Investigating Mechanisms of Viral Impairment of Neurogenesis Using Recombinant AAV

项目摘要 越来越多的证据表明，中枢神经系统（CNS）的病毒感染有助于慢性炎症。 脑部疾病在发育过程中，多种病毒，包括巨细胞病毒、单纯疱疹病毒、风疹 寨卡病毒、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- 由于大脑皮层内神经发生受损而导致的头部较小。这些病毒的后遗症 在神经发育后期和成年期，人们对它们的了解较少，但在这里，它们也会破坏神经发生， 与疾病有关。这些病毒都有一个共同的能力，消除神经祖细胞（NPC）， 发育中的和成年的大脑然而，这些病毒复杂的生物学特性使我们无法识别 这是一种精确的机制，通过这种机制，这些传染性病原体会破坏神经发生。 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）中分裂的NPC和早期有丝分裂后神经元呈剂量依赖性 方式与上述其他病毒不同，rAAV是复制缺陷型的，并且不知道是否会引起感染。 重大病理学这导致了它作为载体在实验生物学和人类基因中的广泛应用 疗法然而，越来越多的证据表明，基于rAAV的基因疗法并非没有重大风险， 过去在儿科rAAV试验中报告了至少7例rAAV相关死亡和大量不良结局 一个人待了三年虽然这些不良反应中的一些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，其中含有两个145个碱基对， 称为反向末端重复序列（ITR）的DNA片段是rAAV毒性的主要来源。我们的初步 实验表明，rAAV ITR结合并耗尽Parp 1，Parp 1是细胞DNA损伤中的第一应答者 核反应（DDR）。此外，rAAV毒性模拟Parp 1的药理学抑制，诱导 细胞周期停滞和细胞死亡，并可通过Parp 1激活部分逆转。我们的目标是利用这些 研究结果，以确定介导ITR诱导的毒性的细胞途径，并辨别是否 这些途径中的治疗靶点在引起小头畸形的病毒中是共享的。

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.