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

基本信息

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
项目类别：
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

项目摘要

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.

项目概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。限制的障碍之一基因治疗的使用是指提供安全有效的载体来广泛传递基因。由于其稳定的转基因表达、广泛的趋向性和适度的免疫原性，重组腺相关病毒 (rAAV) 是人类基因治疗中使用最广泛的病毒载体。已有近 200 种 rAAV 疗法已完成或目前正在进行临床试验，包括 FDA 批准的两种遗传性疾病疗法中枢神经系统。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起与 rAAV 相关的死亡事件和大量不良后果。在最近的一项 Sanfilippo 综合征试验中，1 名患者死亡，其他患者则表现出 MRI 变化 rAAV 注射部位位于大脑内，导致研究停止。其他 rAAV 试验报告了严重的不良反应从血小板减少症到急性肾衰竭再到心肺功能不全。虽然其中一些不良反应被认为是由 AAV 衣壳或转基因的免疫反应引起的，增加了有证据表明，rAAV 基因组包含两个 145 碱基对的 DNA 片段，名为倒置末端重复序列（ITR）是rAAV毒性的主要来源。在进行学习和记忆基础实验时，我们发现rAAV有毒分裂神经祖细胞（NPC）和未成熟神经元，完全消除成年神经发生小鼠海马体。与之前的工作一致，这些实验表明 AAV ITR 似乎对于这种毒性来说是充分且必要的。踏上新的研究方向，我们将利用我们的神经科学、干细胞生物学和工程学方面的互补专业知识，用于开发 rAAV 新方法生产并创建第一个带有工程 ITR 的 rAAV，对人类基因治疗更安全。这些新疗法对于治疗儿童神经发育疾病和其他疾病尤为重要干细胞/祖细胞增殖活跃，对 rAAV 毒性极其敏感。在目前的提案我们的目标是：目标 1. 确定 ITR DNA 序列的哪些成分是 NPC 体内毒性所必需的。目标 2. 开发一种能够用突变 ITR 包装基因组的无细胞合成 rAAV。目标 3. 设计一种 rAAV，以挽救 Rett 综合征小鼠模型的功能丧失，同时表现出比传统 rAAV 更低的毒性。