title: Soluble TNF-α receptor I encoded on plasmid vector and its application in experimental gene therapy of radiation-induced lung fibrosis.
abstract: Post-radiation inflammatory reaction leads to an irreversible pulmonary fibrosis which may cause lethal respiratory insufficiency. Pathological inflammatory and fibrotic changes might be attenuated by inhibiting tumour necrosis factor (TNF)-α activity using TNF-α soluble receptors. Thus, an experimental antifibrotic gene therapy with the plasmid vector encoding a mouse soluble receptor I for TNF-α (psTNFR-I) was assessed. Soluble TNFR-I encoding gene was cloned into pcDNA3.1 plasmid. The ability of psTNFR-I expressing vector to transfect cells, and its biological activity in vitro and in vivo were examined by PCR, RT-PCR, MTT assay and ELISA. The C57Bl/6J mice received single intramuscular injection of psTNFR-I, conjugated with polyetylenimine (PEI) 25 kDa, equally divided to both hind legs, 3 days before irradiation (20 Gy, Co60), and either a single injection or ten injections once a week after irradiation. The data proved the effectiveness of psTNFR-I product to neutralise TNF-α activity in vitro. The in vivo plasmid incorporation and maintenance was confirmed. Measurements of plasma soluble TNFR-I levels showed that the in vivo gene transfer was effective. PEI was found to enhance transfection efficiency in vivo. The psTNFR-I/PEI complexes caused no toxicity in the transfected mice. C57Bl/6J mice that received prolonged psTNFR-I/PEI injections developed lethal fibrotic syndrome and died 8 weeks later than the mice treated with a double plasmid injection and the control mice treated with a control plasmid. Sequential administration of soluble TNFR-I by a nonviral, intramuscular gene transduction in the early and late post-radiation inflammatory phase prolonged survival of irradiated mice and attenuated the symptoms of lung fibrosis. The psTNFR-I gene transduction may provide a safe and simple method to partially neutralise TNF-α activity and prevent radiation-induced lung injury.
authors.affiliation: Cell Biology Department, Maria Skłodowska-Curie Memorial Institute of Oncology and Cancer Centre, Warsaw, Poland. magip@coi.waw.pl
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Przybyszewska
authors.forename: Joanna
authors.initials: J
authors.lastname: Miłoszewska
authors.forename: Sylwia
authors.initials: S
authors.lastname: Rzońca
authors.forename: Halina
authors.initials: H
authors.lastname: Trembacz
authors.forename: Kazimiera
authors.initials: K
authors.lastname: Pyśniak
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Kotlarz
authors.forename: Paweł
authors.initials: P
authors.lastname: Swoboda
authors.forename: Marta
authors.initials: M
authors.lastname: Zalewska
authors.forename: Maciej
authors.initials: M
authors.lastname: Małecki
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003094:Collagen / Q000032:analysis; D004195:Disease Models, Animal; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000627:therapeutic use; D006801:Humans; D008168:Lung / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids; D017564:Radiation Pneumonitis / Q000628:therapy*; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000008:administration & dosage / Q000097:blood / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D047888:Receptors, Tumor Necrosis Factor, Type I; C506236:Tnfrsf1a protein, mouse; D014409:Tumor Necrosis Factor-alpha; D003094:Collagen