Modeling gene therapy of Hemophilia A via liver directed gene expression

肝脏导向基因治疗的一个主要问题是对治疗性转基因的免疫反应的发展。先前，我们发现小鼠cmv驱动因子VIII cDNA通过第一代腺病毒传递给血友病a小鼠，可引起对因子VIII和腺病毒蛋白的大量免疫应答。这种反应可以通过抗cd4抑制t细胞而减弱。在过去的一年中，人们已经清楚地认识到腺相关病毒（AAV）是一种安全且可能比腺病毒更有效的递送载体。在这个项目中，我们的目标是通过AAV载体将FVIII cDNA传递到肝脏，对血友病小鼠和狗进行长期矫正。我们的目标是设计将FVII cDNA以AAV载体传递到肝脏的方法。我们的目标是设计出在不遇到免疫反应的情况下递送FVIII cDNA的人。我们最近克隆了一个小的肝脏特异性启动子（人α -抗胰蛋白酶启动子）驱动的小鼠FVIII cDNA的短SQ版本，作为AAV载体。将对该载体和其他载体进行初步体外和体内治疗效果测试；然后是免疫抑制的fviii缺陷小鼠；最后在具有免疫能力的血友病小鼠中。免疫抑制、fviii缺陷小鼠的总大小；最后在具有免疫能力的血友病小鼠中。今年小鼠FVIII-SQ cDNA的总长度在4.4 kb以下，剩下大约380 bp的启动子/增强子组合。在免疫功能正常的小鼠中，我们将测量FVIII活性、FVIII抗原以及对FVIII的细胞和体液免疫反应。利用从小鼠预期中获得的信息，我们将尝试使用犬FVIII-SQ cDNA通过肝脏定向表达来纠正血友病A犬。我们希望克服对FVIII的任何免疫反应，并为这些动物模型提供成功的长期治疗。这些研究对利用AAV载体肝定向治疗A型血友病的临床试验至关重要。

A major problem in liver-directed gene therapy is the development of an immune response to the therapeutic transgene. Previously, we have found that mouse CMV-driven factor VIII cDNA delivered in a first-generation adenovirus to hemophilia A mice provokes a substantial immune response to both factor VIII and adenoviral proteins. This response can be blunted by suppression of T-cell with anti-CD4. Over the past year, it has become clear that adeno-associated virus (AAV) is a safety and perhaps more effective delivery vehicle than adenovirus. In this project, we aim to carry out long-term correction of hemophilic mice and dogs by delivery of FVIII cDNA to liver in an AAV vector. Our goal is to devise the means to deliver the FVII cDNA to liver in an AAV vector. Our goal is to devise the mans to deliver the FVIII cDNA without encountering an immune response. We have recently cloned a short SQ version of the mouse FVIII cDNA driven by a small liver-specific promoter (human alpha-anti-trypsin promoter) is an AAV vector. This and other vectors will be tested for preliminary for therapeutic effect in vitro and in vivo; then in immunosuppressed, FVIII-deficient mice; and finally in immunocompetent hemophilic mice. The total size of immunosuppressed, FVIII-deficient mice; and finally in immunocompetent hemophilic mice. The total size of the mouse FVIII-SQ cDNA in this year is under 4.4 kb, leaving roughly 380 bp for promoter/enhancer combinations. In immunocompetent mice, we will measure FVIII activity, FVIII antigen, and both cellular and humoral immune responses to FVIII. Using the information gained from mice expectations, we will attempt correction of hemophilia A dogs using canine FVIII-SQ cDNA via liver directed expression. We hope to overcome any immune response to FVIII and provide successful long-term treatment of these animal models. The studies are critical to clinical trials of liver-directed therapy of hemophilia A using AAV vectors.