Supplement to EY021482 to carry out a screen for retinal regeneration using CRISPR-Cas9 gene activation.

描述(申请人提供)：像神经系统的其他区域一样，视网膜会受到许多后天和遗传性神经元退行性疾病的影响。由于视网膜为大脑提供了所有视觉感觉信息的输入，细胞的丧失会导致视觉损伤，并可能导致完全失明。许多视网膜退行性疾病只影响一部分视网膜细胞，尽管在更严重的疾病中，经常会发生整个视网膜的丢失和重组。在哺乳动物中，退化细胞的再生非常有限；然而，在鱼类中，在视网膜损伤后，所有类型的新神经元从Müler神经胶质再生，它们在功能上整合到现有的电路中。然而，所有脊椎动物的视网膜中都存在作为再生细胞来源的Müller胶质细胞。在我们三年前提交的提案中，我们假设哺乳动物Müller神经胶质细胞的再生是有限的，因为它们在损伤后不能表达关键的原神经转录因子Ascl1。我们建议通过病毒介导的Ascl1在小鼠M？ler神经胶质细胞中的表达来检验这一假设。在两年的资助中，我们测试了这一假说，并发现Ascl1的病毒表达足以在分离培养的和完整的视网膜中激活小鼠Müler神经胶质细胞的神经发生程序。重新编程的M？ler胶质细胞产生的细胞在形态、基因表达及其对神经递质的反应上类似于神经元。在下一个资金阶段，我们建议使用其他转录因子和表观遗传学修饰物进一步优化这种重新编程的过程，然后测试Ascl1重新编程的Muller胶质细胞是否可以为新开发的转基因小鼠的体内再生提供来源。

DESCRIPTION (provided by applicant): Like other areas of the nervous system, the retina is subject to many acquired and inherited neuronal degenerative diseases. Since the retina provides the input for all visual sensory information to the brain, the loss of cells results in viual impairment and potentially complete blindness. Many retinal degenerative diseases affect only a subset of the retinal cells, although, frequently in more advanced disease, loss and reorganization of the entire retina can occur. In mammals, there is very limited regeneration of the degenerated cells; however, in fish, new neurons of all types regenerate from M¿ller glia following retinal damage and they are functionally integrated into the existing circuitry. Nevertheless, M¿ller glia, the cellular source for regeneration, is present in all vertebrate retins. In the proposal we submitted three years ago, we hypothesized that regeneration from mammalian M¿ller glia was limited because they fail to express a key proneural transcription factor, Ascl1, after injury. We proposed to test this hypothesis by virally-mediated expression of Ascl1 in mouse M¿ller glia. In the two years of funding, we have tested the hypothesis, and found that viral expression of Ascl1 is sufficient to activate a neurogenic program in mouse M¿ller glia, both in dissociated cultures and in the intact retina. The reprogrammed M¿ller glia generates cells that resemble neurons in morphology, gene expression and their responses to neurotransmitters. In the next funding period, we propose to further optimize this reprogramming process, using other transcription factors and epigenetic modifiers, and then to test whether Ascl1-reprogrammed Muller glia can provide a source for regeneration in vivo in a newly developed line of transgenic mice.