Anti-Viral Gene Delivery in the Nervous System

描述（由申请人提供）：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%将经历潜伏病毒的复发性再激活。大多数与HSV-1感染相关的发病率是宿主对反复病毒再激活的免疫应答的结果。这种再活化引发破坏性炎症反应，表现为基质瘢痕形成和炎性细胞浸润到眼睛基质中，最终导致失明（HSV-1是工业化世界中感染性失明的主要原因）。急性感染后，HSV-1在支配感染组织的感觉神经节的神经元内建立潜伏感染;在眼睛的情况下，三叉神经节。 最近，我们发现将编码干扰素（IFN）-β的有效抗病毒转基因递送到角膜中导致转基因易位到三叉神经节中的神经元。该转基因在三叉神经节中是有活性的，这通过IFN-应答基因、2 '-5'寡腺苷酸合成酶的上调和HSV-1复制的强烈抑制来证明。初步数据还显示，当在培养开始时用IFN-β转基因转导外植体时，在培养物中去除神经节后三叉神经节中潜伏HSV-1的再活化在发生率（再活化百分比）和病毒产量方面均显著降低。然而，目前通过腺病毒载体进行转基因递送的方法受到缺乏延长的转基因表达、载体的免疫原性和靶细胞的阻碍。我们建议开发一种新形式的转基因递送，其使用在扩增子HSV载体中用于延长表达的启动子，以靶向特异性潜伏感染HSV-1的那些神经元并拮抗病毒再活化。为了完成这一任务，我们将产生在β-肌动蛋白启动子下表达IFN-β转基因的盒，并将其克隆到HSV扩增子载体中（仅含有DNA复制起点和DNA切割/包装信号（无与载体相关的抗原性），以便使用该HSV扩增子来测试以这种方式递送的IFN-β转基因将拮抗HSV-1的假设。1体外和体内随时间的再活化。

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.