A novel approach for crispr-dcas9-mediated neuroprotective gene therapies for inherited retinal diseases

Official URL: https://risc01.sabanciuniv.edu/record=b3072977_(Table of contents)

Eye diseases are a prominent group of diseases that are difficult to treat, with a great socioeconomic impact on healthcare systems in the world. It is estimated that more than 190 million people worldwide suffer from visual impairments and 1 in 4000 people is diagnosed with retinitis pigmentosa, one of the most common inherited retinal diseases. Although inherited retinal diseases have been researched for decades, they remain mostly incurable. However, the recent approval of the gene therapy product voretigene neparvovec for the treatment of retinitis pigmentosa and Leber congenital amaurosis showed that gene therapy offers a solution to this decades-old problem. In several cases, these approaches are not sufficient and additional neuroprotective strategies are required for a complete treatment. One possible approach to protect vision is to halt or delay neuronal loss by overexpressing neuroprotective molecules. The recent discovery of CRISPR-Cas9 and its rapid adaptation in translational studies showed that CRISPR-Cas9 will change modern medicine forever. CRISPR-Cas systems were modified to produce a set of gene editing tools that were capable of gene correction and gene regulation. CRISPR- iv dCas9 activation (such as dCas9-SPH) is one of these tools that is capable of transcriptional regulation of genes. With the help of these novel tools, it is now possible to overexpress and control the dosage of neuroprotective molecules in the retina. Here, we generated several multiplexed transgenic mouse lines, including RhoP23H/wt; Tg(Rho-Cre); ROSA26-LSL-tdTomato which is a novel rod reporter mouse model with a slow retinal degeneration. This mouse model was further improved by crossing to CRISPR-dCas9-SPH activator mouse line to generate a neuroprotective test platform, the RhoP23H/wt; Tg(Rho-Cre); ROSA26-LSL-tdTomato; dCas9-SPH-LSL quadruple transgenic mouse line. This model was complemented with AAVs which is the most commonly used gene therapy delivery tool in the ophthalmology field. AAV vectors are engineered to deliver both Cre-recombinase gene to activate the dCas9-SPH and the gRNAs to establish the artificial transcription factors in retina. Establishment of artificial transcription factors within the retina allows the overexpression of neuroprotective factors like Lif, Cntf, Fgf2 and Manf. Furthermore, this model can be used as a platform to test the effects of neuroprotective factors in the degenerating retina. An additional transgenic model, Pde6brd10/rd10; dCas9-SPH, was also established to test the efficacy of neuroprotective factors in a fast retinal degeneration model. Further work is needed to fully characterize these transgenic models and to test the effects of different combinations and dosages of neuroprotective factors in different retinal degeneration models. This will also show whether early intervention of eye diseases with neuroprotective factors can alleviate disease progression and preserve visual function. In addition, the recently generated transgenic mouse models will be further used for several proof-of-concept studies that will open the way to generate new gene therapy products with more complex gene regulatory functions.