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.

项目摘要 医学目前正在经历一场革命，正在开发可行的基因疗法， 多种疾病，包括中枢神经系统（CNS）疾病。其中一个障碍，限制了 基因治疗的使用是安全有效的载体的可获得性，用于基因的广泛递送。由于其 稳定转基因表达、广泛嗜性和适度免疫原性重组腺相关病毒 重组腺相关病毒（rAAV）是用于人类基因治疗的最广泛使用的病毒载体。近200种rAAV疗法已经被应用于临床。 已完成或目前正在进行临床试验，包括两种FDA批准的治疗遗传性疾病的药物。 CNS。然而，越来越多的证据表明，基于rAAV的基因疗法并非没有毒性或重大风险， 仅在过去三年中就报告了几例rAAV相关死亡和许多不良结果。 在最近的一项Sanfilippo综合征试验中，1例患者死亡，其他患者在 rAAV注射部位在脑内，停止研究。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衰竭再到心肺功能不全。虽然其中一些 副作用被认为是由对AAV衣壳或转基因的免疫反应引起的， 有证据表明，rAAV基因组，其中包含两个145个碱基对的DNA片段命名为反向 末端重复序列（ITR）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， 分裂的神经前体细胞（NPC）和未成熟的神经元，完全消除了成年人的神经发生， 小鼠海马。与先前的工作一致，这些实验表明AAV ITR似乎 这是足够的和必要的毒性。在新的研究方向上，我们将利用我们的 在神经科学，干细胞生物学和工程学方面的互补专业知识，以开发rAAV的新方法 生产和创建第一个具有工程ITR的rAAV，这些ITR对于人类基因治疗更安全。这些新 在儿童神经发育和其他疾病的治疗中， 具有活跃的干/祖细胞增殖的人，所述干/祖细胞对rAAV毒性极其敏感。在 目前的建议旨在： 目标1.确定ITR DNA序列的哪些组分是NPC体内毒性所需的。 目标2.开发能够包装具有突变ITR的基因组的无细胞合成rAAV。 目标3.工程化rAAV，其将在Rett综合征的鼠模型中挽救功能丧失，同时 显示出比常规rAAV更低的毒性。