RIBOZYME-MEDIATED IN VIVO PHOTORECEPTOR EXPRESSION

我们建议使用启动子调节的核酶在我们的实验室开发的重组病毒载体作为一个遗传挑战的实验动物的视网膜。具体来说，我们将分析光感受器的表达反应，以在其他正常的啮齿动物视网膜中下调关键的单个光转导mRNA。核酶是RNA酶，具有阻断特定基因表达的潜力。在过去，我们已经成功地使用腺相关病毒（AAV）提供核酶作为治疗在啮齿动物模型的显性遗传疾病的视网膜。我们的计划将采用类似的方法，这一次提供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分析表达反应。该技术允许产生体内体细胞基因敲除，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导致的实验不可接近性、以及限制大多数转基因为已知功能或疾病病因的基因的实际问题。我们的两年目标将是验证AAV-核酶的方法，通过以下杆光感受器的表达反应，以改变两个充分研究的光转导基因，杆cGMP-磷酸二酯酶（PDE）的β-或γ-亚基的mRNA水平。将表达模式与来自同一动物的对照视网膜和来自具有内源性改变的β-或γ-PDE mRNA水平的小鼠视网膜的表达模式进行比较，以确定针对给定mRNA的核酶是否引起与遗传确定的变化相同的全局表达变化。最后，将确定表达谱变化的时间模式，以区分初始细胞应答与更多下游事件，如凋亡途径的激活。需要强调这一战略的普遍性。即使在这项可行性研究中，我们也有可能发现杆中新的基因功能，确定视网膜疾病的新候选基因，更普遍地说，完善在全身各种组织中广泛应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.