Anti-Viral Gene Delivery in the Nervous System

描述(申请人提供)：根据血清阳性率，1.5亿至2亿美国人潜伏感染1型单纯疱疹病毒(HSV-1)，其中20%将经历潜伏病毒的反复激活。与HSV-1感染相关的大多数发病率是宿主对反复病毒重新激活的免疫反应的结果。这种重新激活引起了破坏性的炎症反应，表现为基质结疤和炎症细胞渗入眼睛基质，最终导致失明(HSV-1是工业化世界中传染性失明的主要原因)。在急性感染后，HSV-1在支配受感染组织的感觉神经节的神经元内建立了潜伏感染；对于眼睛，三叉神经节。 最近，我们发现，将编码干扰素-β的有效抗病毒转基因送入角膜后，转基因会转位到三叉神经节中的神经元。转基因在三叉神经节是活跃的，表现为干扰素反应基因、2‘-5’寡腺苷合成酶的上调和对HSV-1复制的强烈抑制。初步数据还显示，三叉神经节内潜伏的HSV-1在培养的三叉神经节移植后的重新激活在培养开始时转导干扰素-β转基因的三叉神经节的发生(再激活百分比)和病毒产量方面都显著降低。然而，目前通过腺病毒载体进行转基因传递的方法由于缺乏长时间的转基因表达、载体的免疫原性和靶细胞而受到阻碍。我们建议开发一种新的转基因传递方式，利用启动子在HSV扩增载体中延长表达时间，以靶向那些潜伏感染HSV-1的神经元，并拮抗病毒的重新激活。为了完成这一任务，我们将产生一个在β-肌动蛋白启动子下表达干扰素-β转基因的盒，并将其克隆到单纯疱疹病毒扩增载体(仅包含DNA复制起点和DNA切割/包装信号(与载体无抗原性)中)，以便使用该扩增子来测试以这种方式传递的干扰素-β转基因将随着时间的推移在体外和体内对抗HSV-1重新激活的假设。

DESCRIPTION (provided by applicant): Based on the seroprevalence rate, between 150 and 200 million Americans are latently infected with herpes simplex virus type 1 (HSV-1) of which 20% will experience recurrent reactivation of latent virus. Most of the morbidity associated with HSV-1 infection is a result of the host's immune response to repeated viral reactivation. The reactivation elicits a destructive inflammatory response manifested by stromal scarring and infiltration of inflammatory cells into the stroma of the eye ultimately leading to blindness (HSV-1 is the leading cause of infectious blindness in the industrialized world). Following acute infection, HSV-1 establishes a latent infection within the neurons of the sensory ganglia innervating the infected tissue; in the case of the eye, the trigeminal ganglia. Recently, we have found the delivery of a potent anti-viral transgene encoding for interferon (IFN)-beta into the cornea results in the translocation of the transgene to neurons in the trigeminal ganglia. The transgene is active in the trigeminal ganglia evident by the up-regulation of the IFN-responsive gene, 2'-5' oligoadenylate synthetase and strong suppression of HSV-1 replication. Preliminary data also shows that the reactivation of latent HSV-1 in the trigeminal ganglia following explantation of the ganglia in culture is significantly reduced in both occurrence (percent reactivation) and viral yield when explants are transduced with the IFN-beta transgene at the time of initiation of culture. However, the current method of transgene delivery via an adenovirus vector is hindered by the lack of prolonged transgene expression, the immunogenicity of the vector, and the targeted cells. We propose to develop a novel form of transgene delivery using a promoter for prolonged expression in an amplicon HSV vector in order to target those neurons that are specifically latently infected with HSV-1 and antagonize virus reactivation. In order to accomplish this task we will generate a cassette expressing the IFN-beta transgene under a beta-actin promoter and clone it into a HSV amplicon vector (contains only the origin of DNA replication and a DNA cleavage/packaging signal (no antigenicity associated with the vector) in order to use this HSV amplicon to test the hypothesis that the IFN-beta transgene delivered in this manner will antagonize HSV-1 reactivation in vitro and in vivo over time.