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Salvatore Botta, Elena Marrocco, Nicola de Prisco, Martina Sofia, Mariangela Lupo, Maria Laura Bacci, Carlo Gesualdo, Settimio Rossi, Francesca Simonelli, Enrico Maria Surace; Modulating transcription factor spatial pattern to generate gene-targeted therapeutics. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3966.
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© ARVO (1962-2015); The Authors (2016-present)
Transcriptional regulation represents the first key step for expression of genetic programs. In the recent years we showed that modulation of transcription via somatic gene transfer of synthetic transcription factors (S-TFs) enables to silence in a potent and specific manner the RHODOPSIN (RHO) gene. Currently, we are exploring a novel strategy to exploit transcription regulation for therapeutic purpose based on the modulation of expression of endogenous TFs.
We identified TFs enabling putative binding to a DNA regulatory sequence of RHO by searching in a TFs repository. We selected KLF15 TF based on its ability to recognize RHO-cis sequence and the absence of expression in rods. We generated an Adeno-associated virus (AAV) vector (AAV8-hGNAT1-hKLF15) and delivered by sub-retinal injection to adult porcine retina. We performed quantitative Real Time PCR to verify the ability of KLF15 to repress endogenous porcine Rho expression. To evaluate the off-target effects of KLF15, we performed RNAseq analysis on porcine injected retina Finally, we tested in a mouse model of autosomal dominant Retinitis Pigmentosa (adRP) the therapeutic effect of KLF15 by electro-retinogram analysis (ERG).
The ectopic expression of KLF15 in porcine rods resulted in the repression of the 40% of mRNA and protein levels of endogenous Rhodopsin. Histological analysis showed depletion of RHO content, while maintaining the integrity of the nuclear layer. The RNAseq showed that KLF15 perturbed 156 genes (differentially expressed genes, DEGs), including RHO demonstrating high selectivity (on-target) over the expected thousands of targets typical of TFs. The injection of KLF15 in a transgenic mouse model of adRP (P347S mouse) showed the preservation of retinal functionality and the preservation of outer nuclear layer (ONL).
Ectopic expression of KLF15 resulted in efficient Rho silencing in wild type porcine retina. Silencing of the severe RHO-P347S gain-of-function mutation in the adRP mouse model translated into structural and functional protection of the retina from degeneration. Coupling Rho transcriptional silencing with replacement may support further development of this strategy for the treatment of adRP and other monogenic diseases. In summary, we provided a proof-of-concept of a novel mode to efficiently and specifically silence a gene by ectopic expression of a TF in a novel cell-specific context.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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