Abstract
Purpose :
Retinitis pigmentosa (RP) is an important cause of visual impairment, characterized by degeneration of retinal photoreceptors (PR). Mutations in pre-mRNA processing factor 31 (PRPF31) are the second most common cause of autosomal dominant RP. Gene therapy holds promise to treat retinal inherited diseases, and AAV-mediated PRPF31 augmentation has been shown to restore critical functions in iPSC-derived PRPF31+/- cells. Currently, there are no satisfying animal models with similar phenotypes to human PRPF31 patients. Our study aims at establishing animal models of this disease by AAV-CRISPR/Cas9 gene editing and testing gene therapy efficiency in vivo in these models.
Methods :
Cas9 and/or guide RNA with/without GFP reporter gene targeting early coding exons of PRPF31 were delivered to the retina of WT and SpCas9-expressing transgenic mice using AAV. Different delivery routes including systemic injection, intravitreal injection (IVT), and sub-retinal injection (SRi) were evaluated. AAV-PRPF31 gene augmentation was achieved using SRi. Retinal conditions were evaluated by fundus imaging, OCT, ERG, and histology.
Results :
Systemic delivery of knockout (KO) vectors to P0 neonatal mice caused low body weights and high early mortality rates. IVT delivery to the retina resulted in reduced thickness of the inner plexiform layer and reduced amplitude of the ERG b-wave. SRi, which led to higher expression levels of KO vectors in PR, induced retinal pallor and formation of black-brown pigmentation, which spread away from the injection site over time. OCT revealed the disappearance of the inner and outer segment layers, followed by loss of the outer nuclear layer, indicating PR degeneration. ERGs showed notable reductions in a-, b- and c-wave amplitudes, with prolonged c-wave implicit time. Co-injection of PRPF31 therapeutic vectors with KO vectors decreased retinal pallor and pigmentation, improved retinal thickness, as well as a-, b- and c-wave responses compared to PBS and KO vectors co-injected eyes.
Conclusions :
PRPF31 RP mouse models with early on-set morphological and functional impairments like those in patients were established, providing new platforms to investigate the pathogenetic mechanisms of PRPF31 and to evaluate treatment efficacy. AAV-mediated gene therapy rescued the function of PRPF31-mutant retinas, further demonstrating proof-of-concept for gene therapy to treat PRPF31 RP.
This is a 2021 ARVO Annual Meeting abstract.