Establishing and Optimizing a Prime Editing Method in Neurons for Treatment of Rett Syndrome

建立和优化用于治疗 Rett 综合征的神经元素数编辑方法

• 批准号: 10607549
• 负责人: David G. Keener
• 金额: $ 3.25万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-15 至 2026-01-14
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607549
• 关键词: Affect; Age Months; Binding; Bioinformatics; Biological Assay; Biology; Brain; CRISPR/Cas technology; Cells; Chemicals; Chemistry; Chimeric Proteins; Chromatin; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; DNA Integration; DNA Methylation; DNA delivery; DNA sequencing; Defect; Development; Effectiveness; Encapsulated; Engineering; Flow Cytometry; Functional disorder; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genomic DNA; Guide RNA; Impairment; In Vitro; Induced pluripotent stem cell derived neurons; Infant; Interphase Cell; Length; Lentivirus; Ligation; Liver; Luciferases; Measures; Mediating; Messenger RNA; Methods; Methyl-CpG-Binding Protein 2; Missense Mutation; Modification; Molecular; Mus; Mutation; Neurites; Neurobiology; Neurodevelopmental Disorder; Neurologic; Neuronal Differentiation; Neurons; Nonsense Mutation; Nuclear; Nucleic Acids; Patients; Pattern; Phenotype; Primer Extension; Productivity; Quality of life; RNA Sequences; RNA chemical synthesis; RNA-Directed DNA Polymerase; Reading Frames; Regulator Genes; Research; Rett Syndrome; Severities; Symptoms; System; Technology; Testing; Therapeutic; Time; Training; Transfection; Viral Vector; Work; career; chemical synthesis; curative treatments; design; flexibility; genome sequencing; immunogenic; improved; in vivo; induced pluripotent stem cell; insight; lipid nanoparticle; loss of function mutation; mRNA delivery; model organism; molecular phenotype; mutant; nervous system disorder; neurite growth; neuron development; novel; nuclease; postmitotic; prime editing; prime editor; technology platform; transcriptome; whole genome

PROJECT SUMMARY Infants with Rett syndrome (Rett) are born with loss-of-function mutations in the gene encoding MeCP2, a global regulator of gene expression. MeCP2 dysfunction in the brain severely affects neurons, leading to developmental deficits of varying severity that manifest after 6 months of age. Current treatments can manage some symptoms, but correcting MECP2 mutations would more effectively restore patients’ quality of life. CRISPR gene editing has made this approach conceivable. Among CRISPR technologies, prime editing is the most flexible, utilizing an RNA-guided Cas9 nuclease fused to reverse transcriptase to “search and replace” mutations in post-mitotic cells. Thus, prime editing is a strong candidate for Rett treatment. Yet, prime editor has only been delivered to neurons via lentivirus (not clinically relevant), and its editing efficiency in cells is low. Previous work demonstrates that delivery of Cas9 mRNA encapsulated in lipid nanoparticles (LNP) is simple, safe, and supports robust editing in mouse liver. LNP-encapsulated mRNA also delivers to brain, but delivery of prime editor mRNA and efficiency of prime editing in neurons remains untested. In addition, chemically modifying the guide RNA of other CRISPR systems can protect against nuclease-mediated degradation and improve gene editing rates in cells. The Watts lab recently developed a method to synthesize chemically modified prime editing guide RNA (pegRNA), something that was considered unfeasible due to the length of pegRNA (~150 nt). The effect of pegRNA modification on prime editing efficiency has not yet been tested. With support from Drs. Jonathan Watts (nucleic acid chemistry), Michael Green (Rett neurobiology), Erik Sontheimer (prime editor biology), Scot Wolfe (gene regulation), and Athma Pai (bioinformatics), this project seeks to establish and chemically optimize mRNA-based prime editors to correct MECP2 mutations and reverse their phenotypes in neurons. Aim 1 will establish baseline effectiveness of mRNA-delivered prime editor (vs. lentiviral) against the most common Rett mutation (a missense mutation) and two clinically severe nonsense mutations in HEK cells expressing each mutant MeCP2, patient-derived induced pluripotent stem cells (iPSCs), and iPSC-derived neurons. This Aim will also probe neurons with and without editing to understand the molecular phenotypes of each MECP2 mutation and extent to which editing reverses them. Aim 2 will iterate on the Watts lab’s pegRNA assembly method to optimize pegRNA yield and synthesis time, and identify editing-compatible pegRNA modification patterns using in vitro and in cellulo assays. The effect of pegRNA modifications on MECP2 editing will be tested and optimized in HEK cells, iPSCs, and iPSC-derived neurons, as in Aim 1. Molecular phenotypes of prime edited vs. unedited neurons will also be characterized as in Aim 1. This work will offer insight into how MeCP2 mutants affect severity of Rett phenotypes in neurons and inform development of a prime-editing platform to treat any form of Rett as well as other neurological disorders. The training provided from this research will prepare the fellow for a productive career in the gene editing and neuro-therapeutics field.

