AAV-mediated PRPF31 Gene Augmentation Prevents Retinal Degeneration In A Mouse Model Of Retinitis Pigmentosa

Purpose: Mutations in pre-m RNA processing factor 31(PRPF31)is a common cause of autosomal dominant retinitis pigmentosa(RP),a currently incurable form of inherited blindness.AAV-mediated gene augmentation therapy holds promise to treat PRPF31-RP.However,currently there are no satisfying animal models in which to develop AAV-PRPF31 gene augmentation therapy.Our study aims at establishing animal models of PRPF31-RP by AAV-CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)gene editing and testing gene therapy efficiency in vivo in these models.Methods: CRISPR-associated protein 9(Cas9)and/or guide RNA(g RNA)targeting early coding exons of Prpf31 were delivered to the retina of WT and Cas9-expressing transgenic mice using AAV.Different delivery routes including systemic injection,intravitreal injection and subretinal injection were tested.The knock out(KO)efficiency,morphological function and biochemical changes in the retina of resulting models were fully evaluated from molecular,cellular,histological and physiological levels using DNA sequencing,RNA sequencing,western blotting,immunohistochemistry,fundoscopy,optical coherence tomography(OCT)and electroretinogram(ERG).We then used subretinal injection to achieve AAV-PRPF31 gene augmentation therapy in the most effective PRPF31-RP mouse model and evaluated the treatment efficiency.To establish PRPF31-RP non-human primate(NHP)model using the method developed here,we further tested the AAVCRISPR/Cas9 PRPF31 KO vectors on human and NHP retina explants,and performed evaluation of KO efficiency and retinal morphology changes.Results: Systemic delivery of Prpf31 KO vectors to neonatal mice caused low body weights and high early mortality rates.Intravitreal delivery of Prpf31 KO vectors to the retina resulted in changes primarily in the inner retina,with reduced thickness and cells of the inner plexiform layer and reduced amplitude of the ERG b-wave.Subretinal injection,which led to the highest expression levels of KO vectors in photoreceptors,induced retinal pallor and formation of black-brown pigmentation,which spread away from the injection site over time.The disappearance of the inner and outer segment layers,loss of the outer nuclear layer and retinal pigment epithelium(RPE)were observed.ERGs showed notable reductions in a-,b-and c-wave amplitudes,with prolonged c-wave implicit times,indicating photoreceptors and RPE degeneration.Co-injection of PRPF31 therapeutic vectors with KO vectors decreased retinal pallor and pigmentation,preserved retinal structure,as well as improved a-,b-and c-wave responses compared to PBS and KO vectors co-injected eyes.The AAV-CRISPR/Cas9 PRPF31 KO vectors resulted in truncated PRPF31 DNA and a substantial reduction of PRPF31 protein in the human and NHP retina explants.Conclusions: In this study,we establish PRPF31-RP mouse models with early on-set retinal morphological and functional impairments like those in patients.Using this model,we demonstrate that AAV-mediated PRPF31 augmentation therapy prevents degeneration of Prpf31 mutant retina in vivo.We further prove that the AAV-CRISPR/Cas9 KO vectors mediated efficient gene editing of PRPF31 in the human and NHP retina ex-vivo culture.