Nonviral delivery techniques for in vivo prime editing

用于体内引物编辑的非病毒传递技术

基本信息

批准号：
10548169
负责人：
DANIEL G ANDERSON
金额：
$ 38.78万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10548169
关键词：
Bar Codes Cells Chimeric Proteins Clinical Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Combinatorial Chemistry Techniques Cystic Fibrosis DNA DNA Double Strand Break Dangerousness Development Disease Disease model Drug Kinetics Encapsulated Endonuclease I Epithelium Event Formulation Fumarylacetoacetase Gene Delivery Genes Genetic Diseases Genome Health Hereditary Disease Human Human Genome Hydrolase In Vitro Inbred CFTR Mice Individual Intravenous Libraries Lipid Inclusion Lipids Liver Liver diseases Lung Lung diseases Measures Mission Mus Mutation Nebulizer Organ Phenotype Property Proteins RNA RNA delivery RNA-Directed DNA Polymerase Reporting Research Somatic Cell Sorting Structure System Techniques Technology Testing Time Tissues Toxic effect Tyrosinemias United States National Institutes of Health Writing base editing combinatorial chemistry design disease-causing mutation effective therapy experimental study follow-up genome editing improved in vitro testing in vivo in vivo evaluation lipid nanoparticle mouse model nanoformulation nanoparticle delivery novel novel strategies pre-clinical prime editing prime editor stem cells tool

项目摘要

Gene editing is a promising strategy for treating or even permanently curing genetic diseases. In particular, a new technique called prime editing has the potential to make small targeted insertions, deletions, and substitutions with very high potential coverage of known disease- causing mutations, and while minimizing dangerous double-stranded breaks in DNA. In order to realize this potential, robust delivery strategies must be developed to deliver prime editing tools efficiently to disease-relevant organs. One such delivery strategy is lipid nanoparticle delivery of RNA and/or protein-based prime editing components. LNPs are nonviral, nontoxic, and clinically validated delivery tools. However, there is an extremely diverse space of possible LNPs, with tens of thousands of potential lipid structures that may be useful for LNP delivery. Selecting the best possible LNP for a prime editing application, therefore, is challenging because in vitro testing is often unreliable and in vivo testing of one LNP at a time is extremely low throughput. Here, we propose to combine two scalable techniques to generate and test safe, potent LNP formulations for performing prime editing. First, we will employ combinatorial chemistry techniques to generate large libraries of biodegradable lipids for inclusion into LNPs. Second, we will introduce a new technique which we term pegRNA barcoding to screen dozens to hundreds of LNPs for successful prime editing in a single mouse. We will employ this technique to identify the best biodegradable LNPs for editing of multiple organs, including in particular the lung and the liver. Having identified the top candidates, we will proceed to use our LNPs to apply prime editing to treat mouse models of two different inherited genetic diseases: hereditary tyrosinemia type I (HTI), a liver disease, and cystic fibrosis (CF), primarily a lung disease. We will evaluate the efficiency of prime editing, the levels of undesired editing events, and phenotypic correction of these mice. The results may identify promising preclinical candidates for the treatment of HTI, CF, and many other lung and liver diseases.

基因编辑是治疗甚至永久治愈遗传疾病的有前途的策略。在特别是，一种称为Prime Editing的新技术有可能使针对性小插入，缺失和取代，具有非常高的已知疾病潜在覆盖范围引起突变，同时最大程度地减少DNA中危险的双链断裂。为了意识到必须制定这种潜力，强大的交付策略来提供主要的编辑工具有效地适用于疾病与疾病的器官。一种这样的输送策略是脂质纳米颗粒的递送 RNA和/或基于蛋白质的Prime编辑组件。 LNP是非病毒，无毒和临床上的经过验证的交付工具。但是，有可能的LNP的多样化空间，数以万计的潜在脂质结构可能可用于LNP递送。选择因此，用于主要编辑应用程序的最佳LNP是具有挑战性的，因为体外测试通常是不可靠的，一次对一个LNP进行体内测试非常低。在这里，我们建议将两种可扩展技术结合起来，以生成和测试安全，有效的LNP 执行主要编辑的配方。首先，我们将采用组合化学生成大量可生物降解脂质库以包含在LNP中的技术。第二，我们将引入一种新技术在单个鼠标中成功编辑数百个LNP。我们将采用这种技术确定用于编辑多个器官的最佳可生物降解的LNP，特别是肺和肝脏。确定了顶级候选人后，我们将继续使用我们的LNP 应用主要编辑以治疗两种不同遗传遗传疾病的鼠标模型：遗传 I型酪氨酸血症（HTI），肝病和囊性纤维化（CF），主要是一种肺部疾病。我们将评估主要编辑效率，不希望的编辑事件的水平以及这些小鼠的表型校正。结果可能会确定有希望的临床前候选人用于治疗HTI，CF以及许多其他肺和肝病。