GENE REPLACEMENT THERAPY AND THE IMMUNE SYSTEM

这个项目的目标是阐明预先存在的腺相关病毒-2特异性T细胞介导的作用 肝脏rAAV载体介导的基因转移的免疫力。重组AAV-2载体具有 在免疫耐受实验动物的基因替代治疗中取得了有希望的结果 这表明它们的免疫原性相对较差。人类一般都接触到AAV-2 通过感染带有另一种病毒的AAV-2病毒，即所谓的辅助病毒， 比如腺病毒或疱疹病毒。在免疫原性差的AAV-2是 引入一种会引起强烈炎症反应的辅助病毒，辅助病毒预计将 提供一种佐剂效应，应有助于诱导强烈的适应性免疫反应 AAV-2的抗原。因此，与实验啮齿动物或犬科动物不同，人类受试者很可能有抗原经验。 在rAAV-2介导的基因转移中更容易激活的T细胞对AAV-2的作用 比到目前为止进行的临床前研究所认识到的更多。可能会混淆这个问题的是 AAV-2以一种表明其基因组可能继续转录的形式在人类体内持续存在。连 非常低水平的持续AAV-2抗原将维持免疫系统处于高度的 激活。先前存在的对AAV-2的T细胞介导的免疫也可能影响猴AAVs对基因的使用 由于人类和猿源AAVs共享T细胞表位而进行的替代治疗。其中一个 这一应用的挑战是在实验小动物中模拟人类的自然AAV感染 适合对免疫反应进行深入研究的物种。为此，将使用近亲繁殖的小鼠 尽管它们不是AAV-2或其天然辅助病毒的自然宿主。为了克服这一限制，我们 在目标1中开发并将继续完善诱导AAV衣壳特异性T细胞的实验系统 小鼠的反应，在功能上类似于人类受试者。在目标2中，我们将描述 采用最合适的小鼠模型，深入研究AAV-2衣壳蛋白的免疫应答。在目标3中，我们将使用 AAV-2预先免疫对甲型肝炎病毒疗效、免疫原性和寿命影响的模型 RAAV-2介导的基因转移。目标4中的实验将扩展到基于Simian导出的矢量系统 评估异源rAAV载体是否能克服先前存在的对AAV-2的免疫。

The goal of this project is to elucidate the effect of pre-existing adeno-associated virus-2-specific T cell-mediated immunity on hepatic rAAV vector-mediated gene transfer. Recombinant (r) AAV-2 vectors have yielded promising results in gene replacement therapy of immunocornpetent experimental animals suggesting that they are comparatively poorly immunogenic. Humans are generally exposed to AAV-2 antigens during childhood through infection by AAV-2 accompanying another virus, a so-called helper virus, such as an adenovirus or a herpes virus. Under circumstances where the poorly immunogenic AAV-2 is introduced with a helper virus that causes a strong inflammatory reaction, the helper virus is expected to provide an adjuvant effect that should facilitate the induction of a strong adaptive immune response to antigens of AAV-2. Human subjects unlike experimental rodents or canines are thus likely to have antigen-experienced T cells to AAV-2 that may become activated more readily upon rAAV-2-mediated gene transfer than has been appreciated by pre-clinical studies conducted thus far. Potentially confounding this problem is that AAV-2 persists in humans in a form that suggests that its genome may continue to be transcribed. Even very low levels of persistent AAV-2 antigen would maintain the immune system at a heightened stage of activation. Pre-existing T cell-mediated immunity to AAV-2 may also affect the use of simian AAVs for gene replacement therapy due to shared T cell epitopes between human and simian origin AAVs. One of the challenges of this application is to mimic natural AAV infection of humans in an experimental small animal species suited to conduct an in depth study of immune responses. For this, inbred mice will be used although they are not natural hosts for AAV-2 or its natural helper viruses. To overcome this limitation we developed and will continue to refine in aim 1 an experimental system to induce AAV capsid-specific T cell responses in mice, which resemble functionally those in human subjects. In aim 2, we will characterize the immune response to AAV-2 capsid in depth using the most suitable mouse model. In aim 3, we will use the model to test the effect of pre-existing immunity to AAV-2 on the efficacy, immunogenicity and longevity of rAAV-2-mediated gene transfer. Experiments will be extended in aim 4 to a vector system based on simianderived AAV-8 to assess if a heterologous rAAV vector can overcome pre-existing immunity to AAV-2.