IMMUNE RESPONSES TO AAV IN GENE TRANSFER FOR HEMOPHILIA

血友病B是由于凝血因子IX（F.IX）缺乏而引起的出血素质。AAV介导的， 肝脏定向基因转移已经产生了治疗水平的长期（>5年）表达， F.IX在该疾病的犬模型中。一项基于这些的临床研究 研究结果揭示了在临床前研究中不明显的障碍。第一个接受治疗的受试者 治疗剂量最初显示约10-12%的F.IX水平持续4周，但随后F.IX水平逐渐升高， 恢复到<1%的基线水平。F.IX水平的下降伴随着轻度的自限性 在载体输注后4周开始，并在几周后完全消退。瞬态 在第二个受试者中观察到转氨酶升高，并且在该受试者中的免疫学研究记录了T细胞 对AAV衣壳的应答。这个应用程序的目标是了解，在细胞和体液方面， 免疫反应，这些受试者发生了什么，以及是否有更详细的免疫反应特征， 对AAV衣壳的应答将使我们能够鉴定可能从AAV介导的、肝脏定向的 基因转移此外，我们准备确定是否免疫调节疗法，或 载体中的变化可以改变有利于延长表达的结果。为了实现这些目标，我们 应在小鼠和非人灵长类动物模型以及正常和血友病受试者中进行研究。 在第一个目标中，我们将检查正常人中T细胞对AAV-2衣壳序列的应答。 人群、血友病患者和已胃肠外注射AAV的血友病受试者 向量。第二个目标将确定AAV衣壳蛋白在免疫学上持续多长时间。 在将载体引入小鼠肝脏后，可检测到的形式。在第三个目标中，我们将检查T 注射AAV-8的非人灵长类动物中对AAV-8和A修饰的恒河猴F.IX的细胞应答 表达F. IX的载体。AAV-8是猿AAV;因为NHP可以在载体之前自然感染， 通过使用AAV-2载体输注，这可以更准确地模拟输注有AAV-2载体的人中的一系列应答。 在这些研究中，将检查肝脏和外周血的淋巴细胞。这些研究报告将 这对于了解预先存在的对AAV的免疫及其对AAV介导的免疫的影响至关重要。 基因转移

Hemophilia B is a bleeding diathesis due to a deficiency of blood coagulation Factor IX (F.IX). AAV-mediated, liver-directed gene transfer has yielded long-term (>5 years) expression of therapeutic levels of F.IX in the canine model of the disease. A clinical study based on these findings uncovered obstacles that had not been evident in pre-clinical studies. The first subject treated at a therapeutic dose initially demonstrated F.IX levels of approximately 10-12% for 4 weeks, but then F.IX levels gradually returned to the baseline level of <1%. The decline in F.IX levels was accompanied by a mild, self-limited transaminitis, that began 4 weeks after vector infusion and fully resolved several weeks later. Transient transaminitis was observed in a second subject and immunologic studies in this subject documented a T cell response to AAV capsid. The goal of this application is to understand, in terms of the cellular and humoral immune response, what happened to these subjects, and whether more detailed characterization of immune responses to AAV capsid will permit us to identify subjects likely to benefit from AAV-mediate, liver-directed gene transfer. In addition, we prepare to determine whether immunomodulatory therapies, or changes in the vector, can alter the outcome in favor of prolonged expression. To accomplish these goals, we shall pursue studies in murine and non-human primate animal models, and in normal and hemophilic subjects. In the first aim, we will examine T cell responses to AAV-2 capsid sequences in the normal human population, in hemophilic patients, and in hemophilic subjects who have been injected parenterally with AAV vectors. The second aim will determine how long AAV capsid proteins persist in an immunologically detectable form following introduction of vector into the livers of mice. In the third aim, we shall examine T cell responses to AAV-8 and to A modified Rhesus F.IX in non-human primates injected with an AAV-8 vector expressing F.IX. AAV-8 is a simian AAV; because NHP may be naturally infected prior to vector infusion, this may more accurately model the series of responses in humans infused with AAV-2 vectors. Lymphocytes from both liver and peripheral blood will be examined in these studies. These studies will be critical for developing an understanding of pre-existing immunity to AAV and its implications for AAV-mediated gene transfer.