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脑疾病的治疗。然而，在这方面， 脑递送的并发症限制了基于CRISPR的治疗剂的效用。处理这个关键 需要，我们开发了一种新的基因编辑递送载体，称为CRISPR-PEG，它由Cas9 与聚乙二醇（PEG）缀合的RNP。CRISPR-PEG作为运载工具具有巨大的前景 由于其优异的生物相容性、PEG良好的临床跟踪记录以及其增强的组织相容性， 与纳米颗粒相比，我们的初步结果表明，CRISPR-PEG提供了 并在小鼠的运动皮层或纹状体中有效地编辑神经元;在颅内注射后， 特异性为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具有作为生物工具的巨大潜力， as a platform平台for developing发展therapeutic治疗.在这项提案中，我们将测试我们的新型运载工具CRISPR-PEG， 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，无治疗方法。此外，FXS具有单基因病因， 即扩增的CGG重复序列>200和FMR 1启动子区域的超甲基化，这导致FMR 1基因沉默。 脆性X智力低下1（FMR 1）基因。因此，本提案的中心目标是（1）测试 CRISPR-PEG通过靶向FXS相关基因在脑疾病中的应用，以及（2）开发新的CRISPR-PEG变体 具有改进的扩散和效率。核心假设是：新型非病毒递送载体CRISPR- PEG将Cas9 RNP递送到大脑中，有效地编辑神经元中的FXS相关基因，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 具体目标。 具体目标1。在FXS小鼠模型中使用CRISPR-PEG敲低mGluR 5作为原理证明。 具体目标2。使用CRISPR-PEG重新激活FMR 1基因表达。 具体目标3。开发CRISPR-PEG，扩散到整个大脑并有效编辑脑组织。 在这项拟议的研究完成时，我们将开发出一种用于基因编辑神经元的有效策略。 使用一种新型的非病毒递送载体CRISPR-PEG。我们提出的研究是重要的，因为结果 将为FXS和FMR 1引起的脆性X相关疾病的治疗提供基础 缺陷此外，我们还将开发一种非病毒载体，它可以在一次注射后编辑大量脑组织。 单次注射。这项建议中的实验是高度创新的，因为我们将开发出一种有效的 和安全的非病毒运载工具，这将大大推进神经科学和基于CRISPR的 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.