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.

摘要 基于腺相关病毒(AAV)的基因转移载体已被证明是安全和转化的 对几种遗传性疾病的治疗效果，包括RPE65-/-视网膜病(FDA批准的2017年)，脊柱 肌肉萎缩(FDA批准，2019年)，以及血友病A和B(关键试验正在进行中)，验证了他们的 巨大的潜力。然而，宿主免疫反应仍然是基于AAV产品成功的主要障碍 发展。尤其值得关注的是，这一应用的焦点是产生衣壳特异的CD8+ 载体注射后的细胞毒性T淋巴细胞(CTL)可导致炎症毒性和丧失 通过破坏载体转导的细胞进行治疗性转基因表达。表达能力的丧失是一个主要问题 问题是，暴露于AAV研究产品的受试者会出现高滴度和广泛交叉 反应性AAV抗体，排除了未来以AAV为基础的治疗方法。新方法，那就是 防止初始启动的CTL反应，是迫切需要的。之前的报道支持AAV载体 CpG二核苷酸胞嘧啶(MenegCpG)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CPGS结合和二聚化Toll样受体9(TLR9)病原体- 相关分子模式(PAMP)感受器蛋白存在于内体/溶酶体隔室 浆细胞样树突状细胞(PDCs)激活MyD88天然途径，导致 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，并可以通过消除MenegCpG- 相关的PAMP，从而防止有害的免疫反应，逆转最初的治疗 从AAV介导的治疗性基因转移中获益。我们将在体外同时使用人类和小鼠 体内模型系统评估通过添加以下任一种方法在AAV载体中增加MeposCpG的免疫学效果 载体基因组生物合成和包装过程中对HEK293细胞甲基转移酶活性的影响 颗粒(目标1)或通过甲基化载体载体DNA在转染和基因组包装到AAV之前 HEK293细胞中的颗粒(目标2)。我们的目标是制定强有力的战略，提高耐用性和有效性 AAV载体介导的转基因表达的改善，从而导致临床试验结果的改善。积极的一面 我们的方法的结果将促进生成AAV矢量的方法的重要改进，从而导致 它们在人类基因治疗中的改进表现。最关键的是，会减少先天免疫刺激 通过这些去免疫的AAV载体在人类体内实现持久的治疗水平的基因表达，这 将直接导致更大的概念验证研究(R01或类似)，以及随后的人体临床试验。