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产生免疫反应。此前，我们已经发现，通过第一代腺病毒向血友病A小鼠传递的小鼠CMV驱动的第VIII因子cDNA会对第VIII因子和腺病毒蛋白产生实质性的免疫反应。这种反应可以通过用抗CD4抗体抑制T细胞来减弱。在过去的一年里，腺相关病毒(AAV)是一种安全的、也许比腺病毒更有效的递送工具，这一点已经变得明显。在这个项目中，我们的目标是通过在AAV载体中将FVIII基因转移到肝脏来进行血友病小鼠和狗的长期矫正。我们的目标是设计一种方法，将FVII基因通过AAV载体运送到肝脏。我们的目标是设计出能够在不引起免疫反应的情况下传递FVIII基因的MAN。我们最近克隆了小鼠FVIII基因的一个短SQ版本，该基因由一个小的肝脏特异性启动子(人α-抗胰蛋白酶启动子)驱动，是一个AAV载体。该载体和其他载体将在体外和体内进行初步治疗效果测试；然后在免疫抑制的FVIII缺陷小鼠中进行测试；最后在免疫活性血友病小鼠中进行测试。免疫抑制、FVIII缺陷小鼠的总大小；最后是免疫功能良好的血友病小鼠。今年小鼠FVIII-SQ基因的总大小不到4.4kb，剩下约380个碱基对用于启动子/增强子组合。在具有免疫能力的小鼠中，我们将检测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，我们将尝试通过肝脏定向表达使用犬FVIII-SQ基因来纠正血友病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。这些研究对使用AAV载体进行肝脏导向治疗血友病A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.