Generation of a reporter-retinitis pigmentosa mouse model for retinal regeneration studies using crispr/dcas9-based artificial transcription factors

Official URL: https://risc01.sabanciuniv.edu/record=b2771034_(Table of contnets)

Many blinding diseases of the retina involve neurodegeneration, including age-related macular degeneration, diabetic retinopathy, glaucoma, and inherited retinal diseases (IRDs). Inherited retinal diseases are one of the most common causes of blindness with the major subtype retinitis pigmentosa (RP) occurring with a worldwide prevalence of 1:4000. More than 200 genes are linked to inherited retinal diseases, which complicates approaches to target all individual blinding conditions. Furthermore, more than 150 mutations have been identified solely in the rod visual pigment, rhodopsin, that causes RP. In the majority of RP cases, the ongoing photoreceptor degeneration cannot be stopped, or the disease is diagnosed at a very late stage. Therefore, in addition to the therapies that will stop the ongoing degeneration, there is also a demanding need for restorative therapies to regain vision in patients that already have a severe neuronal loss. Mammalians have very limited retinal regeneration capacity. However, in zebrafish, the Muller glia has an extraordinary regenerative capacity, which opens up the possibility of Muller glia reprogramming for compensating the neuronal loss in IRDs. To mimic this feature of teleost fish, several studies have been performed in mammalian models to reprogram Muller glia. Here, we established a reporter mouse model for RP using one of the very-well studied rd1 mouse models and the rhodopsin reporter system that labels the newly regenerated rods in vivo which will allow us to elucidate the regenerative potential of candidate drugs. Furthermore, the RP-reporter model was combined with the novel dCas9-SPH system for multiplex gene regulation and to establish synthetic gene regulatory networks using artificial transcription factors for the treatment of neurodegenerative retinal diseases. This system already allowed us to robustly label retinal cells in vivo using our AAVShH10-Cre vectors that mainly target Muller glia. We have also generated and tested stable Muller glia cell lines expressing CRISPR-dCas9 activators for the establishment of artificial transcription factors. These transgenic mice and cell lines will be valuable tools for combining the gene therapy approach with potential candidate drugs.