Investigating Mechanisms of Viral Impairment of Neurogenesis Using Recombinant AAV

项目总结 越来越多的证据表明，中枢神经系统(CNS)的病毒感染导致慢性 脑部疾病。在发育过程中，包括巨细胞病毒、单纯疱疹病毒、风疹在内的多种病毒 病毒、人类免疫缺陷病毒和寨卡病毒每年导致数千例小头畸形症- 由于大脑皮层内的神经发生受损而导致的头部偏小。这些病毒的后遗症 在神经发育和成年期的后期，人们对它们知之甚少，但在这里，它们也扰乱了神经发生，并 与疾病有牵连。这些病毒具有共同的消除神经前体细胞(NPC)的能力 发育中的和成人的大脑。然而，这些病毒的复杂生物学使我们无法识别 一种精确的机制，这些感染源通过它来消融神经发生。 我们最近发现，广泛使用的重组腺相关病毒(RAAV)可以迅速杀死 成年小鼠齿状回(DG)内神经前体细胞和早期有丝分裂后神经元的分离 举止。与上面描述的其他病毒不同，rAAV是复制缺陷的，目前还不知道会导致 重要的病理学。这使得它在实验生物学和人类基因方面都被广泛用作载体。 心理治疗。然而，越来越多的证据表明，基于rAAV的基因治疗并不是没有重大风险，at 过去在儿科rAAV试验中报告的至少7例rAAV相关死亡和众多不良后果 一个人三年了。虽然其中一些不良反应被认为是由免疫反应引起的 衣壳或转基因，越来越多的证据表明，rAAV基因组，其中包含两个145碱基对 被称为反向末端重复序列(ITRs)的DNA片段是rAAV毒性的主要来源。我们的预赛 实验表明，rAAV ITR结合并耗尽PARP1，PARP1是细胞DNA损伤的第一反应 核内反应(DDR)。此外，rAAV的毒性模拟了对PARP1的药理抑制，诱导 细胞周期停滞和细胞死亡，可被PARP1激活部分逆转。我们的目标是利用这些优势 发现介导ITR诱导的毒性的细胞通路，并区分 这些途径中的治疗靶点在导致小头畸形症的病毒之间是共享的。

PROJECT SUMMARY There is growing evidence that viral infections of the central nervous system (CNS) contribute to chronic brain disease. During development, multiple viruses including cytomegalovirus, herpes simplex virus, rubella virus, human immunodeficiency virus, and Zika virus annually cause thousands of cases of microcephaly — small head size resulting from impaired neurogenesis within the cerebral cortex. The sequalae of these viruses later in neurodevelopment and adulthood are less understood, but here they also disrupt neurogenesis and have been implicated in disease. These viruses share a common ability to eliminate neural progenitor cells (NPCs) in the developing and adult brain. However, the complex biology of these viruses has precluded our ability to identify a precise mechanism by which these infectious agents ablate neurogenesis. We recently discovered that the widely used recombinant adeno-associated virus (rAAV) rapidly kills dividing NPCs and early post-mitotic neurons in the adult murine dentate gyrus (DG) in a dose-dependent manner. Unlike the other viruses described above, rAAV is replication defective and is not known to cause significant pathology. This has resulted in its wide use as a vector in both experimental biology and human gene therapy. However, evidence is mounting that rAAV-based gene therapies are not without significant risk, with at least 7 rAAV-related deaths and numerous adverse outcomes reported in pediatric rAAV trials during the past three years alone. While some of these adverse effects are thought to be caused by immune reactions to the capsid or transgene, increasing evidence indicates that the rAAV genome, which contains two 145-base pair DNA segments named inverted terminal repeats (ITRs), is a major source of rAAV toxicity. Our preliminary experiments indicate that rAAV ITRs bind to and deplete Parp1, a first responder in cellular DNA damage response (DDR) within the nucleus. Moreover, rAAV toxicity mimics pharmacological inhibition of Parp1, inducing cell cycle arrest and cell death, and can be partially reversed by Parp1 activation. We aim to capitalize on these findings to identify the cellular pathways that mediate ITR-induced toxicity and discern whether therapeutic targets within these pathways are shared among viruses that cause microcephaly.