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

项目摘要 患有Rett综合征（Rett）的婴儿出生时在编码MeCP 2的基因中存在功能缺失突变，这是一个全球性的遗传缺陷。 基因表达的调节因子。大脑中的MeCP 2功能障碍严重影响神经元，导致发育障碍。 6个月后出现的不同严重程度的缺陷。目前的治疗方法可以控制一些症状， 但纠正MECP 2突变将更有效地恢复患者的生活质量。CRISPR基因编辑 使这种方法变得可行。在CRISPR技术中，prime editing是最灵活的，它利用了 RNA引导的Cas9核酸酶与逆转录酶融合，以“搜索和替换”有丝分裂后细胞中的突变。 因此，prime编辑是Rett治疗的强有力候选者。然而，主要的编辑器只被输送到神经元， 通过慢病毒（临床上不相关），其在细胞中的编辑效率很低。 先前的工作表明，封装在脂质纳米颗粒（LNP）中的Cas9 mRNA的递送是简单的， 安全，并支持小鼠肝脏中的稳健编辑。LNP包封的mRNA也递送到大脑，但LNP包封的mRNA的递送是不可能的。 prime editor mRNA和prime editing在神经元中的效率仍然未经测试。此外，化学修饰 其他CRISPR系统指导RNA可以防止核酸酶介导的降解，并改善基因 编辑单元格中的速率。Watts实验室最近开发了一种合成化学修饰的prime编辑的方法 向导RNA（pegRNA），由于pegRNA的长度（~150 nt），这被认为是不可行的。的 pegRNA修饰对引物编辑效率的影响尚未得到测试。 在Jonathan Watts（核酸化学）、Michael绿色（Rett神经生物学）、Erik Sontheimer（主要编辑生物学），Scot Wolfe（基因调控）和Athma派（生物信息学），该项目 旨在建立和化学优化基于mRNA的主要编辑器，以纠正MECP 2突变和逆转录病毒。 它们在神经元中的表型。目标1将建立mRNA交付的主要编辑器的基线有效性（与 慢病毒）针对最常见的Rett突变（错义突变）和两种临床上严重的无义突变。 表达每种突变MeCP 2的HEK细胞中的突变，患者来源的诱导多能干细胞（iPSC）， 和iPSC衍生的神经元。这个目标还将探测有和没有编辑的神经元，以了解分子水平。 每个MECP 2突变的表型和编辑逆转它们的程度。Aim 2将在Watts上运行 实验室的pegRNA组装方法，以优化pegRNA产量和合成时间，并确定编辑兼容 使用体外和细胞内测定的pegRNA修饰模式。pegRNA修饰对MECP 2的影响 将在HEK细胞、iPSC和iPSC衍生的神经元中测试和优化编辑，如目标1中所述。分子 原始编辑的神经元与未编辑的神经元的表型也将如目的1所述进行表征。这项工作将提供 深入了解MeCP 2突变体如何影响神经元中Rett表型的严重程度，并告知 prim-editing平台来治疗任何形式的Rett以及其他神经系统疾病。提供的培训 这项研究将为该研究员在基因编辑和神经治疗领域的富有成效的职业生涯做准备。

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.