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（由申请人提供）：在临床前和临床试验中，腺相关病毒（AAV）是非常有前途的基因治疗载体。但是，最近的研究表明，AAV2衣壳可以通过经典抗原表现和交叉呈递途径引起细胞毒性T淋巴细胞（CTL）反应，从而引发与AAV载体免疫反应有关的关注点。特别是，已经提出，衣壳特异性CTL消除了AAV2转导的肝细胞，并在血友病B临床试验中导致治疗衰竭。该提案的目的是了解体外和体内AAV CAPSID抗原的表现机制，更重要的是，制定了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矢量的安全性和功效。由于来自动物模型的数据已与上述临床观察结果相矛盾（可能是由于小鼠AAV Capsid的免疫原性差），因此我们将强大的免疫结构域OVA Epatope siinfekl整合到了AAV2 CAPSID（AAV2-ova）的VP3蛋白中。在用AAV2-OVA载体转导后，在记忆OVA CTL的小鼠中，转基因表达降低。在当前的提案中，我们将使用AAV2-OVA载体研究体外和体内转导细胞中AAV Capsid交叉散发的动力学和机制（AIM 1和2）。通过这些研究，我们希望在小鼠中更准确地代表人类获得的数据，以彻底表征对小鼠AAV衣壳蛋白的CTL反应。 在AAV载体通过内体摄取在细胞表面上结合后，在载体基因组转录之前，AAV2衣壳必须不向核纳入细胞核。这种贩运途径表明，转导后的衣壳的抗原表现可能遵循经典的MHC级I途径。许多病毒（例如，疱疹，EB）通过合成称为病毒蛋白的小肽来逃避宿主免疫反应，干扰了抗原呈现（VIPR），我们建议将VIPR整合到AAV Capsid evade host CTL介导的AAV介导的AAV转移靶细胞中的消除（AIM AIM 3）。通过将VIPR工程化为AAV2衣壳蛋白，我们将确保仅在AAV2转导的细胞中减弱抗原表现，而不会对免疫系统进行全身副作用（与免疫抑制药物或调节剂T细胞的应用一样）。这些实验依赖于AAV CAPSID蛋白中的预定域来掺入VIPR结构域。及时的理解对于在当前方案（即没有免疫抑制附录）下继续使用AAV治疗至关重要。 公共卫生相关性：分析腺相关病毒（AAV）已在50多次临床试验中使用，并且由于递送AAV载体后的长期治疗基因表达，对基因治疗非常有前途。最近，来自一项血友病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。但是，小鼠模型研究的结果不支持这些发现。对这些矛盾结果的一种解释可能是小鼠AAV衣壳的免疫原性弱。为了更准确地模仿临床试验并建立适当的小鼠模型，我们建议将强力免疫原插入AAV2 CAPSID中，并使用这些突变体AAV帽固体制成重组病毒。将这些病毒注射到小鼠中后，我们将研究免疫原的特异性CTL是否消除了AAV2转导的小鼠靶细胞。此外，为了减少AAV Capsid引起的任何免疫反应，我们将将Epstein-Barr病毒产物EBNA-1插入AAV Capsid的非必需区域。我们将测试EBNA-1是否会介导对免疫反应的抑制并防止消除AAV感染细胞。该提案的长期目标是批判性地评估对AAV感染细胞的免疫反应，并开发出一种新型的AAV载体，该载体将在不损害功能的情况下逃避免疫反应，从而改善AAV作为基因治疗工具并增强其治疗价值。