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

开发用于神经学应用的基于纳米颗粒的基因编辑技术

• 批准号: 10669525
• 负责人: Krystof S Bankiewicz
• 金额: $ 109.61万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10669525
• 关键词: Address; Adult; Alzheimer' s Disease; Anatomy; Animals; Blood - brain barrier anatomy; Brain; Brain Diseases; CRISPR therapeutics; Charge; Chromosome Mapping; Clinic; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Convection; Corpus striatum structure; DRD2 gene; Data; Development; Diffuse; Diffusion; Dopamine D2 Receptor; Encapsulated; Engineering; Family suidae; Formulation; Genes; Guide RNA; Human; Huntington Disease; Injections; Magnetic Resonance Imaging; Messenger RNA; Miniature Swine; Mus; Neurologic; Neurons; Nuclear Localization Signal; Parkinson Disease; Pathogenicity; Patients; Pharmaceutical Preparations; Phase; Proteins; Rattus; Reagent; Reporter Genes; Rest; Ships; Signal Pathway; Signal Transduction; Site; Technology; Testing; Tissues; Toxic effect; Transgenic Organisms; Translations; Virus; base; base editing; brain tissue; brain volume; copolymer; experimental study; innovation; macromolecule; nanoparticle; nervous system disorder; neuroinflammation; neurotransmission; novel; overexpression; prevent; scale up; translational impact; translational potential

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.

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，如亨廷顿舞蹈病、阿尔茨海默病和帕金森病， 由致病蛋白过度表达引起，可以用基于 CRISPR 的疗法进行治疗。 然而，尽管有潜力，开发基于 CRISPR 的大脑疗法一直具有挑战性 因为交货问题。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。首先，高效、安全地递送 Cas9 和 颅内注射后，需要将 gRNA 培养成神经元。其次，可以采取的策略 颅内注射 CRISPR 试剂后也可转染大量脑组织（> 1 cm） 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传递策略 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) 来递送工程化的 Cas9 RNP，该 RNP 已融合到多个 核定位信号（NLS），并被封装在聚乙二醇化嵌段共聚物中。 C-CRISPR 解决了阻碍 CRISPR 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，避免了病毒的毒性问题， 使用 mRNA 的制造挑战，因此具有巨大的转化潜力。此外， C-CRISPR 使用 CED 将 Cas9 RNP 分布在脑组织的厘米范围内，因此具有 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大脑中的基因，并且 与 PEG 嵌段共聚物复合的 Cas9 RNP 可以被递送至纹状体中数厘米的脑组织， 通过 CED 交付后。因此，工程化 Cas9 RNP 与 PEG 嵌段共聚物复合的 CED 具有 编辑人类患者基因的潜力。因此，我们提出以下目标/里程碑： UG3 具体目标 1. 开发分布在大鼠纹状体中的 C-CRISPR 制剂 UG3 具体目标 2. 开发编辑厘米脑组织的 C-CRISPR 配方 UH3 具体目标 1. 开发可编辑猪脑中数厘米组织的 C-CRISPR 配方 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 可以编辑大型动物大脑中基因的非病毒传递策略。本提案中的实验 之所以具有创新性，是因为 C-CRISPR 是有效整合 3 种递送策略的第一个例子 互补技术，(1) 工程化 Cas9 RNP (2) 聚乙二醇化和 (3) 对流增强扩散， 并将为制定高等动物基因编辑策略提供路线图。