Gene editing in the brain with CRISPR-PEG

基于CRISPR的基因编辑有可能彻底改变遗传性大脑疾病的治疗。然而， 脑部分娩的并发症限制了基于CRISPR的治疗方法的应用。要解决这一关键问题 根据需要，我们开发了一种新的基因编辑载体CRISPR-PEG，它由Cas9组成 RNP与聚乙二醇偶联物。CRISPR-PEG作为一种交付工具具有巨大的前景 由于其良好的生物相容性，良好的临床记录，以及其增强的组织 与纳米粒子相比的扩散能力。我们的初步结果表明，CRISPR-PEG提供了 并有效地编辑小鼠运动皮质或纹状体中的神经元；脑内注射后，神经元 特异度高(45~85%)。值得注意的是，CRISPR-PEG还编辑了嗅球中的神经元 鼻腔给药。这些令人振奋的结果表明，CRISPR-PEG作为生物工具具有巨大的潜力，并且 作为开发治疗学的平台。在这项计划中，我们将测试我们的新型递送工具CRISPR-PEG在 脆性X综合征(FXS)。我们选择FXS作为CRISPR-PEG的测试平台，因为它是最常见的 遗传性智力残疾，没有治疗方法。此外，FXS有一个单一的原因， 即扩增的CGG重复&gt；200和FMR1启动子区域的超甲基化，这导致沉默 脆性X智力低下1(FMR1)基因。因此，该提案的中心目标是(1)测试 通过靶向FXS相关基因治疗脑部疾病的CRISPR-PEG，以及(2)开发新的CRISPR-PEG变异体 有更好的扩散和效率。中心假设是：新的非病毒传递工具CRISPR- PEG将把Cas9 RNPs送入大脑，有效地编辑神经元中与FXS相关的基因，并将小鼠从 多种FXS相关表型。中心目标将通过完成以下工作来实现 明确的目标。 具体目的1.在FXS小鼠模型中用CRISPR-PEG法敲除mGluR5作为原理证据。 具体目的2.用CRISPR-PEG法重新激活FMR1基因表达。 具体目标3.开发CRISPR--扩散到整个大脑并有效编辑脑组织的钉子。 在这项拟议的研究完成后，我们将开发出一种有效的基因编辑神经元策略 使用一种新型的非病毒递送载体CRISPR-PEG。我们提出的研究具有重要意义，因为 将为开发治疗FXS和由FMR1引起的脆性X相关疾病提供基础 缺乏症。此外，我们将开发一种非基于病毒的工具，可以在一年后编辑大量脑组织 单次注射。这项提案中的实验具有很高的创新性，因为我们将开发出一种高效的 和安全的非病毒递送载体，这将极大地推动神经科学和CRISPR领域的发展 治疗学。

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.