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

Abstract

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.

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Acknowledgements

We would like to acknowledge Tiansen Li and Janis Lem for providing the founder mice used to generate the novel mouse model described in this investigation. We would also like to thank Robert Molday for providing the rhodopsin 1D4 and 4D2 antibodies. This study was supported by the NIH/NEI (EY013837), The Ellison Foundation and grants to the Department of Ophthalmology at Tufts University School of Medicine from the Lions Eye Foundation and Research To Prevent Blindness.

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Correspondence to R Kumar-Singh.

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Greenwald, D., Cashman, S. & Kumar-Singh, R. Mutation-independent rescue of a novel mouse model of Retinitis Pigmentosa. Gene Ther 20, 425–434 (2013). https://doi.org/10.1038/gt.2012.53

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