title: An engineered tale-transcription factor rescues transcription of factor VII impaired by promoter mutations and enhances its endogenous expression in hepatocytes.
abstract: Tailored approaches to restore defective transcription responsible for severe diseases have been poorly explored. We tested transcription activator-like effectors fused to an activation domain (TALE-TFs) in a coagulation factor VII (FVII) deficiency model. In this model, the deficiency is caused by the -94C > G or -61T > G mutation, which abrogate the binding of Sp1 or HNF-4 transcription factors. Reporter assays in hepatoma HepG2 cells naturally expressing FVII identified a single TALE-TF (TF4) that, by targeting the region between mutations, specifically trans-activated both the variant (>100-fold) and wild-type (20-40-fold) F7 promoters. Importantly, in the genomic context of transfected HepG2 and transduced primary hepatocytes, TF4 increased F7 mRNA and protein levels (2- to 3-fold) without detectable off-target effects, even for the homologous F10 gene. The ectopic F7 expression in renal HEK293 cells was modestly affected by TF4 or by TALE-TF combinations. These results provide experimental evidence for TALE-TFs as gene-specific tools useful to counteract disease-causing promoter mutations.
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Barbon
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Pignani
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Italy.|LTTA Center, University of Ferrara, Italy.
authors.forename: Alessio
authors.initials: A
authors.lastname: Branchini
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Italy.|LTTA Center, University of Ferrara, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Bernardi
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Italy.|LTTA Center, University of Ferrara, Italy.
authors.forename: Mirko
authors.initials: M
authors.lastname: Pinotti
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Bovolenta
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D005167:Factor VII / Q000235:genetics* / Q000378:metabolism; D005168:Factor VII Deficiency / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D017930:Genes, Reporter; D015316:Genetic Therapy; D057809:HEK293 Cells; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D049409:Luciferases, Firefly / Q000096:biosynthesis / Q000235:genetics; D009154:Mutation; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D005167:Factor VII; D049409:Luciferases, Firefly