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C. Mussolino, D. Sanges, V. Marigo, G. Meroni, E. M. Surace; AAV-Mediated Delivery of an Artificial Zinc Finger Transcription Repressor to P347S Mouse Retinae Improves Visual Function. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5349.
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© ARVO (1962-2015); The Authors (2016-present)
Retinitis Pigmentosa (RP) represents a group of retinal degenerations with different patterns of inheritance, characterized by a wide genetic and clinical heterogeneity. Rhodopsin (Rho) is the gene most commonly associated to the autosomal dominant form of RP (ADRP) with over 150 mutations identified. In this study we use a novel strategy to treat ADRP based on artificial zinc finger transcription factors (ZF-TFs) to selectively silence, at the transcriptional level, the Rho gene in mutation independent fashion
To design Zinc Finger based DNA binding domains targeted to the human Rhodopsin (hRho) promoter we used the algorithm: http:\\www.zincfingertool.org. To generate transcriptional repressors and activators (ZF-TFs) we fused Zinc Fingers to the KRAB or the VP64 domains, respectively. The selection of the most efficient and selective ZF-TF was performed by co-transfection of a plasmid harbouring the luciferase gene under the control of hRho promoter. The therapeutic potential of 2 selected ZF-TFs was assessed by their retroviral delivery in Retinal Stem Cells (RSC) explanted and cultured from adult P347S ADRP transgenic mouse model. The P347S RSC die once differentiated because of the expression of the mutated hRho gene. To test in vivo the ability of ZF-TFs to specifically repress the expression of the hRho gene, subretinal injection of AAV2/8 vector were performed. To evaluate the efficacy of the treatment histological and Electroretinograms (ERG) analysis were used
Two out of ten ZFP-TFs specifically and efficiently transactivate or repress luciferase expression driven by hRho promoter. Delivery of ZF-TFs to RSC resulted in 90% reduction of apoptotic cells compared to controls. In addition, the relative expression levels of the human and murine rhodopsin showed selective ZF-TFs repression of the human transcript. Subretinal delivery of AAV2/8-ZF-TFs to P347S mice retinae resulted in a significant morphological and functional recovery
Taken together these results demonstrate efficient and selective ZF-TFs-mediated repression of hRho gene in vitro and in vivo, underscoring the potential impact of this novel strategy to cure untreatable ADRP in a future clinical setting
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