CTL response to AAV Vector

CTL 对 AAV 载体的反应

• 批准号: 8071320
• 负责人: Chengwen Li
• 金额: $ 1.54万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-17 至 2010-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8071320
• 关键词: Adverse effects; Alanine; Animal Model; Antigen Presentation; Antigens; Attenuated; Binding; Bortezomib; Capsid; Capsid Proteins; Cell Line; Cell Nucleus; Cell surface; Cells; Clinical; Clinical Trials; Cross Presentation; Cytotoxic T-Lymphocytes; Data; Dendritic Cells; Dependovirus; Detection; Dose; Drug Formulations; Engineering; Ensure; Epitopes; Epstein-Barr Virus Nuclear Antigens; Failure; Gene Expression; Gene Transduction Agent; Genetic Transcription; Glycine; Goals; Hemophilia B; Hepatocyte; Human; Human Herpesvirus 4; Immune; Immune response; Immune system; Immunosuppression; Immunosuppressive Agents; In Vitro; Injection of therapeutic agent; Kinetics; Liver; MG132; MHC Class I Genes; Mediating; Memory; Monitor; Mus; Muscle; OVA-8; Pathway interactions; Peptides; Pharmaceutical Preparations; Proteasome Inhibitor; Proteins; Protocols documentation; Route; Safety; Serotyping; T-Lymphocyte; Testing; Therapeutic; Time; Viral Proteins; Virus; adeno-associated viral vector; base; cellular transduction; gene therapy; immunogenic; immunogenicity; improved; in vivo; inhibitor/antagonist; killings; mouse model; mutant; novel; novel strategies; pre-clinical; prevent; public health relevance; recombinant virus; research study; response; therapeutic gene; tool; trafficking; transgene expression; uptake; vector; vector genome

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.

描述（申请人提供）：腺相关病毒（AAV）是一种在临床前和临床试验中非常有前景的基因治疗载体。然而，最近的研究表明，AAV2 衣壳可以通过经典抗原呈递和交叉呈递途径诱导细胞毒性 T 淋巴细胞 (CTL) 反应，从而引起与 AAV 载体免疫反应相关的担忧。特别是，有人提出，衣壳特异性 CTL 消除了 AAV2 转导的肝细胞，并导致 B 型血友病临床试验中的治疗失败。该提案的目的是了解 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型免疫逃避策略来增强 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相矛盾（可能是由于小鼠 AAV 衣壳的免疫原性较差），我们将强免疫结构域 OVA 表位 SIINFEKL 整合到 AAV2 衣壳 (AAV2-OVA) 的 VP3 蛋白中。在用 AAV2-OVA 载体进行肝转导后，具有记忆 OVA CTL 的小鼠中转基因表达减少。在当前的提案中，我们将使用 AAV2-OVA 载体来研究体外和体内转导细胞中 AAV 衣壳交叉呈递的动力学和机制（目标 1 和 2）。通过这些研究，我们希望彻底表征小鼠中 CTL 对 AAV 衣壳蛋白的反应，从而更准确地代表在人类中获得的数据。 AAV 载体通过内体摄取结合在细胞表面后，AAV2 衣壳必须在载体基因组转录之前在通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反应，我们建议将 VIPR 整合到 AAV 衣壳中，逃避宿主 CTL 介导的 AAV 转导靶细胞的消除（目标 3） ）。通过将 VIPR 工程化到 AAV2 衣壳蛋白中，我们将确保仅在 AAV2 转导细胞中减弱抗原呈递，而不会对免疫系统产生系统性副作用（如免疫抑制药物或应用调节性 T 细胞的情况）。这些实验依赖于 AAV 衣壳蛋白中的预定结构域来掺入 VIPR 结构域。及时了解对于在当前方案（即没有免疫抑制附录）下继续使用 AAV 治疗至关重要。 公共健康相关性：简单总结 腺相关病毒 (AAV) 已用于 50 多项临床试验，并且由于 AAV 载体递送后可长期表达治疗性基因，因此被证明对于基因治疗非常有前景。最近，一项血友病 B 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。对这些矛盾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并建立合适的小鼠模型，我们建议将强免疫原插入AAV2衣壳中，并使用这些突变的AAV衣壳来制备重组病毒。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 是否消除小鼠体内 AAV2 转导的靶细胞。此外，为了减少 AAV 衣壳引起的任何免疫反应，我们将 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 感染细胞的免疫反应，并开发一种新型 AAV 载体，在不损害功能的情况下逃避免疫反应，从而改进 AAV 作为基因治疗工具并增强其治疗价值。