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

基本信息

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
项目类别：
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&apos s Disease Alzheimer&apos 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

项目摘要

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.

用于超声辅助脑基因编辑的纳米 CRISPR 核糖核蛋白抽象的新兴的 CRISPR 技术为推进基因疗法治疗许多疾病提供了新的机会鉴于临床试验的失败，包括神经元变性疾病在内的顽固性遗传疾病。通过直接靶向 β 淀粉样蛋白和 tau 蛋白来治疗阿尔茨海默氏病，开发一种未满足的需求这一领域有不同的策略，基因编辑技术可能具有巨大的潜力，但有一个关键障碍。开发 CRISPR 疗法的关键在于 CRISPR 成分的大脑传递可能是有效的，但这些载体的使用可能会引起免疫原性和毒性的担忧，这可能会导致相比之下，传统的非病毒系统可能更安全，但副作用要小得多。有效，可能是由于尺寸不理想，限制了它们向目标大脑区域的运输。针对这些挑战，我们建议探索筛选更利于运输、有效的非病毒的可行性与传统的纳米粒子设计不同，大脑基因编辑的载体可以治疗阿尔茨海默病。我们将首先通过脂质分离和池化创建一个大型纳米配方 CRISPR/Cas9 核糖核蛋白文库涂层并优化聚焦超声（FUS）介导的血脑屏障开口，以筛查所有可能的脂质成分（目标1）与传统的纳米颗粒制剂相比，直接脂质。通过对每个分裂中的每种脂质进行条形码编码，涂层可以产生更小、更利于运输的“纳米编辑器”。和池轮，所有纳米配方的Cas9核糖核蛋白都可以直接在同一动物中进行筛选，这最大限度地减少了动物和操作的差异。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一旦获得最有效的脂质成分，我们将验证其基因编辑性能和在报告基因和阿尔茨海默病治疗小鼠模型中的疗效（目标 2）。用于病毒脑基因编辑的 FUS 交付帮助我们建立了所需的能力和所有管道在这项拟议的研究中，我们的目标是扩展 CRISPR 递送工具包。从病毒到非病毒系统，并探索非病毒 CRISPR 基因编辑治疗的潜力阿尔茨海默病的发现和发现将帮助我们为更大规模的、潜在的 IND 获得足够的支持。赋能研究。