Mussolino, Claudio (2010) Mutation-independent treatment of autosomal dominant Retinitis Pigmentosa (adRP). [Tesi di dottorato] (Inedito)

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Abstract

Viral-mediated gene therapy holds great promise for the treatment of severe inherited retinal diseases, such as Retintitis Pigmentosa (RP), which is caused by mutations in genes preferentially expressed in photoreceptor cells. The availability of vectors derived from the small adeno-associated virus (AAV) which efficiently and stably transduce the retina of animal models after intraocular administration strongly support the possibility to develop novel strategies for the treatment of such severe retinal degenerations otherwise incurable thus far. The main goals of my PhD project were: - generate artificial transcription repressors (ZFPs) targeted to the human rhodopsin promoter to silence at the transcriptional level the rhodopsin gene; - assess the efficacy of the treatment and the impact on the disease progression in the RP mouse model. Retinitis pigmentosa is by far the most studied inherited retinal disease. It is clinically and genetically heterogeneous recognizing autosomal recessive (arRP), autosomal dominant (adRP), X-linked, and digenic patterns of inheritance. More than 30 diseases genes have been identified so far and 12 of these have been associated with (adRP), representing between 15% and 35% of all cases. Despite recent success of the gene-based complementation approach for genetic recessive traits, the development of therapeutic strategies for gain-of-function mutations poses great challenges. General therapeutic principles to correct these genetic defects mostly rely on post-transcriptional gene regulation (RNA silencing). Engineered zinc finger protein (ZFP)-based-repression of transcription may represent a novel and alternative mutation independent therapeutic approach for treating gain-of-function mutations, but proof-of-concept of this use is still lacking. In my PhD project we used a novel strategy to treat adRP based on zinc-finger-based artificial transcription factors (ZF-ATFs). These molecules can be engineered to silence genes carrying gain-of-function mutations that cause toxic effects into the cell where they are expressed. We generated ten artificial transcriptional repressors targeted to the human Rhodopsin which is the gene most commonly associated with adRP (20–30% of cases) with more than 150 mutations identified throughout its sequence, representing the most commonly mutated gene in RP. We characterized in vitro the ability of artificial transcriptional repressors to bind specifically the human rhodopsin promoter in order to exert a specific transcriptional control and we selected two out of ten functional zinc-finger-based repressors of rhodopsin. One of this was selected as the most efficient and was enclosed in an AAV2/8 for in vivo experiments. We demonstrated that the selected artificial zinc-finger-based repressors (ZFRs) resulted in a robust transcriptional repression of hRHO impacting disease progression in a mouse model of adRP over-expressing the P347S mutation. The data obtained support the use of ZFP-mediated silencing as a potentially relevant therapeutic strategy to treat gain of function mutations.

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