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ERC

ALLELECHOKER Report Summary

Project ID: 311682
Funded under: FP7-IDEAS-ERC
Country: Italy

Final Report Summary - ALLELECHOKER (DNA binding proteins for treatment of gain of function mutations)

In the ERC consolidator Project No: 311682 “ALLELECHOKER” we propose to generate RHO DNA binding silencers (“AlleleChoker”), to inactivate the RHODOPSIN gene (RHO). RHO is associated with mutations causing the blinding disease autosomal dominant retinitis pigmentosa (adRP).
In particular, we took the challenge to design and develop therapeutics based on transcriptional repression, a novel therapeutic paradigm. We were the first to demonstrate that pairing a ZF DNA binding protein to the transcriptional repressor Kruppel-associated box (KRAB) enables in vivo the transcriptional repression of the rhodopsin gene (RHO) by somatic gene transfer (Mussolino et al., EMBO Mol. Med. 2011 Mar;3(3):118-28. doi: 10.1002/emmm.201000119). Built on this promising result, we implemented the proof of concept of transcriptional repression through the “ALLELECHOKER” ERC grant. In particular, we demonstrated two additional modes of RHO transcriptional repression by using i) a synthetic DNA-binding protein without repressor domains and ii) by showing that the ectopic expression of an endogenous TF, lead to targeted silencing of RHO. We demonstrated in vivo that these three modes of transcriptional repression: i- synthetic transcription factor (TF; Mussolino et al., EMBO Mol. Med. 2011 Mar;3(3):118-28; Botta S. et al. Elife. 2016 Mar 14;5), ii- synthetic DNA-binding protein (Botta S. et al. Elife. 2016 Mar 14;5) and iii- the ectopic expression of an endogenous TF (Botta S, et al. JCI Insight. 2017 Dec 21;2(24) of RHO gene in a gain-of-function mutant transgenic mouse (modelling adRP) and the pre-clinical pig retina.
We showed that in vivo somatic retinal gene transfer by Adeno-associated virus (AAV) vectors of transcriptional repressors based on synthetic ZF DNA-binding proteins with or without canonical repressor domain, enable the complete RHO transcriptional silencing and in turn therapy in the P347S mouse model of RHO adRP. Notably, we established that the synthetic DNA binding proteins without a repressor domain, thus, acting only by DNA binding (literally RHO allele chokers) may be use as therapeutics (Rhodopsin targeted transcriptional silencing by DNA-binding. Botta S. et al. Elife. 2016 Mar 14;5. pii: e12242. doi: 10.7554/eLife.12242). Furthermore, to additionally implement transcriptional modulation, we challenged the canonical multi-targeting activity of endogenous TFs, hypothesizing that the ectopic expression of a selected TF might be directed to a single gene target (the RHO promoter elements) by the novel cell-specific context in which the TF is expressed. 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). AAV vector-mediated ectopic expression of KLF15, similarly to the above mentioned synthetic TFs, generates robust RHO silencing (Targeting and silencing of rhodopsin by ectopic expression of the transcription factor KLF15. Botta S, et al. JCI Insight. 2017 Dec 21;2(24). pii: 96560. doi: 10.1172/jci.insight.96560). We were also able to determine how the use of these factors impact the retinal transcriptome in vivo, a key point to evaluate the safety of transcriptional repression. Notably, the synthetic DNA binding proteins and the ectopically expressed KLF15 showed limited off targeting and thus, a safe profile (Botta S. et al. Elife. 2016 Mar 14;5. and Botta S, et al. JCI Insight. 2017 Dec 21;2(24). Collectively, the data generated with the ERC “ALLELECHOKER” grant enabled to support transcriptional repression as a novel paradigm to generate transcriptional-based therapies for treatment of human inherited disorders due to gain of function mutations and in particular, the proof of concept that transcriptional repression coupled with replacement strategy may be used in translational medicine for the treatment of the incurable blinding disorder adRP due to the RHO mutations.

Reported by

FONDAZIONE TELETHON
Italy
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