Gene editing in the brain with CRISPR-PEG

使用 CRISPR-PEG 对大脑进行基因编辑

• 批准号: 10378044
• 负责人: Hye Young Lee
• 金额: $ 64.61万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10378044
• 关键词: Address; Advanced Development; Beds; Brain; Brain Diseases; Brain region; CGG repeat; CRISPR therapeutics; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Corpus striatum structure; DNA Damage; Diffuse; Diffusion; Disease; Etiology; Exposure to; FMR1; FMRP; Formulation; Fragile X Syndrome; Gene Expression; Genes; Genetic; Genetic Diseases; Genomic DNA; Gold; Hypermethylation; Inherited; Injections; Intellectual functioning disability; Intranasal Administration; Knock-in Mouse; Knockout Mice; Length; Morphology; Motor Cortex; Mus; Neurons; Neurosciences; Other Genetics; Patients; Phenotype; Polyethylene Glycols; Promoter Regions; Proteins; Publishing; Reagent; Ribonucleoproteins; Signal Transduction; Solubility; Specificity; Testing; Therapeutic; Time; Tissues; Toxic effect; Transfection; Variant; Vertebral column; Viral; base; base editing; behavior test; behavioral phenotyping; biomaterial compatibility; brain tissue; brain volume; delivery vehicle; design; experimental study; immunogenicity; improved; in vivo; in vivo evaluation; induced pluripotent stem cell; innovation; knock-down; mouse model; nanoGold; nanoparticle; neurotoxicity; novel; olfactory bulb; promoter; repetitive behavior; restoration; tool; translational impact; translational potential

CRISPR-based gene editing has the potential to revolutionize the treatment of genetic brain disorders. However, complications with brain delivery have limited the utility of CRISPR-based therapeutics. To address this critical need, we have developed a new gene editing delivery vehicle, termed CRISPR-PEG, which is composed of Cas9 RNP conjugated to polyethylene glycol (PEG). CRISPR-PEG has tremendous promise as a delivery vehicle because of its excellent biocompatibility, the well-established clinical track record of PEG, and its enhanced tissue diffusion capability in comparison to nanoparticles. Our preliminary results demonstrate that CRISPR-PEG delivers and edits neurons efficiently in the motor cortex or striatum in mice; after an intracranial injection, neurons were edited with a high specificity (45~85%). Notably, CRISPR-PEG also edited neurons in the olfactory bulb after intranasal administration. These exciting results demonstrate that CRISPR-PEG has great potential as bio-tool, and as a platform for developing therapeutics. In this proposal we will test our novel delivery vehicle CRISPR-PEG in fragile X syndrome (FXS). We have selected FXS as a test bed for CRISPR-PEG because it is the most common inherited cause of intellectual disability with no treatment available. In addition, FXS has a monogenic cause, namely expanded CGG repeats>200 and hypermethylation in the FMR1 promoter region, which causes silencing of the fragile X mental retardation 1 (FMR1) gene. Therefore, the central objectives of this proposal are (1) to test CRISPR-PEG in brain disorders by targeting FXS-associated genes, and (2) to develop new CRISPR-PEG variants with improved diffusion and efficiency. The central hypothesis is: the novel non-viral delivery vehicle CRISPR- PEG will deliver Cas9 RNPs into the brain, efficiently edit FXS-associated genes in neurons, and rescue mice from multiple FXS-associated phenotypes. The central objective will be accomplished by completing the following specific aims. Specific Aim 1. Knock down mGluR5 using CRISPR-PEG in the mouse model of FXS as proof of principle. Specific Aim 2. Reactivate FMR1 gene expression using CRISPR-PEG. Specific Aim 3. Develop CRISPR-PEGs that diffuse throughout the brain and edit brain tissue efficiently. At the completion of this proposed study, we will have developed an efficient strategy for gene editing neurons using a novel non-viral delivery vehicle CRISPR-PEG. Our proposed studies are significant because the results will provide the basis for developing therapeutics for FXS and fragile X-associated disorders caused by FMR1 deficiency. Moreover, we will develop a non-viral-based vehicle that can edit large volumes of brain tissue after a single injection. The experiments in this proposal are highly innovative because we will have developed an efficient and safe non-viral delivery vehicle, which will greatly advance the field of neuroscience and CRISPR-based therapeutics.

基于CRISPR的基因编辑有可能彻底改变遗传脑疾病的治疗。然而， 大脑分娩并发症限制了基于CRISPR的治疗剂的实用性。解决这个关键 需要，我们开发了一种新的基因编辑递送工具，称为CRISPR-PEG，该工具由Cas9组成 RNP与聚乙二醇（PEG）结合。 CRISPR-PEG作为送货工具具有巨大的希望 由于其出色的生物相容性，PEG的临床记录及其增强的组织 与纳米颗粒相比，扩散能力。我们的初步结果表明CRISPR-PEG提供 并在小鼠运动皮质或纹状体中有效地编辑神经元；颅内注射后，神经元为 具有高特异性（45〜85％）。值得注意的是，CRISPR-PEG还编辑了嗅球中的神经元 鼻内给药。这些令人兴奋的结果表明，CRISPR-PEG作为生物工具具有巨大的潜力，并且 作为开发治疗剂的平台。在此提案中，我们将测试我们新颖的送货车CRISPR-PEG 脆弱的X综合征（FXS）。我们选择了FXS作为CRISPR-PEG的测试床，因为它是最常见的 智力残疾的继承原因，没有可用的治疗。此外，FXS具有单一原因， 即FMR1启动子区域中扩展的CGG重复> 200和高甲基化，这导致沉默 脆弱的X智力低下1（FMR1）基因。因此，该提案的核心目标是（1）测试 CRISPR-PEG通过靶向FXS相关基因，以及（2）开发新的CRISPR-PEG变体 随着扩散和效率的提高。中心假设是：新型的非病毒递送工具crispr- PEG会将Cas9 RNP传递到大脑中，有效地编辑神经元中的FXS相关基因，并从 多个与FXS相关的表型。中心目标将通过完成以下内容来实现 具体目标。 具体目标1。在FXS的小鼠模型中使用CRISPR-PEG作为原理证明。 特定目标2。使用CRISPR-PEG重新激活FMR1基因表达。 特定目的3。开发CRISPR-PEG在整个大脑中扩散并有效地编辑大脑组织。 这项拟议的研究完成后，我们将制定有效的基因编辑神经元的策略 使用新型的非病毒递送车CRISPR-PEG。我们提出的研究很重要，因为结果 将为FXS开发FXS和脆弱的X相关疾病的疗法提供基础 不足。此外，我们将开发一种基于非病毒的车辆，该车辆可以在 单个注射。该提案中的实验具有很高的创新性，因为我们将发展有效 和安全的非病毒递送工具，这将大大推动神经科学和基于CRISPR的领域 疗法。