CTL response to AAV Vector

说明书(申请人提供)：腺相关病毒(AAV)在临床前和临床试验中是一种非常有前途的基因治疗载体。然而，最近的研究表明，AAV2衣壳可以通过经典的抗原提呈和交叉提呈途径诱导细胞毒性T淋巴细胞(CTL)反应，从而引起人们对AAV载体免疫应答的关注。特别是，在血友病B临床试验中，有人认为衣壳特异性CTL消除了AAV2转导的肝细胞，并导致治疗失败。这项建议的目的是了解AAV衣壳抗原在体外和体内的呈递机制，更重要的是，设计逃避免疫反应的策略。我们的长期目标是通过制定新的免疫逃避策略来提高AAV载体的安全性和有效性。由于动物模型的数据与上述临床观察结果相矛盾(可能是由于AAV衣壳在小鼠中的免疫原性较差)，我们将一个强大的免疫结构域OVA表位SIINFEKL整合到AAV2衣壳的VP3蛋白中(AAV2-OVA)。AAV2-OVA载体肝转导记忆性OVA CTL小鼠后，转基因表达降低。在目前的方案中，我们将使用AAV2-OVA载体来研究AAV衣壳在体外和体内转导细胞中交叉呈递的动力学和机制(目标1和2)。通过这些研究，我们希望彻底描述小鼠对AAV衣壳蛋白的CTL反应，以更准确地代表在人类中获得的数据。AAV载体结合到细胞表面后，通过内体摄取，在载体基因组转录之前，AAV2衣壳必须在前往细胞核的途中脱壳。这一转运途径表明，衣壳蛋白转导后的抗原提呈可能遵循经典的MHC-I类途径。许多病毒(如疱疹、EB)通过合成称为干扰抗原提呈(VIPR)的病毒蛋白来逃避宿主免疫反应，我们提出将VIPR整合到AAV衣壳中可以逃避宿主CTL介导的AAV转导靶细胞的消除(AIM 3)。通过将VIPRs基因工程到AAV2衣壳蛋白中，我们将确保只在AAV2转导细胞中减弱抗原递呈，而不会对免疫系统产生系统性副作用(就像免疫抑制药物或调节性T细胞的应用一样)。这些实验依赖于AAV衣壳蛋白中的预定结构域来整合VIPR结构域。及时了解对于在当前方案(即不使用免疫抑制附录)下继续使用AAV疗法至关重要。 公共卫生相关性：腺相关病毒(AAV)已被用于50多个临床试验，并被证明是非常有希望的基因治疗，因为AAV载体交付后，长期的治疗性基因表达。最近，一项针对血友病B的临床试验的数据表明，AAV2可以诱导免疫反应，从而消除AAV2感染的肝细胞。然而，小鼠模型研究的结果并不支持这些发现。对这些相互矛盾的结果的一种解释可能是AAV衣壳在小鼠体内的免疫原性较弱。为了更准确地模拟临床试验，建立合适的小鼠模型，我们建议在AAV2衣壳中插入一种强大的免疫原，并用这些突变的AAV衣壳制作重组病毒。将这些病毒注射到小鼠体内后，我们将研究免疫原特异性CTL是否能消除AAV2转导的小鼠靶细胞。此外，为了减少AAV衣壳引起的任何免疫反应，我们将把Epstein-Barr病毒产物EBNA-1插入AAV衣壳的非必需区域。我们将测试EBNA-1是否会介导免疫反应的抑制和防止AAV感染细胞的根除。这项建议的长期目标是严格评估AAV感染细胞的免疫反应，并开发一种新型的AAV载体，在不影响功能的情况下逃避免疫反应，从而改进AAV作为基因治疗工具，提高其治疗价值。

DESCRIPTION (provided by applicant): Adeno-associated virus (AAV) is a very promising gene therapy vector in pre-clinical and clinical trials. However, recent studies have demonstrated that the AAV2 capsid can induce a cytotoxic T lymphocyte (CTL) response via both classical antigen presentation and cross-presentation pathways, thereby raising concerns associated with immune response to AAV vectors. In particular, it has been suggested that capsid specific CTLs eliminated AAV2 transduced liver cells and resulted in therapeutic failure in a hemophilia B clinical trial. The goal of this proposal is to understand the mechanisms of presentation of AAV capsid antigens in vitro and in vivo and more importantly, to devise strategies to evade the immune response. Our long term goals are to enhance the safety and efficacy of AAV vectors through formulation of novel immune evasion strategies. Since data from animal models have contradicted clinical observations outlined above (possibly due to poor immunogenicity of the AAV capsid in mice), we have integrated a strong immune domain OVA epitope SIINFEKL into the VP3 protein of the AAV2 capsid (AAV2-OVA). Decreased transgene expression was seen in mice with memory OVA CTLs following liver transduction with AAV2-OVA vector. In the current proposal, we will use AAV2-OVA vector to investigate the kinetics and mechanisms of AAV capsid cross-presentation in transduced cells in vitro and in vivo (Aim 1 and 2). Through these studies, we expect to thoroughly characterize the CTL response to AAV capsid proteins in mice that more accurately represents data obtained in humans. After AAV vector binds on cell surface, via endosomal uptake, AAV2 capsids must uncoat enroute to the nucleus prior to vector genome transcription. This trafficking route suggests that antigen presentation of capsids after transduction may follow a classical MHC-class I pathways. Many viruses (For example herpes, EB) evade host immune response by synthesizing small peptides called viral proteins interfering with antigen presentation (VIPR), we propose that integration of VIPR into AAV capsid evade host CTL mediated elimination of AAV transduced target cells (Aim 3). By engineering VIPRs into AAV2 capsid proteins, we will ensure that antigen presentation will be attenuated only in AAV2 transduced cells without systemic side effects on the immune system (as would be the case with immunosuppressive drugs or application of regulator T cells). These experiments rely on predetermined domains in AAV capsid proteins for incorporation of VIPR domains. A timely understanding is critical for the continued use of AAV therapy under the current protocols (i.e. without immunosuppression addendums). PUBLIC HEALTH RELEVANCE: Lay summary Adeno-associated virus (AAV) has been used in over 50 clinical trials and proves to be very promising for gene therapy, due to long-term therapeutic gene expression after delivery of AAV vectors. Recently, data from one clinical trial for hemophilia B suggested that AAV2 could induce an immune response and thus eliminate AAV2 infected liver cells. However, the results from a mouse model study did not support these findings. One explanation of these contradictory results could be the weak immunogenic activity of AAV capsids in mice. To more accurately mimic the clinical trial and establish an appropriate mouse model, we propose to insert a strong immunogen into AAV2 capsid and use these mutant AAV capsids to make recombinant virus. After injection of these viruses into mouse, we will investigate whether immunogen specific CTLs eliminate AAV2 transduced target cells in mice. Additionally, to reduce any immune response elicited by the AAV capsid, we will insert the Epstein-Barr virus product EBNA-1 into a non-essential region of the AAV capsid. We will test whether EBNA-1 will mediate inhibition of an immune response and prevent eradication of AAV infected cells. The long-term goals of this proposal are to critically evaluate the immune response to AAV-infected cells and to develop a novel AAV vector that will evade an immune response without compromising function, thus improving AAV as a gene therapy tool and enhancing its therapeutic value.