Targeting and silencing of rhodopsin by ectopic expression of the transcription factor KLF15
Salvatore Botta, Nicola de Prisco, Elena Marrocco, Mario Renda, Martina Sofia, Fabiola Curion, Maria Laura Bacci, Domenico Ventrella, Cathal Wilson, Carlo Gesualdo, Settimio Rossi, Francesca Simonelli, Enrico Maria Surace
Salvatore Botta, Nicola de Prisco, Elena Marrocco, Mario Renda, Martina Sofia, Fabiola Curion, Maria Laura Bacci, Domenico Ventrella, Cathal Wilson, Carlo Gesualdo, Settimio Rossi, Francesca Simonelli, Enrico Maria Surace
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Research Article Ophthalmology Therapeutics

Targeting and silencing of rhodopsin by ectopic expression of the transcription factor KLF15

Abstract

The genome-wide activity of transcription factors (TFs) on multiple regulatory elements precludes their use as gene-specific regulators. Here we show that ectopic expression of a TF in a cell-specific context can be used to silence the expression of a specific gene as a therapeutic approach to regulate gene expression in human disease. We selected the TF Krüppel-like factor 15 (KLF15) based on its putative ability to recognize a specific DNA sequence motif present in the rhodopsin (RHO) promoter and its lack of expression in terminally differentiated rod photoreceptors (the RHO-expressing cells). Adeno-associated virus (AAV) vector–mediated ectopic expression of KLF15 in rod photoreceptors of pigs enables Rho silencing with limited genome-wide transcriptional perturbations. Suppression of a RHO mutant allele by KLF15 corrects the phenotype of a mouse model of retinitis pigmentosa with no observed toxicity. Cell-specific-context conditioning of TF activity may prove a novel mode for somatic gene–targeted manipulation.

Authors

Salvatore Botta, Nicola de Prisco, Elena Marrocco, Mario Renda, Martina Sofia, Fabiola Curion, Maria Laura Bacci, Domenico Ventrella, Cathal Wilson, Carlo Gesualdo, Settimio Rossi, Francesca Simonelli, Enrico Maria Surace

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Figure 1

KLF15 is not expressed in rods and binds the human rhodopsin promoter.

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KLF15 is not expressed in rods and binds the human rhodopsin promoter. (...
(A) TRANSFAC analysis of the human rhodopsin promoter identifies transcription factors (TFs) predicted to bind the rhodopsin regulatory motif hRHO-cis (–88 to –58 from the transcription start site, TSS; Figure 2A and refs. 12, 13) including the TF KLF15 (orange arrow, minus strand). (B) Immunofluorescence analysis of Klf15 in C57BL6/J retina shows its absence in photoreceptors in the outer nuclear layer (ONL) and expression in the inner nuclear layer (INL) and in the ganglion cell layer (GCL). Scale bar: 50 μm. (C) qPCR of mRNA (2–ΔCt) shows that Klf15 is not expressed in porcine rods. Porcine rods transduced with AAV8-hGNAT1-eGFP (1 × 1012 genome copies [gc]) and FACS sorted show lack of expression of Klf15. For comparison the retina-specific cone-rod homeobox (Crx) and rod-specific neural retina leucine zipper (Nrl) TFs are shown. (D) Gel mobility shift titrations of hKLF15 and artificial ZF6-DB TF with the hRHO 65-bp oligonucleotide. In the saturation-binding experiments the nanomolar concentration of specific binding data were plotted against nanomolar increasing concentration of DNA ligand. KLF15 and the synthetic TF ZF6-DB show similar binding affinity for the target sequence (12, 13). (E) qPCR ChIP analysis of the human rhodopsin TSS region, after the transfection of hKLF15 in HEK293 cells, shows enrichment of binding in the Rho promoter region compared with eGFP-transfected cells. Data are shown as the mean ± SEM. **P < 0.01 by 2-tailed Student’s t test. n = 3 independent experiments.