1{alpha} (PhEF1{alpha}), (iii) PhCMV– or PPGK– (phosphoglycerate kinase promoter) driven G418 resistance markers or fluorescent protein-based expression tracers and (iv) tricistronic expression cassettes for coordinated expression of up to three transgenes. In addition, we have designed a size-optimized series of highly modular lentiviral expression vectors (pLenti Module) which contain, besides the extensive central polylinker, unique restriction sites flanking any of the 5'U3, R-U5-{psi}+-SD, cPPT-RRE-SA and 3'LTR{Delta}U3 modules or placed within the 5'U3 (–78 bp) and 3'LTR{Delta}U3 (8666 bp). pLentiModule enables straightforward cassette-type module swapping between lentiviral expression vector family members and facilitates the design of Tat-independent (replacement of 5'LTR by heterologous promoter elements), regulated and self-excisable proviruses (insertion of responsive operators or LoxP in the 3'LTR{Delta}U3 element). We have validated our lentiviral expression vectors by transduction of a variety of insect, chicken, murine and human cell lines as well as adult rat cardiomyocytes, rat hippocampal slices and chicken embryos. The novel multi-purpose construction kit-like vector series described here is compatible with itself as well as many other (non-viral) mammalian expression vectors for straightforward exchange of key components (e.g. promoters, LTRs, resistance genes) and will assist the gene therapy and tissue engineering communities in developing lentiviral expression vectors tailored for optimal treatment of prominent human diseases.


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