外泌体相关AAV载体通过调控下游MAPK信号通路提高先天性视网膜劈裂症基因治疗的疗效及其机制研究

AAV介导的先天性视网膜劈裂症基因治疗在动物中取得良好效果。临床试验证实了其安全性，但视网膜形态和功能未达到预期疗效。推测与基因转染不足及下游相关通路功能未恢复有关。新发现:1.RS1调控Na-K-ATP酶下游MAPK信号通路; 2.外泌体相关载体AAV(exo-AAV)可增加靶组织转染效率。研究假设exo-AAV-RS1可通过提高基因转染效率并抑制MAPK通路提高疗效。将证实:1.AAV-RS1和ERK1/2激酶抑制剂均可通过抑制MAPK信号通路改善Rs1h-/γ小鼠视网膜的形态和功能；2.exo-AAV-RS1可提高视网膜的转染效率并抑制MAPK通路增加治疗效果; 3.exo-AAV-RS1联合ERK1/2激酶抑制剂可对MAPK通路产生协同作用而提高疗效。项目从解决国际热点新问题和瓶颈出发，探讨治疗先天性视网膜劈裂症的分子机制。成果有望很快转化临床并对基因治疗其他视网膜病也有借鉴意义。

AAV mediated gene therapy of congenital retinoschisis achieved promising results in animal studies. Clinical trials have also proved the safety of AAV mediated gene therapy, nevertheless, the expected corrections in retinal morphology and functions have not been reached. Low gene transduction efficacy and failure in rekindling the downstream signal pathways may attribute to the different therapeutic outcomes observed in animals and in human. More recent studies showed: 1. RS1 protein regulated MAPK signaling pathway related with Na-K-ATPase and influence the activation of ERK1/2-MAPK pathway; 2. exosome-associated AAV(exo-AAV) vector improved the transfection of the target tissues. We hypothesis that by increasing the transfection efficiency of RS1 and inhibiting the downstream MAPK signaling pathway, exo-AAV-RS1 will increase the efficacy of gene therapy in retinoschisis mouse model. We will confirm: 1. Both AAV-RS1 and an inhibitor of ERK1/2 kinase (SCH772984) will improve the retinal morphology and functions in Rs1h-/γ mice by inactivating MAPK pathway; 2. Combination of AAV-RS1 and SCH772984 will enhance the therapeutic effects; 3. exo-AAV-RS1 will increase gene transfection and inhibit MAPK signaling, and consequently improve the therapeutic effects of gene therapy. This proposal aimed to solve the emerging problems in on-going clinical trials and further explore the underlying molecular mechanisms of congenital retinoschisis and its genetic intervention. The proposed novel techniques are promising to be translated in clinical trials. The results will not only provide direct information for guiding gene therapy of retinoschisis, but also are of great significance for developing novel gene therapies for other inherited retinal diseases.