Gene Therapy for Urea Cycle Disorders

描述（由申请人提供）：本申请要求对题为“尿素循环障碍的基因治疗”的计划项目的项目I提供两年的修订支持，该计划项目已进入第二年的资助。 P0 I的所有三个项目都在推进应用程序的目标方面取得了成效，并按照最初的时间表如期进行。 项目I的具体目标3是评估炎症在AAV载体性能中的作用。 假设是使AAV转导的肝细胞对细胞毒性T淋巴细胞（CTL）的杀伤敏感的关键步骤是MHC I类的上调和改善的抗原呈递。 一系列的过继转移研究证实了这一假设。 将lacZ特异性CTL转移到肝脏用AAV-LacZ转导的小鼠中不能清除转基因表达，除非在过继转移时用Toll样受体（TLR）的配体处理动物;在这些条件下，转基因表达完全丧失，载体基因组清除，同时MHC I类上调。 研究人员最近将这些研究扩展到该计划的范围之外，以评估先天免疫可能足以消除不依赖于衣壳或转基因T细胞的转基因表达的可能性。 事实上，他们表明，在AAV基因转移后两周注射低剂量的表达具有TLR配体的不相关转基因的腺病毒载体足以完全消除AAV编码的转基因表达;载体基因组减少但未消除，表明转录关闭可能起作用以及载体基因组的部分不稳定。 该修订旨在进一步评估炎症在抑制肝脏中AAV载体转基因表达中的作用。 修订后的具体目标1将评估一些重要的问题，关于研究人员的最新观察，包括：低剂量的广告有助于通过产生的CTL或通过直接激活的APC？ TLR配体是否通过其同源受体的参与起作用？ 促炎细胞因子是必要的和/或足够的吗？ 最后，通过肝细胞与常驻APC的信号传导的相对作用是什么？ 修订后的特定目标2将利用小鼠模型来评估慢性肝炎感染对AAV载体性能的影响，当肝炎在基因转移时或基因转移后存在时。 相关性：腺相关病毒有望用于肝脏定向基因治疗，治疗各种遗传性和获得性疾病。 本申请评估了肝脏炎症在肝脏定向基因转移的有效性和安全性中的作用。 其结果将对候选疾病的选择和临床试验的设计具有重要意义。

DESCRIPTION (Provided by Applicant): This application requests two years of revised support for Project I of a program project entitled "Gene Therapy for Urea Cycle Disorders" which is in its second year of funding. All three projects of the P0I have been productive in advancing the aims of the application and remain on schedule according to the original timelines. Specific Aim 3 of Project I was to evaluate the role of inflammation in the performance of AAV vectors. The hypothesis was that a critical step in rendering AAV-transduced hepatocytes susceptible to killing by cytotoxic T lymphocytes (CTLs) was up-regulation of MHC class I and improved presentation of antigen. A series of adoptive transfer studies did confirm this hypothesis. Transfer of lacZ-specific CTLs into mice whose livers were transduced with AAV-LacZ failed to clear transgene expression unless the animals were treated with ligands for Toll-like receptors (TLRs) at the time of the adoptive transfer; under these conditions there was complete loss of transgene expression and clearance of the vector genome concurrent with up-regulation of MHC class I. The investigators recently extended these studies beyond the scope of the program to evaluate the possibility that innate immunity may be sufficient to extinguish transgene expression independent of capsid or transgene T cells. In fact, they showed that injection of low doses of an adenoviral vector expressing an irrelevant transgene with TLR ligands two weeks subsequent to AAV gene transfer was sufficient to completely abolish AAV-encoded transgene expression; vector genomes were diminished but not eliminated, suggesting transcriptional shut off may play a role as well as partial destabilization of the vector genome. The revision aims to further evaluate the role of inflammation in inhibiting transgene expression from AAV vectors in liver. The revised Specific Aim 1 will evaluate a number of important questions regarding the investigators' recent observation, including: Does the low dose Ad contribute through the generation of CTLs or via direct activation of APCs? Do the TLR ligands contribute through engagement of their cognate receptors? Are pro-inflammatory cytokines necessary and/or sufficient? And finally, what is the relative role of signaling via the hepatocyte versus resident APCs? The revised Specific Aim 2 will utilize a mouse model to evaluate the impact of chronic hepatitis infection on AAV vector performance when hepatitis is present at the time of gene transfer or subsequent to gene transfer. RELEVANCE: Adeno-associated viruses hold promise for liver-directed gene therapy in the treatment of a variety of inherited and acquired disorders. This application evaluates the role of liver inflammation in the efficacy and safety of liver-directed gene transfer. The results will be important in the selection of candidate diseases and design of clinical trials.