Allele-specific editing ameliorates dominant retinitis pigmentosa in a transgenic mouse model

Clarissa Patrizi,Manel Llado,Daniela Benati,Carolina Iodice,Elena Marrocco,Rosellina Guarascio,Enrico M Surace,Michael E Cheetham,Alberto Auricchio,Alessandra Recchia

doi:10.1016/j.ajhg.2021.01.006

Abstract

SummaryRetinitis pigmentosa (RP) is a group of progressive retinal degenerations of mostly monogenic inheritance, which cause blindness in about 1:3,500 individuals worldwide. Heterozygous variants in the rhodopsin (RHO) gene are the most common cause of autosomal dominant RP (adRP). Among these, missense variants at C-terminal proline 347, such as p.Pro347Ser, cause severe adRP recurrently in European affected individuals. Here, for the first time, we use CRISPR/Cas9 to selectively target the p.Pro347Ser variant while preserving the wild-type RHO allele in vitro and in a mouse model of adRP. Detailed in vitro, genomic, and biochemical characterization of the rhodopsin C-terminal editing demonstrates a safe downregulation of p.Pro347Ser expression leading to partial recovery of photoreceptor function in a transgenic mouse model treated with adeno-associated viral vectors. This study supports the safety and efficacy of CRISPR/Cas9-mediated allele-specific editing and paves the way for a permanent and precise correction of heterozygous variants in dominantly inherited retinal diseases.

Highlights

Retinitis pigmentosa (RP) is a group of genetically heterogeneous retinal diseases afflicting three million people across the globe with an incidence of 1 in 3,500 live births.[1,2,3,4] Individuals affected by RP initially manifest night-blindness with gradual constriction of the visual field but sparing of central vision
Pro347Ser transgenic mice were maintained as F0 by crossing them with themselves and were crossed with C57BL/6J mice purchased from Envigo Italy SRL (Udine, Italy) to generate experimental F1 mice
One-week-old mice were anesthetized with an intraperitoneal injection of 2 mL/100 g of body weight of ketamine/medetomidine, AAV2/8 vectors were delivered subretinally via a trans-scleral trans-choroidal approach, as described by Liang et al.[38]

Summary

Introduction

Retinitis pigmentosa (RP) is a group of genetically heterogeneous retinal diseases afflicting three million people across the globe with an incidence of 1 in 3,500 live births.[1,2,3,4] Individuals affected by RP initially manifest night-blindness with gradual constriction of the visual field but sparing of central vision. More than 3,000 disease-associated variants in approximately 70 disease-causing genes have been causally associated with RP5 and currently more than 150 documented missense/nonsense variants in the rhodopsin (RHO) gene are associated with an autosomal dominant RP (adRP) phenotype (RP4 [MIM: 613731]).[6,7] The RHO gene encodes for rhodopsin (RHO), a visual pigment found in rod photoreceptors, responsible for converting photons into chemical signals initiating vision. Rhodopsin is a 348 amino acid G-protein-coupled receptor characterized by an extracellular N-terminal domain needed to stabilize the protein, seven transmembrane-spanning a helices hosting the binding site for the chromophore 11-cisretinal, and an intracellular C-terminal domain, involved in vectorial transport of rhodopsin to rod outer segments (OSs).[8,9,10,11] approximately half of the RHO-associated adRP cases in the US are caused by the substitution of proline to histidine at position 23 (p.Pro23His)[12] in the extracellular N-terminal domain, class I variants clustered in the C-terminal domain[6] give rise to a defect in post-

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