Mutation-independent rescue of a novel mouse model of Retinitis Pigmentosa.
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.
Department of Ophthalmology, Tufts University School of Medicine, Boston, MA 02111, USA.
D L
DL
Greenwald
S M
SM
Cashman
R
R
Kumar-Singh
D000818:Animals; D000229:Dependovirus / Q000235:genetics; D004195:Disease Models, Animal; D020868:Gene Silencing; D015316:Genetic Therapy; D051379:Mice / Q000235:genetics*; D035683:MicroRNAs / Q000378:metabolism; D020125:Mutation, Missense; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism; D012174:Retinitis Pigmentosa / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism; D019076:Transgenes
D035683:MicroRNAs; D012243:Rhodopsin