title: Tailor-made fibroblast-specific and antibiotic-free interleukin 12 plasmid for gene electrotransfer-mediated cancer immunotherapy.
abstract: Electrotransfer mediated delivery of interleukin-12 (IL-12) gene, encoded on a plasmid vector, has already been demonstrated to have a potent antitumor efficacy and great potential for clinical application. In the present study, our aim was to construct an optimized IL-12-encoding plasmid that is safe from the regulatory point of view. In light of previous studies demonstrating that IL-12 should be released in a tumor localized manner for optimal efficacy, the strong ubiquitous promoter was replaced with a weak endogenous promoter of the collagen 2 gene, which is specific for fibroblasts. Next, to comply with increasing regulatory demands for clinically used plasmids, the expression cassette was cloned in a plasmid lacking the antibiotic resistance gene. The constructed fibroblast-specific and antibiotic-free IL-12 plasmid was demonstrated to support low IL-12 expression after gene electrotransfer in selected cell lines. Furthermore, the removal of antibiotic resistance did not affect the plasmid expression profile and lowered its cytotoxicity. With optimal IL-12 expression and minimal transgene non-specific effects, i.e., low cytotoxicity, the constructed plasmid could be especially valuable for different modern immunological approaches to achieve localized boosting of the host's immune system.
authors.affiliation: Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia. Electronic address: ukamensek@onko-i.si.
authors.forename: Urska
authors.initials: U
authors.lastname: Kamensek
authors.affiliation: University of Primorska, Faculty of Health Sciences, Polje 42, SI-6310 Izola, Slovenia. Electronic address: natasa.tesic@fvz.upr.si.
authors.forename: Natasa
authors.initials: N
authors.lastname: Tesic
authors.affiliation: Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia. Electronic address: gsersa@onko-i.si.
authors.forename: Gregor
authors.initials: G
authors.lastname: Sersa
authors.affiliation: Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia. Electronic address: skos@onko-i.si.
authors.forename: Spela
authors.initials: S
authors.lastname: Kos
authors.affiliation: Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia; University of Primorska, Faculty of Health Sciences, Polje 42, SI-6310 Izola, Slovenia. Electronic address: mcemazar@onko-i.si.
authors.forename: Maja
authors.initials: M
authors.lastname: Cemazar
mesh_terms: D000818:Animals; D018274:Electroporation / Q000379:methods; D005347:Fibroblasts / Q000378:metabolism*; D015870:Gene Expression; D023061:Gene Order; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D006801:Humans; D007167:Immunotherapy; D018664:Interleukin-12 / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D009369:Neoplasms / Q000235:genetics / Q000276:immunology / Q000473:pathology / Q000628:therapy; D009928:Organ Specificity / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D019076:Transgenes
chemical_list: D018664:Interleukin-12
keywords: Antibiotic-free plasmid; Fibroblast-specific expression; Gene electrotransfer; Interleukin 12