Design and in vivo characterization of self-inactivating human and non-human lentiviral expression vectors engineered for streptogramin-adjustable transgene expression.
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(DeltaS), 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 (P(PIR8)) 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 P(PIR)-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.
Mitta
Barbara
B
Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Hoenggerberg, HPT D74, CH-8093 Zurich, Switzerland.
Weber
Cornelia C
CC
Rimann
Markus
M
Fussenegger
Martin
M
D017667:Adipocytes / Q000166:cytology; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D002454:Cell Differentiation; D002478:Cells, Cultured; D002642:Chick Embryo; D006224:Cricetinae; D005786:Gene Expression Regulation; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D006678:HIV / Q000235:genetics; D006801:Humans; D004860:Infectious Anemia Virus, Equine / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D032446:Myoblasts / Q000166:cytology; D010006:Osteoblasts / Q000166:cytology; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D025361:Streptogramins / Q000494:pharmacology*; D014161:Transduction, Genetic; D019076:Transgenes; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D025361:Streptogramins; D042461:Vascular Endothelial Growth Factor A; C054562:yellow fluorescent protein, Bacteria