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疾病的治疗。 大量无法治愈的大脑疾病，如亨廷顿氏症、阿尔茨海默氏症和帕金森氏症， 由致病蛋白过度表达引起，可用以CRISPR为基础的治疗方法治疗。 然而，尽管有潜力，开发基于CRISPR的大脑疗法一直是具有挑战性的 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 大型动物的大脑和人类的大脑是可能的。第一，高效、安全地交付Cas9和 在颅内注射后，需要开发进入神经元的gRNA。第二，能够实现 脑内注射CRISPR试剂后需要大量转基因的脑组织(&gt；1 cm)也 需要被开发。 这项提议的中心目标是开发一种用于大型动物大脑基因编辑的交付策略 在颅内注射后，称为对流增强CRISPR(C-CRISPR)。C-CRISPR基于使用 对流增强输送(CED)，以交付已融合到多个 核定位信号(NLS)，并已被包裹在聚乙二醇化嵌段共聚物中。C-CRISPR 解决阻碍CRISPR在以下方面产生翻译影响的关键翻译瓶颈 大脑。特别是，由于它直接传递Cas9 RNP，它避免了病毒和 使用信使核糖核酸的制造挑战，因此具有巨大的翻译潜力。此外, C-CRISPR使用CED将Cas9 RNP分布在厘米厚的脑组织中，因此具有 有可能编辑大型动物的大脑。C-CRISPR基于我们的初步数据，表明 融合了多个NLS信号的Cas9 RNP可以在脑内注射后编辑小鼠大脑中的基因，并且 与聚乙二醇嵌段共聚物复合的Cas9 RNP可以被输送到厘米长的脑组织中，在纹状体， 通过CED交付后。因此，与聚乙二醇嵌段共聚物络合的工程Cas9 RNP的CED具有 有可能编辑人类患者的基因。因此，我们提出以下目标/里程碑： UG3特定目标1.研制分布于大鼠纹状体的C-CRISPR制剂 UG3特定目标2.开发可编辑厘米长脑组织的C-CRISPR配方 UH3特定目标1.开发编辑猪脑中厘米组织的C-CRISPR配方 这项提议中的实验意义重大，因为如果成功，C-CRISPR将成为 非病毒传递策略，可以编辑大型动物大脑中的基因。这项提案中的实验 是创新的，因为C-CRISPR是有效集成3个 补充技术，(1)工程Cas9 RNPs(2)聚乙二醇化和(3)对流增强扩散， 并将为开发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.