Jakobsen, Maria9; Stenderup, Karin10; Rosada, Cecilia11; Moldt, Brian2; Kamp, Søren6; Dam, Tomas Norman11; Jensen, Thomas G.12; Mikkelsen, Jacob Giehm13
1 The Department of Dermatology and Venereology, Faculty of Health Sciences, Aarhus University, Aarhus University2 Department of Human Genetics, Faculty of Health Sciences, Aarhus University, Aarhus University3 Department of Molecular Biology and Genetics - Gene Expression and Gene Medicine, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University4 Aarhus University School of Engineering - Bioprocess Engineering, Aarhus University School of Engineering, Science and Technology, Aarhus University5 Department of Clinical Medicine - The Department of Dermatology and Venereology, Department of Clinical Medicine, Health, Aarhus University6 Dermatologisk Afdeling, Roskilde Hospital7 Department of Biomedicine, Health, Aarhus University8 Department of Biomedicine - Forskning og uddannelse, Øst, Department of Biomedicine, Health, Aarhus University9 Department of Molecular Biology and Genetics - Gene Expression and Gene Medicine, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University10 Aarhus University School of Engineering - Bioprocess Engineering, Aarhus University School of Engineering, Science and Technology, Aarhus University11 Department of Clinical Medicine - The Department of Dermatology and Venereology, Department of Clinical Medicine, Health, Aarhus University12 Department of Biomedicine, Health, Aarhus University13 Department of Biomedicine - Forskning og uddannelse, Øst, Department of Biomedicine, Health, Aarhus University
The proinflammatory cytokine Tumor Necrosis Factor alpha (TNF- ) is upregulated in inflammatory psoriatic skin. The increased level of TNF- protein is thought to cause keratinocyte hyperproliferation, leukocyte infiltration as well as growth and dilation of superficial blood vessels, which are all characteristics of human psoriasis skin. Blockade of TNF- function with specific inhibitors at the protein level has resulted in a rapid clinical improvement in psoriasis patients, demonstrating that TNF- inhibition offers a promising therapy of psoriasis. Whether TNF- -encoding RNA is a valid therapeutic target, however, is still a matter of speculation, as recent findings have suggested that the level of TNF- is not increased in psoriatic skin. To test the hypothesis that TNF- in skin can be stably down-regulated by RNA interference (RNAi), we designed a panel of short hairpin RNAs (shRNAs) targeting human TNF- . Their efficiency in down-regulating TNF- protein expression was evaluated using a Renilla luciferase screening-assay based upon targeting of luc-TNF- fusion RNAs and a transient co-transfection assay. The three most potent shRNAs, which at most reduced the expression from target templates to 15% of the control samples treated with irrelevant shRNAs, were selected and cloned into lentiviral vectors. The lentiviral vectors expressing TNF- shRNAs were used to transduce HEK-293 cells and verify vector-derived knock-down of stable TNF- expression in vitro. The most efficient TNF- -directed shRNA, which in cell lines reduced the amount of released TNF- more than 50% upon viral transduction, was selected for in vivo studies. In vivo studies were carried out in a xenograft mouse model in which human psoriatic plaques keratome skin biopsies were transplanted onto SCID mice. Initial studies using eGFP-encoding lentiviral vectors demonstrated efficient transduction of human psoriatic skin. Grafted psoriatic skin was exposed to viral vector-encoded TNF- shRNAs by a single intradermal injection of purified VSV-G-pseudotyped lentiviral vectors (150 l containing 46.4 ng p24/ l was injected at a single site). Biopsies were taken three weeks after injection. qPCR-based measurements of TNF- mRNA in skin treated with lentiviral vector-encoded TNF- shRNA demonstrated a 50% reduction in the level of TNF- mRNA. Most interestingly, the epidermal thickness of the human psoriatic plaques was reduced relative to mice treated with lentiviral vectors encoding an irrelevant shRNA. In conclusion, our results demonstrate that lentiviral vector-encoded TNF- shRNAs have the potential to down-regulate TNF- production both in vitro and in vivo. Phenotypic changes in shRNA-treated psoriatic skin suggest that TNF- -encoding RNA is a valid therapeutic target in psoriasis treatment.
Lentivirus VectorsRNAi; Oligonucleotide Based Therapies; shRNA