Gene Therapy for Urea Cycle Disorders

描述（由申请人提供）：本申请为一项名为“尿素循环障碍的基因治疗”的项目I申请为期两年的修订资助，该项目已进入第二年获得资助。所有三个项目在推进申请目标方面都取得了成效，并按照原定时间表如期进行。项目1的具体目的3是评估炎症在AAV载体表现中的作用。该假设认为，使aav转导的肝细胞易受细胞毒性T淋巴细胞（ctl）杀伤的关键步骤是MHC I类的上调和抗原呈递的改善。一系列的过继性迁移研究确实证实了这一假设。将laz特异性ctl转移到用AAV-LacZ转导的小鼠肝脏中不能清除转基因表达，除非动物在过继转移时用toll样受体（TLRs）配体处理；在这些条件下，转基因表达完全丧失，载体基因组被清除，同时MHC i类上调。研究人员最近将这些研究扩展到项目范围之外，以评估先天免疫是否足以消除独立于衣壳或转基因T细胞的转基因表达。事实上，他们表明，在AAV基因转移两周后注射低剂量的腺病毒载体，表达与TLR配体无关的转基因，足以完全消除AAV编码的转基因表达；载体基因组减少了，但没有完全消除，这表明转录关闭可能在载体基因组的部分不稳定中起作用。该修订旨在进一步评估炎症在抑制AAV载体在肝脏中的转基因表达中的作用。修订后的特异性目标1将评估研究人员最近观察到的一些重要问题，包括：低剂量Ad是通过ctl的产生还是通过apc的直接激活起作用？TLR配体是否通过其同源受体的参与而起作用？促炎细胞因子是必要和/或充分的吗？最后，通过肝细胞和常驻APCs的信号传导的相对作用是什么？修订后的Specific Aim 2将利用小鼠模型来评估在基因转移时或基因转移后存在肝炎时慢性肝炎感染对AAV载体性能的影响。

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.