CTL response to AAV Vector

CTL 对 AAV 载体的反应

• 批准号: 7783065
• 负责人: Chengwen Li
• 金额: $ 36.68万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7783065
• 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）是临床前和临床试验中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，从而引起了与AAV载体免疫反应相关的关注。特别是，在血友病B临床试验中，有研究表明衣壳特异性ctl消除了AAV2转导的肝细胞并导致治疗失败。本研究的目的是了解AAV衣壳抗原在体外和体内的呈递机制，更重要的是制定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（可能是由于小鼠AAV衣壳的免疫原性较差），我们将一个强大的免疫结构域OVA表位SIINFEKL整合到AAV2衣壳（AAV2-OVA）的VP3蛋白中。用AAV2-OVA载体进行肝转导后，记忆性OVA ctl小鼠的转基因表达降低。在目前的提案中，我们将使用AAV2-OVA载体在体外和体内研究AAV衣壳在转导细胞中的交叉呈递动力学和机制（目的1和2）。通过这些研究，我们希望彻底表征小鼠对AAV衣壳蛋白的CTL反应，从而更准确地代表在人类中获得的数据。在AAV载体通过内体摄取与细胞表面结合后，AAV2衣壳必须在通往细胞核的途中脱壳，才能进行载体基因组转录。这种转运途径表明，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（如疱疹病毒、EB病毒）通过合成称为病毒蛋白干扰抗原呈递（VIPR）的小肽逃避宿主免疫应答，我们提出VIPR整合到AAV衣壳逃避宿主CTL介导的AAV转导靶细胞的消除（Aim 3）。通过将vipr改造成AAV2衣壳蛋白，我们将确保抗原呈递仅在AAV2转导的细胞中减弱，而不会对免疫系统产生全身性副作用（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衣壳蛋白中预先确定的结构域来整合VIPR结构域。及时的了解对于在当前方案下继续使用AAV治疗（即没有免疫抑制附录）至关重要。