Oligonucleotide Repair of a Retinal Degeneration Model

描述（由申请人提供）：许多致盲疾病涉及光感受器和视网膜色素上皮（RPE）细胞的丧失。通常，这种细胞死亡是由DNA畸变引起的，无论是遗传的还是环境获得的。修复DNA有几个生化和分子过程，统称为DNA损伤反应（DDR）。当在基因组DNA中检测到损伤时，DDR被内源性激活。它也可以通过实验诱导非特异性或序列特异性DNA损伤来激活。出乎意料的是，最近令人兴奋的发展表明，即使没有DNA损伤，DDR也可以被激活，并且无论DNA损伤与否，DDR的激活都可能是一种先决条件或保护机制。与未分化细胞相比，对终末分化细胞（如视网膜细胞）的DDR知之甚少。本项目旨在增加我们对光感受器和RPE细胞内源性DNA修复机制的理解。通过了解DDR如何在视网膜细胞中起作用以及如何通过实验激活它，我们可以开发新的策略来增强寡核苷酸定向DNA修复，这是一种基因治疗方法，在之前的资助期内在视网膜变性模型中被证明是有效的。此外，可能类似于缺氧或循环光预处理，实验激活DDR增强了视网膜细胞承受压力的能力，否则会导致DNA损伤和死亡的积累。因此，利用自然DNA修复过程可以通过增强特定基因突变的修复和保护视网膜DNA免受环境损害来减缓或预防失明。本项目的具体目的是验证以下假设：1)视网膜细胞中的DDR可以通过实验激活，并可以增强寡核苷酸导向的基因修复；2)实验激活的DDR对随后的DNA损伤或其影响具有保护作用。

DESCRIPTION (provided by applicant): Many blinding diseases involve loss of photoreceptor and retinal pigment epithelium (RPE) cells. Often, this cell death results from DNA aberrations, whether inherited or environmentally acquired. There are several biochemical and molecular processes for repairing DNA, in aggregate called the DNA Damage Response (DDR). The DDR is endogenously activated when damage is detected in genomic DNA. It can also be activated by experimentally inducing nonspecific or sequence-specific DNA damage. Unexpectedly, recent exciting developments show that the DDR can be activated even without DNA damage and that DDR activation with or without DNA damage can be a preconditioned, or protective, mechanism. Compared to non-differentiated cells, little is known about the DDR in terminally-differentiated cells such as those of the retina. This project is designed to increase our understanding of the endogenous DNA repair mechanisms of photoreceptor and RPE cells. By learning how DDR functions in retinal cells and how it can be activated experimentally, we can develop new strategies to enhance oligonucleotide-directed DNA repair, a gene therapy approach proven efficacious in retinal degeneration models in the previous funding period. Further, it may be that, analogous to hypoxic or cyclic light preconditioning, experimentally activating the DDR enhances the ability of retinal cells to withstand stresses that would otherwise lead to accumulation of DNA damage and death. Exploiting natural DNA repair processes thus could slow or prevent blindness by enhancing repair of specific gene mutations and by protecting retinal DNA from environmental insult. The specific aims of this project are to test the hypotheses that: 1) DDR in retinal cells can be experimentally- activated and can enhance oligonucleotide-directed gene repair; and 2) experimentally-activated DDR is protective against subsequent DNA damage or its effects. PUBLIC HEALTH RELEVANCE: Many blinding diseases involve loss of photoreceptor and retinal pigment epithelium (RPE) cells due to DNA aberrations, whether inherited or acquired. There are several natural processes that cells possess to repair DNA damage, but little is known about these processes in retinal cells compared to other cells of the body. This project is designed to increase our understanding of the endogenous DNA repair mechanisms of retinal cells. By learning how these natural processes work in retinal cells, we may be able to increase their activities, prevent DNA damage or ameliorate its effects, and so slow or prevent blindness.