Oligonucleotide Repair of a Retinal Degeneration Model

描述(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)细胞的丢失。通常，这种细胞死亡是由DNA异常引起的，无论是遗传的还是环境获得性的。有几个修复DNA的生物化学和分子过程，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。出人意料的是，最近令人兴奋的发展表明，即使在没有DNA损伤的情况下，DDR也可以被激活，并且无论有没有DNA损伤，DDR的激活都可以是一种预适应或保护性机制。与未分化细胞相比，对视网膜等终末分化细胞中的DDR知之甚少。这个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在视网膜细胞中的功能以及它如何在实验中被激活，我们可以开发新的策略来增强寡核苷酸导向的DNA修复，这是一种基因治疗方法，在之前的资助期被证明在视网膜变性模型中是有效的。此外，与低氧或周期性光预适应类似，通过实验激活DDR可能会增强视网膜细胞抵御压力的能力，否则会导致DNA损伤和死亡的积累。因此，利用自然DNA修复过程可以通过加强特定基因突变的修复和保护视网膜DNA免受环境伤害来减缓或防止失明。这个项目的具体目的是检验这样的假设：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指导的基因修复；以及2)实验激活的DDR可以保护DNA免受后续的DNA损伤或其影响。

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.