title: Effects of APC De-targeting and GAr modification on the duration of luciferase expression from plasmid DNA delivered to skeletal muscle.
abstract: Immune responses to expressed foreign transgenes continue to hamper progress of gene therapy development. Translated foreign proteins with intracellular location are generally less accessible to the immune system, nevertheless they can be presented to the immune system through both MHC Class I and Class II pathways. When the foreign protein luciferase was expressed following intramuscular delivery of plasmid DNA in outbred mice, expression rapidly declined over 4 weeks. Through modifications to the expression plasmid and the luciferase transgene we examined the effect of detargeting expression away from antigen-presenting cells (APCs), targeting expression to skeletal muscle and fusion with glycine-alanine repeats (GAr) that block MHC-Class I presentation on the duration of luciferase expression. De-targeting expression from APCs with miR142-3p target sequences incorporated into the luciferase 3'UTR reduced the humoral immune response to both native and luciferase modified with a short GAr sequence but did not prolong the duration of expression. When a skeletal muscle specific promoter was combined with the miR target sequences the humoral immune response was dampened and luciferase expression persisted at higher levels for longer. Interestingly, fusion of luciferase with a longer GAr sequence promoted the decline in luciferase expression and increased the humoral immune response to luciferase. These studies demonstrate that expression elements and transgene modifications can alter the duration of transgene expression but other factors will need to overcome before foreign transgenes expressed in skeletal muscle are immunologically silent.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Subang
authors.forename: Rewas
authors.initials: R
authors.lastname: Fatah
authors.forename: Ying
authors.initials: Y
authors.lastname: Wu
authors.forename: Drew
authors.initials: D
authors.lastname: Hannaman
authors.forename: Jason
authors.initials: J
authors.lastname: Rice
authors.forename: Claire F
authors.initials: CF
authors.lastname: Evans
authors.forename: Yuti
authors.initials: Y
authors.lastname: Chernajovsky
authors.affiliation: Bone & Joint Research Unit, Queen Mary University of London, William Harvey Research Institute, Charterhouse Square, London EC1M 6BQ, UK. d.j.gould@qmul.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Gould
mesh_terms: D000818:Animals; D000938:Antigen-Presenting Cells / Q000166:cytology*; D004151:Dipeptides / Q000378:metabolism*; D004926:Escherichia coli; D005260:Female; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D018895:Microsatellite Repeats; D018482:Muscle, Skeletal / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D019076:Transgenes; D020298:U937 Cells
chemical_list: D004151:Dipeptides; D035683:MicroRNAs; C529102:Mirn142 microRNA, mouse; C028872:alanylglycine; D008156:Luciferases