RESUMEN
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.
Asunto(s)
Modelos Animales de Enfermedad , Terapia Genética , Ratones/genética , Retinitis Pigmentosa/terapia , Animales , Dependovirus/genética , Silenciador del Gen , MicroARNs/metabolismo , Mutación Missense , Células Fotorreceptoras de Vertebrados/metabolismo , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/patología , Rodopsina/genética , Rodopsina/metabolismo , TransgenesRESUMEN
The alpha7 receptor agonist dimethoxybenzylidene anabaseine (DMXB) protected rat neocortical neurons against excitotoxicity administered 24 h before, but not concomitantly with, NMDA. This action was blocked by nicotinic but not muscarinic antagonists. DMXB (1 mg/kg i.p.) also reduced infarct size in rats when injected 24 h before, but not during, focal ischemic insults. In a mecamylamine-sensitive manner, alpha7 receptors appear neuroprotective in non-apoptotic model.