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: 10620770
• 负责人: John Fraser Wright
• 金额: $ 25.08万
• 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 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10620770
• 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; Dimerization; 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; Immunologic Stimulation; Immunologics; Immunosuppression; In Vitro; Infection; Inflammatory; Interferons; Kinetics; Lead; Life; Lysosomes; Measures; Mediating; Methods; Methylation; Methyltransferase; 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; Transduction Gene; Transfection; Transferase; Transgenes; Transgenic Model; Ultracentrifugation; Variant; Viral Genome; Virion; adeno-associated viral vector; adverse outcome; bisulfite; cellular transduction; cesium chloride; clinical development; comparative; comparison control; cross reactivity; cytokine; cytotoxic CD8 T cells; de-immunization; 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; seroconversion; 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）的基因转移载体已被证明安全性和变革性 对多种遗传性疾病有治疗作用，包括 RPE65-/- 视网膜病（2017 年 FDA 批准）、脊髓病 肌萎缩症（FDA 批准，2019 年）以及血友病 A 和 B（关键试验正在进行中），验证了它们的疗效 巨大的潜力。然而，宿主免疫反应仍然是基于 AAV 的产品成功的主要障碍 发展。特别值得关注的也是本应用的重点是衣壳特异性 CD8+ 的生成 载体施用后的细胞毒性 T 淋巴细胞 (CTL) 可导致炎症毒性和损失 通过破坏载体转导的细胞来实现治疗性转基因表达。表达能力丧失是一个重大问题 问题是因为暴露于 AAV 研究产品的受试者会产生高滴度并广泛交叉 反应性 AAV 抗体阻止未来基于 AAV 的治疗的施用。新方法，即 防止 CTL 反应的初始启动是迫切需要的。之前的报告支持 AAV 载体 CpG 二核苷酸 (MenegCpG) 胞嘧啶的基因组低甲基化是导致形成的关键触发因素 衣壳特异性 CTL。这些未甲基化的 CpG 结合并二聚化 Toll 样受体 9 (TLR9​​) 病原体 相关分子模式（PAMP）传感器蛋白存在于内体/溶酶体区室中 浆细胞样树突状细胞 (pDC) 激活 MyD88 先天通路，从而产生 触发适应性细胞免疫反应的炎症细胞因子。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，并且可以通过消除 MenegCpG- 来纠正这个问题。 相关的 PAMP，从而防止逆转初始治疗的有害免疫反应 从 AAV 介导的治疗性基因转移中获得的益处。我们将在体外使用人类和小鼠 体内模型系统通过添加来评估增加 AAV 载体中 MeposCpG 的免疫效果 在载体基因组生物合成和包装到 AAV 过程中 HEK293 细胞的甲基转移酶活性 颗粒（目标 1）或在转染和基因组包装到 AAV 之前甲基化载体质粒 DNA HEK293 细胞中的颗粒（目标 2）。我们的目标是制定强有力的策略来提高耐久性和功效 AAV 载体介导的转基因表达，从而改善临床试验的结果。积极的 我们的方法的结果将促进生成 AAV 载体的方法的重要改进，从而导致 他们在人类基因治疗中的表现得到了改善。最关键的是，先天免疫刺激会减少 通过这些去免疫的 AAV 载体，可以在人类中实现持久的治疗水平的基因表达，这 将直接导致更大规模的概念验证研究（R01 或类似研究）以及后续的人体临床试验。