title: An adaptable system for improving transposon-based gene expression in vivo via transient transgene repression.
abstract: Transposons permit permanent cellular genome engineering in vivo. However, transgene expression falls rapidly postdelivery due to a variety of mechanisms, including immune responses. We hypothesized that delaying initial transgene expression would improve long-term transgene expression by using an engineered piggyBac transposon system that can regulate expression. We found that a 2-part nonviral Tet-KRAB inducible expression system repressed expression of a luciferase reporter in vitro. However, we also observed nonspecific promoter-independent repression. Thus, to achieve temporary transgene repression after gene delivery in vivo, we utilized a nonintegrating version of the repressor plasmid while the gene of interest was delivered in an integrating piggyBac transposon vector. When we delivered the luciferase transposon and repressor to immunocompetent mice by hydrodynamic injection, initial luciferase expression was repressed by 2 orders of magnitude. When luciferase expression was followed long term in vivo, we found that expression was increased >200-fold compared to mice that received only the luciferase transposon and piggyBac transposase. We found that repression of early transgene expression could prevent the priming of luciferase-specific T cells in vivo. Therefore, transient transgene repression postgene delivery is an effective strategy for inhibiting the antitransgene immune response and improving long-term expression in vivo without using immunosuppression.
authors.affiliation: Medical Scientist Training Program, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Doherty
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Woodard
authors.forename: Adham S
authors.initials: AS
authors.lastname: Bear
authors.forename: Aaron E
authors.initials: AE
authors.lastname: Foster
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Wilson
mesh_terms: D000818:Animals; D004251:DNA Transposable Elements / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D019076:Transgenes / Q000235:genetics*; D019895:Transposases / Q000235:genetics / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D019895:Transposases
keywords: gene transfer; inducible expression; piggyBac