Mutation-independent rescue of a novel mouse model of Retinitis Pigmentosa

• Published in: Gene therapy Vol. 20; no. 4; pp. 425 - 434
• Main Authors: Greenwald, D L, Cashman, S M, Kumar-Singh, R
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.04.2013; Nature Publishing Group
• Subjects: 631/154/51/201; 631/1647/2300/1514; 631/337/384/331; 692/1807/1482; Adeno-associated virus; Animals; Biomedical and Life Sciences; Biomedicine; Blindness; Cell Biology; Dependovirus - genetics; Disease Models, Animal; Electroretinograms; Expression vectors; Eye; Eye diseases; Gene Expression; Gene mutations; Gene Silencing; Gene Therapy; Genetic aspects; Genetic Therapy; Health aspects; Human Genetics; Mice - genetics; MicroRNAs; MicroRNAs - metabolism; miRNA; Mutation; Mutation, Missense; Nanotechnology; original-article; Photoreceptor Cells, Vertebrate - metabolism; Photoreceptors; Physiological aspects; Retinal degeneration; Retinitis; Retinitis pigmentosa; Retinitis Pigmentosa - genetics; Retinitis Pigmentosa - pathology; Retinitis Pigmentosa - therapy; Rhodopsin; Rhodopsin - genetics; Rhodopsin - metabolism; Rodents; Serotypes; Transgenes; Tropism; United States; AAV2/9; microRNA; P347S; RNAi; rhodopsin (Rho); Retinitis Pigmentosa (RP)
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/gt.2012.53

Retinitis Pigmentosa (RP) is the leading cause of inherited blindness in the developed world, affecting approximately 1 in 3000 individuals. Although there is currently no cure for RP, the genetic pathology has been well established. In this study, we developed a novel mouse model of RP (huRhoP347S) expressing a pathogenic human rhodopsin gene with a Pro347Ser (P347S) mutation on a rhodopsin knockout background. These mice undergo severe retinal degeneration at 1 month of age. In contrast to prior studies, this model was administered a gene therapy treatment at 19 days postnata. We evaluated several self-complementary adeno-associated virus (AAV) serotypes for photoreceptor tropism, including scAAV2/2, scAAV2/5, scAAV2/6.2 and scAAV2/9, and found that scAAV2/9 transduced photoreceptors with greater efficiency and expression than other vectors. We engineered an scAAV2/9 vector to contain a microRNA sequence specifically targeting the human rhodopsin gene and demonstrated its ability to silence rhodopsin by 60.2±8.2% in vitro . In addition, we constructed an scAAV2/9 vector to contain a replacement ‘codon-modified’ rhodopsin transgene (RhoR2) that was resistant to degradation by the microRNA. We found that delivery of the RhoR2 by scAAV2/9 is capable of restoring vision to rhodopsin knockout mice, and rescuing our novel transgenic huRhoP347S mouse model of dominant RP. Average a-wave responses of RhoR2-injected eyes were 1.8-fold higher than those of control-injected eyes. We found that delivery of the microRNA and replacement rhodopsin in a 1:2 ratio produced an average electroretinography (ERG) a-wave response of 17.4±2.9 compared to 6.5±2.8 μV for eyes injected with negative control virus.