Developing a Synthetic Adeno-Associated Virus (AAV) for Engineering Safer Gene Therapies

开发合成腺相关病毒（AAV）以设计更安全的基因疗法

• 批准号: 10629902
• 负责人: Matthew Shtrahman
• 金额: $ 42万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10629902
• 关键词: Ablation; Acute Kidney Failure; Adverse effects; Attenuated; Base Pairing; Brain; Capsid; Cardiopulmonary; Cell Death; Cells; Central Nervous System Diseases; Cessation of life; Child; Clinical Trials; DNA; DNA Damage; DNA Sequence; Dependovirus; Disease; Electroporation; Embryo; Engineering; FDA approved; Functional disorder; Gene Delivery; Genes; Genetic Diseases; Genome; Hippocampus; Histology; Human; Injections; Inverted Terminal Repeat; Learning; Learning Disabilities; Magnetic Resonance Imaging; Mediating; Medicine; Memory; Methods; Methyl-CpG-Binding Protein 2; Mucopolysaccharidosis III; Mus; Mutation; Names; Neurons; Neurosciences; Nucleotides; Pathologic; Patients; Production; Proteins; Publishing; R7 Virus; RNA; Recombinant adeno-associated virus (rAAV); Recombinants; Reporting; Research; Rett Syndrome; Risk; Single-Stranded DNA; Site; Source; Structure; Symptoms; Terminal Repeat Sequences; Testing; Therapeutic; Thrombocytopenia; Toxic effect; Transgenes; Tropism; Viral; Viral Genome; Viral Vector; Virus; Work; adult neurogenesis; adverse outcome; c9FTD/ALS; digital; experimental study; gene product; gene therapy; immunogenicity; immunoreaction; in utero; in vivo; loss of function; mouse model; mutant; nerve stem cell; novel therapeutics; response; stem cell biology; stem cell proliferation; transgene expression; vector

PROJECT SUMMARY Medicine is currently undergoing a revolution, where viable gene therapies are being developed for multiple disorders, including diseases of the central nervous system (CNS). One of the obstacles that limits the use of gene therapy is the availability of safe and effective vectors for widespread delivery of genes. Due to its stable transgene expression, broad tropism, and modest immunogenicity, recombinant adeno-associated virus (rAAV) is the most widely used viral vector for human gene therapy. Almost 200 rAAV therapies have been completed or are currently in clinical trials, including two FDA-approved therapies for genetic diseases of the CNS. However, evidence is mounting that rAAV-based gene therapies are not without toxicity or significant risk, with several rAAV-related deaths and numerous adverse outcomes reported during the past three years alone. In a recent trial for Sanfilippo syndrome, 1 patient died and others demonstrated concerning MRI changes at rAAV injection sites within the brain, halting the study. Other rAAV trials have reported serious adverse effects ranging from thrombocytopenia to acute kidney failure to cardio-pulmonary insufficiency. While some of these adverse effects are thought to be caused by immune reactions to the AAV capsid or transgene, increasing evidence indicates that the rAAV genome, which contains two 145-base pair DNA segments named inverted terminal repeats (ITRs), is a major source of rAAV toxicity. While conducting fundamental experiments on learning and memory, we discovered that rAAV was toxic to dividing neural progenitor cells (NPCs) and immature neurons, completely ablating adult neurogenesis in the mouse hippocampus. Consistent with previous work, these experiments indicate that the AAV ITRs appear to be sufficient and necessary for this toxicity. Embarking on a new research direction, we will utilize our complimentary expertise in neuroscience, stem cell biology, and engineering to develop new methods for rAAV production and create the first rAAVs with engineered ITRs that are safer for human gene therapy. These new therapies will be particularly important in the treatment of neurodevelopmental and other diseases in children who have active proliferation of stem/progenitor cells, which are exquisitely sensitive to rAAV toxicity. In the current proposal we aim to: Aim 1. Determine which components of the ITR DNA sequence are required for toxicity in NPCs in vivo. Aim 2. Develop a cell-free synthetic rAAVs capable of packaging genomes with mutant ITRs. Aim 3. Engineer an rAAV that will rescue loss of function in a murine model of Rett syndrome while demonstrating less toxicity than conventional rAAVs.

项目总结 医学目前正在经历一场革命，正在开发可行的基因疗法来治疗 多发性疾病，包括中枢神经系统疾病。限制美国经济发展的障碍之一 基因治疗的使用是安全和有效的载体的可用性，以广泛传递基因。由于其 转基因表达稳定、嗜性广、免疫原性适中的重组腺相关病毒 重组腺相关病毒(RAAV)是目前应用最广泛的人类基因治疗病毒载体。近200种rAAV疗法已经被 已完成或目前正在进行临床试验，包括FDA批准的两种治疗老年人遗传病的方法 中枢神经系统。然而，越来越多的证据表明，基于rAAV的基因治疗并不是没有毒性或重大风险， 仅在过去三年中就报告了几例与rAAV相关的死亡和许多不良后果。 在最近的一项关于Sanfilippo综合征的试验中，1名患者死亡，其他人表现出与MRI的变化有关 RAAV注射部位在大脑内，暂停了研究。其他rAAV试验报告了严重的不良反应。 从血小板减少到急性肾功能衰竭再到心肺功能不全。而其中的一些 不良反应被认为是由对AAV衣壳或转基因的免疫反应引起的， 证据表明，rAAV基因组包含两个145碱基对的DNA片段，命名为Inverted 末端重复序列(ITRs)是rAAV毒性的主要来源。 在进行学习和记忆的基础实验时，我们发现rAAV是有毒的 分裂神经前体细胞和未成熟神经元，完全消融成体神经发生 小鼠海马体。与以前的工作一致，这些实验表明AAV ITR似乎 对于这种毒性是充分和必要的。在一个新的研究方向上，我们将利用我们的 免费提供神经科学、干细胞生物学和工程学方面的专业知识，以开发rAAV的新方法 生产并创造第一个具有对人类基因治疗更安全的工程ITR的rAAVs。这些新的 治疗方法在儿童神经发育和其他疾病的治疗中将特别重要。 干细胞/祖细胞增殖活跃，对rAAV毒性非常敏感。在 我们目前的建议目标是： 目的1.确定在体内对鼻咽癌细胞的毒性所需的ITR DNA序列的哪些成分。 目的2.开发一种无细胞合成rAAVs，该rAAVs能够包装带有突变的ITR的基因组。 目的3.设计一种rAAV，它将挽救Rett综合征小鼠模型的功能丧失，同时 显示出比传统rAAVs毒性更小的特点。