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Enrico Maria Surace, Salvatore Botta, Elena Marrocco, Nicola de Prisco, Martina Sofia, Mariangela Lupo, Carlo Gesualdo, Settimio Rossi, Francesca Simonelli; RHODOPSIN transcriptional silencing & replacement for the treatment of autosomal dominant Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2018;59(9):378.
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The RHODOPSIN (RHO) gene is the most common gene involved in autosomal dominant retinitis pigmentosa (adRP). To silence RHO gain-of-function mutations in a mutation-independent manner we designed and successfully tested a series of ZF transcriptional repressors specifically targeted to a unique human RHO regulatory promoter region. We coupled transcriptional RHO silencing with wild type hRHO replacement cassette and we tested in different animal models. Built on these results, we are currently investigating transcriptional RHO silencing by evaluating the two expression cassettes enclosed in the single vector (ZF DNA-binding repression & replacement). In particular, we generated a set of promoters, which enable the modulation of the repression & replacement to tailor the strategy for clinical translation purposes.
We perform dose-escalation study in porcine retina starting with doses similar to those used in the RPE65 gene therapy clinical trials (2x1010-1x1011 vg of Adeno-associated viral vector, AAVs). To tune the simultaneous expression of ZF and RHO gene, we generated a set of promoters derived from the human RHO proximal promoter. To test dose-effects and their relation with promoter strength we performed quantitative Real Time PCR and western blot analysis to evaluate mRNA and protein expression levels. In addition, we evaluated cellular localization by immunofluorescence and histopathology. Furthermore, we generated a transgenic mouse currying the ZF DNA-binding repressor to assess toxicity by Electroretinography (ERG) and histology.
The injection of a dose five times higher of AAV8-ZF6-DB, resulted in reduction of porcine Rhodopsin mRNA of 60%, compared with the 45% obtained with a 2x1010 gc. The comparison analysis of the hRHO mutagenized promoters showed rod-specificity and different strength of expression according to the site of mutagenesis. Transgenic mice currying the ZF DNA-binding repressor developed normally without any sign of toxicity.
These data suggest the high efficiency and safety of the ZF DNA-binding repressor & replacement strategy for the treatment of autosomal dominant Retinitis Pigmentosa due to RHO mutations in mutation-independent manner. Currently, to complete the pre-clinical studies we are performing long-term assessments.
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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