CTL response to AAV Vector

描述（由申请人提供）：腺相关病毒（AAV）是一种非常有前途的基因治疗载体，正在进行临床前和临床试验。然而，最近的研究表明，AAV 2衣壳可以通过经典的抗原呈递和交叉呈递途径诱导细胞毒性T淋巴细胞（CTL）应答，从而引起了与对AAV载体的免疫应答相关的担忧。特别地，已经表明衣壳特异性CTL消除了AAV 2转导的肝细胞，并导致血友病B临床试验中的治疗失败。该提案的目标是了解AAV衣壳抗原在体外和体内的呈递机制，更重要的是，设计逃避免疫应答的策略。我们的长期目标是通过制定新的免疫逃避策略来增强AAV载体的安全性和有效性。由于来自动物模型的数据与上述临床观察结果相矛盾（可能是由于小鼠中AAV衣壳的免疫原性差），我们将强免疫结构域OVA表位SIINFEKL整合到AAV 2衣壳（AAV 2-OVA）的VP 3蛋白中。在用AAV 2-OVA载体肝转导后，在具有记忆性OVA CTL的小鼠中观察到转基因表达降低。在本提案中，我们将使用AAV 2-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（目的1和2）。通过这些研究，我们期望彻底表征小鼠中对AAV衣壳蛋白的CTL应答，从而更准确地代表在人类中获得的数据。 在AAV载体通过内体摄取结合在细胞表面上之后，在载体基因组转录之前，AAV 2衣壳必须在去往细胞核的途中脱壳。这种运输途径表明，转导后衣壳的抗原呈递可能遵循经典的MHC I类途径。许多病毒（例如疱疹病毒，EB）通过合成称为干扰抗原呈递的病毒蛋白（VIPR）的小肽来逃避宿主免疫应答，我们提出将VIPR整合到AAV衣壳中逃避宿主CTL介导的AAV转导的靶细胞的消除（目的3）。通过将VIPRs工程化到AAV 2衣壳蛋白中，我们将确保抗原呈递将仅在AAV 2转导的细胞中减弱，而对免疫系统没有全身性副作用（如免疫抑制药物或调节T细胞的应用的情况）。这些实验依赖于AAV衣壳蛋白中的预定结构域用于掺入VIPR结构域。及时了解对于在当前方案下继续使用AAV治疗至关重要（即没有免疫抑制补充）。 公共卫生相关性：腺相关病毒（AAV）已被用于超过50个临床试验，并被证明是非常有前途的基因治疗，由于长期的治疗基因表达后，交付的AAV载体。最近，来自血友病B的一项临床试验的数据表明，AAV 2可以诱导免疫应答，从而消除AAV 2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。这些矛盾结果的一种解释可能是小鼠中AAV衣壳的免疫原性活性弱。为了更准确地模拟临床试验并建立合适的小鼠模型，我们建议将强免疫原插入AAV 2衣壳并使用这些突变的AAV衣壳制备重组病毒。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是否消除小鼠中的AAV 2转导的靶细胞。此外，为了减少由AAV衣壳引起的任何免疫应答，我们将EB病毒产物EBNA-1插入AAV衣壳的非必需区。我们将测试EBNA-1是否会介导免疫应答的抑制并阻止AAV感染细胞的根除。该提案的长期目标是严格评估对AAV感染细胞的免疫应答，并开发一种新的AAV载体，该载体将逃避免疫应答而不损害功能，从而改善AAV作为基因治疗工具并提高其治疗价值。

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.