T Cells To AAV And AAV-Encoded Transgene Products

该申请寻求用于研究的资金，该研究旨在解剖T细胞对以下病毒的衣壳抗原的应答： 腺相关病毒（AAV）、不同类型的AAV载体和这些载体的转基因产物。的 这项更新申请的目的是进一步了解有害的免疫反应， 在AAV基因转移后，干扰治疗性蛋白质的持续表达。 在目标1中，我们将确定CD 4 T细胞识别不同AAV载体的衣壳表位的时间长短 使用当前供资期间开发的方法。 在目标2中，我们将表征CD 8 * T细胞对双链（ds）AAV的转基因产物的应答 向量。正如我们在上一个资助期所显示的，转基因产物特异性CD 8 + T细胞诱导的CD 8 + T细胞在小鼠中表达。 通过单链（ss）AAV载体对小鼠的免疫功能是受损的。在最近的研究中，我们观察到， dsAAV载体在较低剂量下刺激远更有效的CDS* T细胞应答，这似乎与CD 4 + T细胞应答相似。 与ssAAV载体诱导的那些不同。我们将进行额外的研究，以评估免疫 本发明的目的是为了阐明dsAAV载体的转基因产物的应答以及这种应答的程度 干扰持续的基因转移。 在目标3中，我们将表征非人灵长类动物（NHP）中对天然AAV感染的T细胞应答。 在人类中也是如此。正如我们在目前的资助期间所展示的那样，人类和NHP都有记忆T 细胞到AAV衣壳。这种T细胞的频率在NHP中非常稳健，在人类中明显较低。 这最初似乎与这个程序的基本假设相矛盾，即预先存在的记忆CDS* T 在AAV介导的基因转移后，细胞与AAV衣壳的结合被重新激活，然后引起细胞排斥 用表达治疗性蛋白质的AAV载体转导，因为这种潜在的问题仅仅是 在人类中发现，但在NHP中没有。我们的假设是，来自于腺相关病毒衣壳特异性CDS* T细胞 人类和NHP在功能上不同，来自人类的NHP可以进行有效的回忆， 反应，而那些从恒河猴是无法做到这一点，因为将深入调查使用 国家卫生中心的样本（目标3.1），健康成人（目标3.2.1.）和儿童康复从一个 腺病毒和可能的AAV感染（目的3.2.2）。一项临床研究中的另一个令人困惑的发现 AAV基因转移患者将在目标3.2.3中进行随访。在这项试验中，一名患者接受了肝移植治疗。 在免疫抑制（IS）下输注AAV 2-hF.IX载体。他的大脑中 基因转移后循环AAV衣壳特异性T细胞没有显示肝细胞 杀伤性这种明显功能失调的AAV captain特异性CDS* T增加的潜在途径 细胞将用IS开始之前和/或之后取自移植患者的样品进一步阐明。

This application seeks funding for studies designed to dissect T cell responses to the capsid antigens of adeno-associated viruses (AAV), different types of AAV vectors and transgene products of such vectors. The goal of this renewal application is to further our understanding on detrimental immune responses that arise upon AAV gene transfer and interfere with sustained expression of therapeutic proteins. In aim 1 we will determine how long CDS"^ T cells recognize capsid epitopes of different AAV vectors using methods developed during the current funding period. In aim 2 we will characterize CD8* T cell responses to the transgene product of double-stranded (ds)AAV vectors. As we showed during the last funding period, transgene product-specific CD8'^ T cells induced in mice by single-stranded (ss)AAV vectors are functionally Impaired. In more recent studies we observed that dsAAV vectors stimulate by far more potent CDS* T cell responses at lower doses, which appear to be functional unlike those Induced by ssAAV vectors. We will conduct additional studies to assess immune responses to transgene products of dsAAV vectors and to elucidate if and to what degree such responses interfer with sustained gene transfer. In aim 3 we will characterize T cell responses to natural AAV infections in non-human primates (NHPs) and in humans. As we showed during the current funding period, both humans and NHPs have memory T cells to AAV capsid. Frequencies of such T cells are very robust in NHPs and markedly lower in humans. This initially appears to contradict the basic hypothesis of this program that pre-existing memory CDS* T cells to AAV capsid are reactivated upon AAV-mediated gene transfer and then cause rejection of cells transduced with AAV vectors expressing a therapeutic protein, as this potential problem was only encountered in humans but not In NHPs. Our hypothesis is that AAV capsid-specific CDS* T cells from humans and NHPs differ in their functionality and that those from humans can mount an effective recall response, while those from rhesus macaques are unable to do so, as will be investigated In depth using samples from NHPs (aim 3.1.), healthy human adults (aim 3.2.1.) and children convalescent from an infection with an adenovirus and potentially AAV (aim 3.2.2). An additional puzzling finding from a clinical AAV gene transfer patient will be followed up in aim 3.2.3. In this trial a patient was treated by hepatic infusion of an AAV2-hF.IX vector while under Immunosuppression (IS). He developed an increase In circulating AAV capsid-specific T cells following gene transfer without showing evidence of liver cell destruction. The pathways underlying this increase of apparently dysfunctional AAV capsid-specific CDS* T cells will be elucidated further with samples from transplant patients taken before and/after initiation of IS.