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.

用于超声促进脑基因编辑的纳米CRISPR核糖核蛋白 摘要 新兴的CRISPR技术为推进基因治疗提供了新的机会， 难治性遗传疾病，包括神经元变性疾病。考虑到临床试验的失败 虽然通过直接靶向淀粉样蛋白β和tau蛋白治疗阿尔茨海默病，但开发一种 在这一领域，基因编辑技术可能具有巨大的潜力。然而，一个关键的障碍 在开发CRISPR疗法中，最重要的是CRISPR成分的大脑递送。病毒载体可能是有效的， 但这些载体的使用可能会引起免疫原性和毒性的担忧， 严重的副作用。相比之下，传统的非病毒系统可能更安全， 有效，可能是由于次优的尺寸，这限制了它们向目标大脑区域的运输。鉴于 针对这些挑战，我们建议探索筛选更有利于转运的、有效的非病毒药物的可行性。 大脑基因编辑的载体来治疗阿尔茨海默病。与传统的纳米颗粒设计不同， 我们将首先通过分离和合并脂质， 涂层和优化聚焦超声（FUS）介导的血脑屏障开放，以筛选所有的 可能的脂质组合物（目标1）。与传统的纳米粒制剂相比，直接脂质 涂层可以产生更小和更有利于运输的“纳米编辑器”。把每一次分裂中的每一种脂质都标上条形码- 和汇集轮，所有纳米配制的Cas9核糖核蛋白可以直接在同一动物中筛选， 其最小化来自动物和操作的变化。我们的初步研究， 不同模型中的纳米制剂已经证明了我们筛选的可行性和可重复性 approach.一旦拥有最有效的脂质成分，我们将验证其基因编辑性能， 在报告基因和阿尔茨海默病小鼠模型中的治疗功效（目的2）。我们以前在开发 用于病毒大脑基因编辑的FUS交付帮助我们建立了所需的能力和所有管道 用于编辑性能验证。在这项拟议的研究中，我们的目标是扩展CRISPR交付工具包， 从病毒系统到非病毒系统，并探索非病毒CRISPR基因编辑治疗癌症的潜力。 老年痴呆症这些发现和发现将帮助我们为更大的、潜在的IND获得足够的支持， 赋能研究。