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

描述（由申请人提供）：像神经系统的其他区域一样，视网膜容易受到许多获得性和遗传性神经退行性疾病的影响。由于视网膜为大脑提供了所有视觉感官信息的输入，因此细胞的损失会导致视力障碍，甚至可能导致完全失明。许多视网膜退行性疾病只影响视网膜细胞的一个子集，尽管在更严重的疾病中，整个视网膜的损失和重组经常发生。在哺乳动物中，退化细胞的再生能力非常有限；然而，在鱼类中，视网膜损伤后，所有类型的新神经元都从M¿ller神经胶质中再生出来，并在功能上整合到现有的电路中。尽管如此，所有脊椎动物视网膜中都存在再生的细胞来源——米勒神经胶质。在我们三年前提交的提案中，我们假设哺乳动物M¿ller胶质细胞的再生受到限制，因为它们在损伤后不能表达关键的前神经转录因子Ascl1。我们提出通过病毒介导的Ascl1在小鼠meller胶质细胞中的表达来验证这一假设。在两年的资助中，我们已经测试了这一假设，并发现Ascl1的病毒表达足以激活小鼠M¿ller胶质细胞中的神经原性程序，无论是在分离培养物中还是在完整的视网膜中。经过重新编程的神经胶质细胞产生的细胞在形态、基因表达和对神经递质的反应上都与神经元相似。在接下来的资助期内，我们建议进一步优化这一重编程过程，使用其他转录因子和表观遗传修饰因子，然后测试ascl1重编程Muller胶质细胞是否可以在新开发的转基因小鼠系中提供体内再生的来源。

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.