Novel AAV vector generation methods to prevent immunogenic unmethylated CpGs that trigger efficacy-limiting CTLs in human gene therapy

新型 AAV 载体生成方法可防止免疫原性未甲基化 CpG 触发人类基因治疗中功效限制的 CTL

• 批准号: 10452898
• 负责人: John Fraser Wright
• 金额: $ 19.68万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-15 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10452898
• 关键词: Address; Affinity Chromatography; Anabolism; Antibodies; Binding; Biological Models; Capsid; Cells; Clinical Data; Clinical Research; Clinical Trials; Codon Nucleotides; CpG dinucleotide; Cytosine; Cytotoxic T-Lymphocytes; DNA Packaging; DNA biosynthesis; Data; Dendritic Cells; Dependovirus; Dose; Endosomes; Eukaryota; Exposure to; FDA approved; Factor IX; Future; Gene Expression; Gene Transfer; Generations; Genetic Diseases; Genome; Goals; Hemophilia A; Hemophilia B; Human; Immune response; Immunity; Immunization; Immunologics; Immunosuppression; In Vitro; Infection; Inflammatory; Interferons; Investigation; Kinetics; Lead; Life; Lysosomes; Measures; Mediating; Methods; Methylation; Methyltransferase; Modeling; Modification; Molecular; Mus; Open Reading Frames; Outcome; Pathway interactions; Patients; Pattern; Peptides; Performance; Plasmid Cloning Vector; Plasmids; Predisposition; Process; Production; Prokaryotic Cells; Proteins; Quality Control; RPE65 protein; Recombinant adeno-associated virus (rAAV); Reporting; Retinal Diseases; Risk; Route; Safety; Serotyping; Spinal Muscular Atrophy; TLR9 gene; Testing; Therapeutic; Therapeutic Effect; Toxic effect; Transfection; Transferase; Transgenes; Ultracentrifugation; Variant; Viral Genome; Virion; adeno-associated viral vector; adverse outcome; base; bisulfite; cellular transduction; cesium chloride; clinical development; comparative; cross reactivity; cytokine; cytotoxic CD8 T cells; de-immunization; dimer; gene therapy; gene transfer vector; human DNA; immunogenic; immunotoxicity; improved; improved outcome; in vivo; in vivo Model; innate immune pathways; novel; novel strategies; pathogen; plasmid DNA; preclinical trial; prevent; primary outcome; product development; response; sensor; therapeutic gene; therapeutic transgene; transgene expression; treatment effect; vector; vector control; vector genome; viral genomics

SUMMARY Gene transfer vectors based on adeno-associated virus (AAV) have demonstrated safety and transformative therapeutic effects for several genetic diseases including RPE65-/- retinopathy (FDA-approved 2017), spinal muscular atrophy (FDA-approved, 2019), and hemophilia A and B (pivotal trials ongoing), validating their enormous potential. However, host immune responses remain a major barrier to successful AAV-based product development. Of particular concern, and the focus of this application, is the generation of capsid-specific, CD8+ cytotoxic T-lymphocytes (CTLs) following vector administration that can lead to inflammatory toxicities and loss of therapeutic transgene expression by destruction of vector-transduced cells. Loss of expression is a major problem because subjects exposed to an AAV investigational product develop high titer and broadly cross reactive AAV antibodies that preclude future administration of AAV-based therapeutics. New approaches, that prevent initial priming of the CTL response, are urgently needed. Previous reports support that AAV vector genome hypomethylation at the cytosine of CpG dinucleotides (MenegCpG) is a key trigger leading to formation of capsid specific CTLs. These unmethylated CpGs bind and dimerize Toll-like receptor 9 (TLR9) pathogen- associated molecular pattern (PAMP) sensor proteins present in the endosome / lysosome compartments of plasmacytoid dendritic cells (pDCs) to activate the MyD88 innate pathway, leading to the generation of inflammatory cytokines that trigger adaptive cellular immune responses. We will test whether increasing CpG methylation in AAV vector genomes is feasible and can correct this problem by eliminating the MenegCpG- associated PAMPs and thereby preventing the deleterious immune responses that reverse the initial therapeutic benefit achieved from AAV-mediated therapeutic gene transfer. We will use both human in vitro and murine in vivo model systems to evaluate the immunological effect of increasing MeposCpG in AAV vectors by either adding methyltransferase activity to HEK293 cells during the biosynthesis and packaging of vector genomes into AAV particles (Aim 1) or by methylating vector plasmid DNA prior to transfection and genome packaging into AAV particles in HEK293 cells (Aim 2). Our goal is to develop robust strategies that improve the durability and efficacy of AAV vector mediated transgene expression, thus leading to improved outcomes in clinical trials. A positive outcome of our approach will facilitate important improvements in methods to generate AAV vectors leading to their improved performance in human gene therapy. Most critically, will be reduced innate immune stimulation by these de-immunized AAV vectors to achieve durable therapeutic levels of gene expression in humans, which will lead directly into a larger proof of concept study (R01 or similar), and subsequent human clinical trials.

概括 基于腺相关病毒（AAV）的基因转移载体已证明安全性和变革性 多种遗传疾病的治疗作用，包括RPE65 - / - 视网膜病（FDA批准2017），脊柱 肌肉萎缩（FDA批准，2019年）和血友病A和B（正在进行的关键试验），验证了它们 巨大潜力。但是，宿主免疫反应仍然是成功基于AAV产品的主要障碍 发展。特别关注的是，本应用的重点是产生带帽特异性的CD8+ 载体给药后的细胞毒性T淋巴细胞（CTL）可能导致炎症毒性和损失 通过破坏载体转导的细胞的治疗转基因表达。表达丧失是主要的 问题是因为暴露于AAV研究产品的受试者会发展出高滴度并广泛交叉 反应性的AAV抗体，这些抗体排除了未来的基于AAV的治疗剂的抗体。新方法 迫切需要防止最初的CTL响应启动。以前的报告支持AAV矢量 CpG二核苷酸（MenegCPG）的胞嘧啶基因组低甲基化是一种关键触发因素，导致形成 capsid特异性CTL。这些未甲基化的CPG结合并二聚Toll样受体9（TLR9）病原体 - 存在于内体 /溶酶体室内的相关分子模式（PAMP）传感器蛋白 质子乳清细胞树突状细胞（PDC）激活MyD88先天途径，导致产生 引发适应性细胞免疫反应的炎性细胞因子。我们将测试是否增加CPG AAV载体基因组中的甲基化是可行的，可以通过消除Menegcpg-来纠正该问题 相关的弹丸，从而防止有害免疫反应扭转初始治疗 通过AAV介导的治疗基因转移获得的益处。我们将在体外和鼠类中同时使用人类 Vivo模型系统，以通过添加来评估增加AAV向量中MEPOSCPG的免疫学效果 在载体基因组的生物合成和包装中，甲基转移酶的活性在HEK293细胞中 转染前颗粒（AIM 1）或通过甲基化载体质粒DNA和基因组包装到AAV HEK293细胞中的颗粒（AIM 2）。我们的目标是制定可提高耐用性和功效的强大策略 AAV载体介导的转基因表达，从而导致临床试验的结果改善。积极 我们方法的结果将有助于改进方法，以产生AAV向量，从而导致 他们在人类基因疗法方面的表现提高。最关键的是，将减少先天免疫刺激 通过这些取消免疫的AAV载体，以实现人类中持久的基因表达水平 将直接进入更大的概念验证研究（R01或类似），然后进行随后的人类临床试验。