IMMUNE RESPONSES TO AAV IN GENE TRANSFER FOR HEMOPHILIA

血友病B是由于凝血因子IX(F.IX)缺乏而导致的出血性素质。AAV介导的， 肝脏导向的基因转移已经产生了治疗水平的长期(&gt；5年)表达 F.IX在犬病模型中的表达。以此为基础的临床研究 结果发现了在临床前研究中并不明显的障碍。第一个受试者在一个 治疗剂量最初显示，在4周内，F.IX水平约为10%-12%，但随后逐渐 已恢复到1%的基准水平。FIX水平的下降伴随着温和的、自我限制的 转氨炎，开始于载体输注后4周，几周后完全消失。瞬变 在第二个受试者中观察到了转氨炎，该受试者的免疫学研究记录了T细胞 对AAV衣壳病毒的反应。这个应用程序的目标是从细胞和体液的角度理解 免疫反应，这些受试者发生了什么，以及是否更详细地描述了免疫 对AAV衣壳的反应将使我们能够识别可能受益于AAV介导性、肝脏导向的受试者 基因转移。此外，我们准备确定免疫调节疗法，或 载体的变化，可以改变结果，有利于延长表达。为了实现这些目标，我们 应在小鼠和非人类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，我们将检测正常人对AAV-2衣壳序列的T细胞反应 在血友病患者和非肠道注射AAV的血友病受试者中 向量。第二个目标将决定AAV衣壳蛋白在免疫学上持续多长时间。 将载体引入小鼠肝脏后可检测到的形式。在第三个目标中，我们将考察T 注射AAV-8的非人灵长类动物对AAV-8和改良恒河猴的细胞反应 表达F.IX AAV-8的载体是猿猴AAV；因为NHP可能在载体之前自然感染 输注，这可能会更准确地模拟注射AAV-2载体的人的一系列反应。 在这些研究中，将对肝脏和外周血液中的淋巴细胞进行检测。这些研究将是 对理解先前存在的对AAV的免疫及其对AAV介导性的影响至关重要 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.