RIBOZYME-MEDIATED IN VIVO PHOTORECEPTOR EXPRESSION

我们建议使用由我们实验室开发的重组病毒载体传递的启动子调控的核酶作为对实验动物视网膜的遗传挑战。具体地说，我们将描述在正常的啮齿动物视网膜中，光感受器对关键的单个光转导mRNAs下调的表达反应。核酶是一种核糖核酸酶，有可能阻止特定基因的表达。在过去，我们已经成功地使用腺相关病毒(AAV)携带核酶来治疗显性视网膜遗传性疾病的啮齿动物模型。我们的计划将使用类似的方法，这次交付针对野生型基因序列的核酶，以创建最初在单个正常光感受器mRNA中改变的视网膜，然后使用光感受器基因特异性阵列技术在全球范围内描述表达反应。这项技术允许产生体内的体细胞基因敲除，避免了通常与转基因或生殖系敲除动物相关的问题，包括胚胎致死性、由于早期发育表达而无法获得实验，以及将大多数转基因限制在已知功能或疾病病因的基因上的实际问题。我们的两年目标将是通过跟踪杆状光感受器对两个研究良好的光转导基因之一的mRNAs水平变化的表达反应来验证AAV-ribozyme方法，这两个光传导基因是杆状cGMP-磷酸二酯酶(PDE)的β或伽马亚基。为了确定针对给定mRNA的核酶是否会引起与基因决定的变化相同的全局表达变化，将与同一动物的对照视网膜和内源性β-或γ-PDE mRNA水平发生变化的小鼠的视网膜的表达模式进行比较。最后，将确定表达谱变化的时间模式，以便将初始细胞反应与更多下游事件区分开来，例如激活凋亡通路。需要强调这一战略的一般性。即使在这项可行性研究中，我们也有可能在视杆细胞中发现新的基因功能，识别视网膜疾病的新候选基因，更广泛地说，完善一种在全身各种组织中广泛使用的体内表达谱技术。

We propose to use promoter-regulated ribozymes delivered by recombinant virus vectors developed in our laboratories as a genetic challenge to the retina of experimental animals. Specifically, we will profile the expression response of photoreceptors to having key individual phototransduction mRNAs down regulated in an otherwise normal rodent retina. Riboenzymes are RNA enzymes that have the potential to block the expression of specific genes. In the past, we have successfully used Adeno-associated virus (AAV) to deliver ribozymes as therapy in rodent models of dominant genetic diseases of the retina. Our plan will employ the analogous approach, this time delivering ribozymes against wild type gene sequences, to create retinas altered initially in a single normal photoreceptor mRNA, and then to profile the expression response globally using photoreceptor gene-specific array technology. This technique permits the generation of in vivo somatic gene knock- outs, avoiding problems commonly associated with transgenic or germ line knock-out animals, including embryonic lethality, experimental inaccessibility due to early developmental expression, and a practical problem limiting most transgenics to genes of known function or disease etiology. Our two-year goal will be to validate the AAV-ribozyme approach by following the expression response of rod photoreceptors to altered levels of mRNAs for either of two well-studied phototransduction genes, the beta- or gamma-subunits of rod cGMP- phosphodiesterase (PDE). Expression patterns will be compared with those from both control retina in the same animal and from retina in mice with endogenously altered levels of beta- or gamma-PDE mRNA in order to determine whether ribozymes against a given mRNA cause the same global expression changes as genetically determined changes. Finally, the temporal pattern of expression profile changes will be determined in order to distinguish initial cellular responses from more downstream events such as activation of apoptotic pathways. The generality of this strategy needs to be emphasized. Even in this feasibility study, we are likely to uncover new gene functions in rods, identify new candidate genes for retinal disease and, more generally, perfect an in vivo expression profiling technology of wide utility in a variety of tissues throughout the body.