Gene and mutation independent therapy via CRISPR-Cas9 mediated cellular reprogramming in rod photoreceptors

Abstract

Purpose : Retinitis pigmentosa (RP) is one of the most common degenerative diseases of the eye, characterized by primary rod photoreceptor death and degeneration, followed by secondary cone death. It can be caused by numerous mutations in over 200 genes, which limits the therapeutic impact of conventional gene therapy strategies. Nrl and Nr2e3 function in concert to activate a rod-specific gene transcription network and control rod differentiation and fate. Loss of function in either Nrl or Nr2e3 reprograms rods to a cone cell fate. In this study, we aim to develop a general therapeutic strategy for treatment of RP in a mutation or gene independent way.

Methods : We designed a gene therapy strategy using CRISPR/Cas9 mediated targeted inactivation of Nrl or Nr2e3 in the retina using adeno-associated virus (AAV) delivery. And then we investigated in vivo rod to cone reprogramming in two RP mice models. Electroretinography (ERG) responses were tested in eyes of live mice to measure the electrical activity of cone photoreceptors (photopic response), followed by T7E1 assay to test editing efficiency, qPCR and histology analysis to show gene expression and ONL thickness.

Results : All eyes treated with AAV-gRNA/Cas9 exhibited significantly improved photopic b-wave values, which suggest improved cone function. qPCR showed that there was down-regulation of rod specific genes with concomitant upregulation of cone specific genes. Histology analysis of the AAV-gRNAs/Cas9 treated retinas showed preservation of a number of mCAR⁺ cells and led to significantly improved preservation of the ONL thickness, indicating AAV-gRNA/Cas9 treatment prevented photoreceptor cell degeneration.

Conclusions : These results suggested that our approach could in situ reprogram rod to cone photoreceptors by inactivating Nrl or Nr2e3. It showed that an increase in cone like cells with concomitant preservation of both cone and rod photoreceptors and visual function in two RP models. Our approach shows promise for cellular reprogramming in preventing degeneration and preserving tissue and function, and points to a novel approach in treating human diseases in a gene and mutation independent manner.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.