title: Assembly PCR synthesis of optimally designed, compact, multi-responsive promoters suited to gene therapy application.
abstract: Gene therapy has the potential to provide innovative treatments for genetic and non-genetic diseases, with the ability to auto-regulate expression levels of therapeutic molecules so that they are produced locally and in direct response to disease activity. Generating disease responsive gene therapy vectors requires knowledge of the activation profile of transcription factors (TFs) during active disease, in order to assemble binding sites for these TFs into synthetic promoters, which can be appropriately activated by the disease process. In this study, we optimised a PCR random assembly approach to generate promoters with optimal spacing between TF binding sites (TFBSs) and their distance from the TATA box. In promoters with optimal spacing, it was possible to demonstrate activation by individual transcription pathways and either additive or synergistic promoter activation when transfected cells were treated with combined stimuli. The kinetics and sensitivity of promoter activation was further explored in transduced cells and when lentivirus was directly delivered to mouse paws a synthetic promoter demonstrated excellent activation by real-time imaging in response to local inflammation.
authors.affiliation: Queen Mary University of London, William Harvey Research Institute, Bone &Joint Research Unit, United Kingdom.
authors.forename: H
authors.initials: H
authors.lastname: Mohamed
authors.affiliation: Queen Mary University of London, William Harvey Research Institute, Bone &Joint Research Unit, United Kingdom.
authors.forename: Y
authors.initials: Y
authors.lastname: Chernajovsky
authors.affiliation: Queen Mary University of London, William Harvey Research Institute, Bone &Joint Research Unit, United Kingdom.
authors.forename: D
authors.initials: D
authors.lastname: Gould
mesh_terms: D000818:Animals; D001665:Binding Sites; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000008:administration & dosage; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D016385:TATA Box; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D014157:Transcription Factors