Nanoformulated CRISPR Ribonucleoproteins for Ultrasound-Facilitated Brain Gene Editing

用于超声辅助脑基因编辑的纳米 CRISPR 核糖核蛋白

• 批准号: 10727386
• 负责人: Yeh-Hsing Lao
• 金额: $ 44.2万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10727386
• 关键词: Adverse effects; Alzheimer' s Disease; Alzheimer' s disease model; Amyloid beta-Protein; Animal Model; Animal Testing; Animals; Bar Codes; Biological Products; Blood - brain barrier anatomy; Brain; Brain region; CRISPR therapeutics; CRISPR/Cas technology; Charge; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Development; Diffusion; Disease; Encapsulated; Exploratory/Developmental Grant for Diagnostic Cancer Imaging; Failure; Focused Ultrasound; Formulation; Genes; Genetic Diseases; Guide RNA; Libraries; Ligase; Ligation; Light; Lipids; Liposomes; Liver; Mediating; Methods; Modeling; Mouse Strains; Mus; National Institute of Mental Health; National Institute of Neurological Disorders and Stroke; Nature; Nerve Degeneration; Neurons; Nucleic Acids; Performance; Race; Reporter; Reproducibility; Research; Ribonucleoproteins; Running; Safety; Sampling; Surface; System; TLR2 gene; Techniques; Technology; Time; Toxic effect; Transfection; Transgenic Organisms; Translations; Treatment Efficacy; United States National Institutes of Health; Validation; Variant; Viral; Viral Genes; Viral Vector; adeno-associated viral vector; behavior test; blood-brain barrier crossing; brain tissue; design; efficacy evaluation; experience; gene therapy; immunogenicity; improved; mouse model; nano; nanoformulation; nanoparticle; operation; post-doctoral training; screening; somatic cell gene editing; tau Proteins; ultrasound; vector

Nanoformulated CRISPR Ribonucleoproteins for Ultrasound-facilitated Brain Gene Editing Abstract Emerging CRISPR technologies provide new opportunities to advance gene therapy in treating many intractable genetic diseases, including neuronal degeneration disorders. Given the failures of clinical trials in treating Alzheimer's disease through directly targeting amyloid β and tau, there is an unmet need to develop a different strategy in this space, and gene editing technologies may be of great potential. However, one key barrier in developing CRISPR therapeutics is the brain delivery of CRISPR components. Viral vectors could be effective, but the use of these vectors could potentially raise the concerns in immunogenicity and toxicity, which may lead to severe adverse effects. Conventional nonviral systems, in contrast, could be safer but significantly less effective, possibly due to the suboptimal size, which limits their transport to the target brain region. In light of these challenges, we propose to explore the feasibility of screening more transport-favorable, effective nonviral carriers for brain gene editing to tackle Alzheimer's disease. Different from the conventional nanoparticle designs, we will first create a large nanoformulated CRISPR/Cas9 ribonucleoprotein library through split-and-pool lipid coating and optimize the focused ultrasound (FUS)-mediated blood-brain barrier opening to screen all the possible lipid compositions (Aim 1). Compared with the conventional nanoparticle formulations, direct lipid coating may generate smaller and more transport-favorable “nano editors.” By barcoding each lipid in each split- and-pool round, all the nanoformulated Cas9 ribonucleoproteins can be screened directly in the same animal, which minimizes the variations from animals and operations. Our preliminary studies with a small set of nanoformulations in different models have demonstrated the feasibility and reproducibility of our screening approach. Once having the most potent lipid composition, we will validate its gene editing performance and therapeutic efficacy in both reporter and Alzheimer’s mouse models (Aim 2). Our previous efforts in developing FUS delivery for viral brain gene editing have helped us established the capability and all the pipelines needed for editing performance validations. In this proposed research, we aim to expand the CRISPR delivery toolkits from viral to nonviral systems and to explore the potential of nonviral CRISPR gene editing for treating Alzheimer’s disease. The discoveries and findings will help us gain enough supports for larger, potentially IND- enabling studies.

纳米成型CRISPR核糖核蛋白用于超声处理的脑基因编辑 抽象的 新兴的CRISPR技术为推进基因疗法提供了新的机会，以治疗许多 棘手的遗传疾病，包括神经元变性疾病。考虑到临床试验的失败 通过直接靶向淀粉样β和tau来治疗阿尔茨海默氏病，有未满足的需求 在这个领域的不同策略，基因编辑技术可能具有很大的潜力。但是，一个关键障碍 在开发CRISPR理论中，CRISPR组件的大脑传递。病毒载体可能有效， 但是这些向量的使用可能会引起对免疫原性和毒性的关注，这可能导致 严重不利影响。相比之下，常规的非病毒系统可能是安全的，但要少得多 有效，由于次优尺寸而可能导致，这限制了其运输到目标大脑区域。鉴于 这些挑战，我们建议探索筛查更有效，有效的非病毒的可行性 用于解决阿尔茨海默氏病的脑基因编辑的载体。与常规纳米颗粒设计不同， 我们将首先通过分裂脂质脂质创建一个大型纳米纳米制造的CRISPR/CAS9核糖核蛋白库 涂层并优化聚焦超声（FUS）介导的血脑屏障开口，以筛选所有 可能的脂质组成（目标1）。与常规纳米颗粒配方相比，直接脂质 涂层可能会产生更小，更有利的“纳米编辑器”。通过对每个分裂中的每种脂质进行条形码 和池塘圆，所有纳米成型的Cas9核糖核蛋白可以直接在同一动物中筛选 这可以最大程度地减少动物和手术的变化。我们的初步研究，一小部分 不同模型中的纳米制剂证明了我们筛选的可行性和可重复性 方法。一旦具有最有效的脂质组成，我们将验证其基因编辑性能和 记者和阿尔茨海默氏症小鼠模型的治疗效率（AIM 2）。我们以前在发展方面的努力 病毒脑基因编辑的FUS输送帮助我们确定了能力和所有所需的管道 用于编辑性能验证。在这项拟议的研究中，我们旨在扩大CRISPR交付​​工具包 从病毒到非病毒系统，并探索非病毒CRISPR基因编辑的潜力 阿尔茨海默氏病。这些发现和发现将有助于我们获得足够的支持 启用研究。