CTL response to AAV Vector

CTL 对 AAV 载体的反应

• 批准号: 8384860
• 负责人: Chengwen Li
• 金额: $ 34.13万
• 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/8384860
• 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).

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）。