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 2

KLF15 ectopically expressed in porcine rod photoreceptors represses Rho expression with limited off-target effects.

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KLF15 ectopically expressed in porcine rod photoreceptors represses Rho ...
(A) Alignment of human, porcine, and murine rhodopsin (Rho) proximal promoter around the hRHO-cis. In red, the sequence recognized by KLF15 retrieved by TRANSFAC analysis (Figure 1A and Supplemental Table 1). (B) qPCR of mRNA levels (2–ΔΔCt) of adult porcine retina injected subretinally with AAV8-hGNAT1-hKLF15 (n = 6) or AAV8-hGNAT1-eGFP (n = 6) at a vector dose of 2 × 1010 genome copies (gc) 15 days after vector delivery shows significant repression of the Rho transcript; Gnat1, guanine nucleotide–binding protein 1; Arr3, arrestin 3. Data are shown as the mean ± SEM. ***P < 0.001; 2-tailed Student’s t test. (C) Western Blot analysis of porcine retinae injected with AAV8-hGNAT1-hKLF15 and AAV8-hGNAT1-eGFP shows the decrease in Rho protein consequent to KLF15 expression. (D) Rho (cyan) and KLF15 (red) immunofluorescence confocal analysis shows expression of hKLF15 in the outer nuclear layer (ONL) of injected retina (coinjected with AAV8-hGNAT1-eGFP, green) toward the nuclear interior of rod photoreceptor nuclei (euchromatin; see ref. 33), the collapse of the Rho-deprived outer-segment (OS), and partial retention of Rho in the cytoplasm. (E) Histological confocal immunofluorescence analysis of Gnat1 (red), which marks the soma of rods, confirmed rod-specific expression of hKLF15 upon transduction with AAV8-hGNAT1-hKLF15. Scale bars: 50 μm. (F) Venn diagrams showing pairwise intersection of differentially expressed genes (DEGs) between hKLF15 and ZF6-DB (12, 13). An adjusted P value (false discovery rate < 0.1), without filtering on fold change levels, resulted in 156 and 19 DEGs, in hKLF15- and ZF6-DB–treated retinae, respectively (12, 13). (G) Transcriptional activation and repression concordances among log fold changes of the genes in common between ZF6-DB and hKLF15.