项目摘要 RETT综合征（RETT）的婴儿天生具有编码MECP2基因的功能丧失突变，这是全球 基因表达的调节剂。大脑中的MECP2功能障碍严重影响神经元，导致发育 当前治疗可以管理一些符号， 但是，纠正MECP2突变将更有效地恢复患者的生活质量。 CRISPR基因编辑有 使这种方法可以想象。在CRISPR技术中，主要编辑是最灵活的 RNA引导的CAS9核酸酶融合到逆转录酶中，以“搜索和替换”有丝分裂细胞中的突变。 那是主要的编辑是RETT治疗的有力候选者。但是，主要编辑只交付给神经元 通过慢病毒（与临床无关），其细胞的编辑效率很低。 以前的工作表明，封装在脂质纳米颗粒（LNP）中的Cas9 mRNA的递送很简单， 安全，并支持小鼠肝脏中的强大编辑。 LNP封装的mRNA也会传递给大脑，但交付 主要编辑mRNA和神经元中的主要编辑效率尚未经过测试。另外，化学修饰 其他CRISPR系统的指南RNA可以预防核酸酶介导的降解并改善基因 细胞中的编辑速率。 Watts Lab最近开发了一种合成化学修改的Prime编辑的方法 引导RNA（Pegrna），由于Pegrna的长度（〜150 nt）而被认为是不可行的。 Pegrna修饰对主要编辑效率的影响尚未测试。 在博士的支持下。乔纳森·瓦茨（Jonathan Watts）（核酸化学），迈克尔·格林（Rett Neurobiology），Erik Sontheimer（Prime Editor Biology），Scot Wolfe（基因调节）和Athma Pai（生物信息学），该项目 寻求建立并化学优化基于mRNA的主要编辑器以纠正MECP2突变并反向 它们在神经元中的表型。 AIM 1将建立mRNA递送的Prime编辑器的基线有效性（VS。 针对最常见的RETT突变（错义突变）和两个临床上严重的胡说八道的慢病毒） 表达每个突变体MECP2的HEK细胞中的突变，患者衍生的诱导多能干细胞（IPSC）， 和IPSC衍生的神经元。该目标还将在没有编辑的情况下探测神经元以了解分子 每个MECP2突变的表型和编辑的程度逆转它们。 AIM 2将在瓦特上迭代 实验室的Pegrna组装方法优化Pegrna产量和合成时间，并确定兼容编辑 使用体外和纤维素测定中的Pegrna修饰模式。 PEGRNA修饰对MECP2的影响 编辑将在HEK细胞，IPSC和IPSC衍生的神经元中进行测试和优化，如AIM 1。 Prime编辑与未编辑的神经元的表型也将被描述为AIM 1。这项工作将提供 深入了解MECP2突变体如何影响神经元中RETT表型的严重程度，并为 用于治疗任何形式的RETT以及其他神经系统疾病的主要编辑平台。提供的培训 从这项研究中，将为这位基因编辑和神经治疗领域的富有成效的职业做好准备。