Development of a Nanoparticle-Based Gene Editing Technology for Neurological Applications

基于CRISPR的大脑基因编辑有可能彻底改变神经系统疾病的治疗。 大量无法治愈的脑部疾病，例如亨廷顿舞蹈症、阿尔茨海默氏症和帕金森氏症，都是 这是由致病蛋白的过度表达引起的，并且可以用基于CRISPR的疗法来治疗。 然而，尽管有潜力，开发基于CRISPR的大脑治疗方法一直具有挑战性 因为送货问题特别是，在基因编辑之前，需要解决两个关键挑战。 大型动物和人类的大脑是可能的。第一，有效和安全地递送Cas9和 在颅内注射后，需要开发gRNA进入神经元。第二，可以使 在颅内注射CRISPR试剂后待转染的大体积脑组织（> 1cm）也 需要开发。 该提案的中心目标是开发一种对大型动物大脑进行基因编辑的递送策略 在颅内注射后，称为对流增强CRISPR（C-CRISPR）。C-CRISPR基于使用 对流增强递送（CED）以递送工程化的Cas9 RNP，其已经融合到多个 核定位信号（NLS），并已封装在聚乙二醇化的嵌段共聚物。C-CRISPR 解决了阻碍CRISPR在生物学中产生翻译影响的关键翻译瓶颈， 大脑特别地，因为它直接递送Cas9 RNP，所以它避免了病毒的毒性问题， 使用mRNA的制造挑战，因此具有巨大的翻译潜力。此外，本发明还提供了一种方法， C-CRISPR使用CED将Cas9 RNP分布在几厘米的脑组织中，因此具有良好的免疫原性。 编辑大型动物大脑的潜力。C-CRISPR基于我们的初步数据，表明 与多个NLS信号融合的Cas9 RNP可以在颅内注射后编辑小鼠脑中的基因，并且 与PEG-嵌段共聚物复合的Cas9 RNP可以被递送到纹状体中的数厘米的脑组织， 通过CED交付后。因此，与PEG嵌段共聚物复合的工程化Cas9 RNP的CED具有 在人类患者中编辑基因的潜力。因此，我们提出以下目标/里程碑： UG 3具体目标1.开发分布在大鼠整个纹状体的C-CRISPR制剂 UG 3具体目标2.开发C-CRISPR配方，编辑厘米的脑组织 UH 3具体目标1.开发C-CRISPR制剂，编辑猪脑中的厘米组织 该提案中的实验意义重大，因为如果成功，C-CRISPR将成为第一个 非病毒递送策略，可以编辑大型动物大脑中的基因。该提案中的实验 是创新的，因为C-CRISPR是第一个有效整合3 互补技术，（1）工程化的Cas9 RNP，（2）PEG化和（3）对流增强扩散， 并将为高等动物的基因编辑策略提供路线图。

CRISPR-based gene editing of the brain has the potential to revolutionize the treatment of neurological diseases. A large number of incurable brain diseases, such as Huntington's, Alzheimer's and Parkinson's disease, are caused by the over-expression of pathogenic proteins and could be treated with CRISPR based therapeutics. However, despite its potential, developing CRISPR based therapeutics for the brain has been challenging because of delivery problems. In particular, two key challenges need to be solved before gene editing in the brains of large animals and in humans is possible. First, strategies for efficiently and safely delivering Cas9 and gRNA into neurons, after an intracranial injection, need to be developed. Second, strategies that can enable a large volume of brain tissue (> 1 cm) to be transfected after an intracranial injection of CRISPR reagents also need to be developed. The central objective of this proposal is to develop a delivery strategy for gene editing the brains of large animals after an intracranial injection, termed convection-enhanced CRISPR (C-CRISPR). C-CRISPR is based on using convection-enhanced delivery (CED) to deliver an engineered Cas9 RNP, which has been fused to multiple nuclear localization signals (NLS), and has been encapsulated in PEGylated block copolymers. C-CRISPR addresses the key translational bottlenecks that have prevented CRISPR from having a translational impact in the brain. In particular, because it delivers the Cas9 RNP directly, it avoids the toxicity problems of viruses and the manufacturing challenges of using mRNA, and consequently has great translational potential. In addition, C-CRISPR uses CED to distribute the Cas9 RNP across centimeters of brain tissue, and therefore has the potential to edit the brains of large animals. C-CRISPR is based on our preliminary data demonstrating that the Cas9 RNP fused to multiple NLS signals can edit genes in murine brains after an intracranial injection, and that Cas9 RNP complexed to PEG-block copolymers can be delivered to centimeters of brain tissue, in the striatum, after delivery via CED. CED of engineered Cas9 RNP complexed to PEG block copolymers, therefore, has the potential to edit genes in human patients. We propose therefore the following aims/milestones: UG3 Specific Aim 1. Develop C-CRISPR formulations that distribute throughout the striatum of rats UG3 Specific Aim 2. Develop C-CRISPR formulations that edit centimeters of brain tissue UH3 Specific Aim 1. Develop C-CRISPR formulations that edit centimeters of tissue in pig brains The experiments in this proposal are significant because, if successful, C-CRISPR will be the first example of a non-viral delivery strategy that can edit genes in the brains of large animals. The experiments in this proposal are innovative because C-CRISPR is the first example of a delivery strategy that effectively integrates 3 complementary technologies, (1) engineered Cas9 RNPs (2) PEGylation and (3) convective enhanced diffusion, and will provide a roadmap for developing strategies for gene editing in higher animals.