title: Rhodopsin targeted transcriptional silencing by DNA-binding.
abstract: Transcription factors (TFs) operate by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs) enabling the coordination of gene expression on a genomic scale. Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain can produce efficient and gene-specific transcriptional silencing. To interfere with RHODOPSIN (RHO) gain-of-function mutations we engineered the ZF6-DNA-binding protein (ZF6-DB) that targets 20 base pairs (bp) of a RHOcis-regulatory element (CRE) and demonstrate Rho specific transcriptional silencing upon adeno-associated viral (AAV) vector-mediated expression in photoreceptors. The data show that the 20 bp-long genomic DNA sequence is necessary for RHO expression and that photoreceptor delivery of the corresponding cognate synthetic trans-acting factor ZF6-DB without the intrinsic transcriptional repression properties of the canonical ED blocks Rho expression with negligible genome-wide transcript perturbations. The data support DNA-binding-mediated silencing as a novel mode to treat gain-of-function mutations.
authors.affiliation: Telethon Institute of Genetics and Medicine, Napoli, Italy.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Botta
authors.affiliation: Telethon Institute of Genetics and Medicine, Napoli, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Marrocco
authors.affiliation: Telethon Institute of Genetics and Medicine, Napoli, Italy.
authors.forename: Nicola
authors.initials: N
authors.lastname: de Prisco
authors.affiliation: Telethon Institute of Genetics and Medicine, Napoli, Italy.
authors.forename: Fabiola
authors.initials: F
authors.lastname: Curion
authors.affiliation: Telethon Institute of Genetics and Medicine, Napoli, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Renda
authors.affiliation: Telethon Institute of Genetics and Medicine, Napoli, Italy.
authors.forename: Martina
authors.initials: M
authors.lastname: Sofia
authors.affiliation: Telethon Institute of Genetics and Medicine, Napoli, Italy.
authors.forename: Mariangela
authors.initials: M
authors.lastname: Lupo
authors.affiliation: Telethon Institute of Genetics and Medicine, Napoli, Italy.
authors.forename: Annamaria
authors.initials: A
authors.lastname: Carissimo
authors.affiliation: Department of Veterinary Medical Sciences, University of Bologna, Bologna, Italy.
authors.forename: Maria Laura
authors.initials: ML
authors.lastname: Bacci
authors.affiliation: Multidisciplinary Department of Medical, Surgical and Dental Sciences, Eye Clinic, Second University of Naples, Naples, Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Gesualdo
authors.affiliation: Multidisciplinary Department of Medical, Surgical and Dental Sciences, Eye Clinic, Second University of Naples, Naples, Italy.
authors.forename: Settimio
authors.initials: S
authors.lastname: Rossi
authors.affiliation: Multidisciplinary Department of Medical, Surgical and Dental Sciences, Eye Clinic, Second University of Naples, Naples, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Simonelli
authors.affiliation: Telethon Institute of Genetics and Medicine, Napoli, Italy.|Department of Translational Medicine, University of Naples Federico II, Naples, Italy.
authors.forename: Enrico Maria
authors.identifier: http://orcid.org/0000-0002-2975-942X
authors.initials: EM
authors.lastname: Surace
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D020868:Gene Silencing; D005822:Genetic Vectors; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012243:Rhodopsin / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014161:Transduction, Genetic
chemical_list: D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D004247:DNA; D012243:Rhodopsin
keywords: AAV; gene expression; gene therapy; human biology; medicine; mouse; neurodegeneration; neuroscience; retina; rhodopsin; transcription; transcription factors; zinc finger