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

项目总结 婴儿Rett综合征(Rett)出生时编码MeCP2的基因发生功能丧失突变，这是一种全球 基因表达的调节器。大脑中的MeCP2功能障碍严重影响神经元，导致发育 不同严重程度的缺陷，在6个月后表现出来。目前的治疗方法可以控制一些症状， 但纠正MECP2突变将更有效地恢复患者的生活质量。CRISPR基因编辑具有 使这种方法成为可能。在CRISPR技术中，Prime编辑是最灵活的，使用了 RNA引导的Cas9核酸酶融合到逆转录酶中，以“寻找和替换”有丝分裂后细胞中的突变。 因此，Prime剪辑是Rett治疗的有力候选者。然而，主要的编辑程序只提供给神经元 通过慢病毒(与临床无关)，其在细胞中的编辑效率低。 先前的工作表明，包裹在脂质纳米粒(LNP)中的Cas9 mRNA的传递很简单， 安全，并支持在小鼠肝脏中进行强大的编辑。LNP包裹的mRNA也可以传递到大脑，但 主要编辑基因的信使核糖核酸和神经元中主要编辑的效率仍未得到测试。此外，通过化学修饰， 其他CRISPR系统的引导RNA可以防止核酸酶介导的降解，并改善基因 编辑单元格中的汇率。瓦茨实验室最近开发了一种合成化学修饰素材编辑的方法 引导RNA(PegRNA)，由于pegRNA的长度(~150个核苷酸)而被认为是不可行的东西。这个 PegRNA修饰对Prime编辑效率的影响尚未得到测试。 在乔纳森·瓦茨博士(核酸化学)、迈克尔·格林(Rett神经生物学)、Erik的支持下 Sonthemer(生物学主编)，Scot Wolfe(基因调控)，Athma Pai(生物信息学)，这个项目 寻求建立和化学优化基于mRNA的初级编辑程序，以纠正MECP2突变并逆转 它们在神经元中的表型。目标1将建立信使核糖核酸传递的初级编辑者的基线有效性(与 慢病毒)对抗最常见的Rett突变(一种错义突变)和两个临床上严重的胡说八道 表达每个突变体MeCP2的HEK细胞、患者来源的诱导多能干细胞(IPSCs)、 和IPSC来源的神经元。这个目标还将在编辑和不编辑的情况下探测神经元，以了解分子 每个MECP2突变的表型以及编辑逆转它们的程度。目标2将在瓦茨上迭代 Lab的pegRNA组装方法优化pegRNA产量和合成时间，并识别编辑兼容 在体外和纤维分析中使用的pegRNA修饰模式。PegRNA修饰对MECP2基因的影响 编辑将在HEK细胞、IPSCs和IPSC来源的神经元中进行测试和优化，就像在Aim 1中一样。 原始编辑过的神经元和未编辑过的神经元的表型也将如目标1所示。 洞察MeCP2突变体如何影响神经元中Rett表型的严重程度并影响A Prime-编辑平台，用于治疗任何形式的Rett以及其他神经疾病。提供的培训 这项研究将为这位研究员在基因编辑和神经治疗学领域的富有成效的职业生涯做好准备。

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.