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：
10344605
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/10344605
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 Epithelial 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 - Cell Movement Structure System Techniques Technology Testing Time Tissues Toxic effect Tyrosinemias United States National Institutes of Health Writing base 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 insertion/deletion mutation lipid nanoparticle mouse model nanoformulation nanoparticle delivery novel novel strategies pre-clinical prime editing prime editor programs 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一种有前景的策略。在特别是，一种称为“prime edit”的新技术有可能使小目标插入、删除和替换对已知疾病具有非常高的潜在覆盖率- 引起突变，同时最大限度地减少 DNA 中危险的双链断裂。为了认识到这一潜力，必须制定强大的交付策略来提供优质的编辑工具有效地作用于疾病相关器官。一种这样的递送策略是脂质纳米颗粒递送基于 RNA 和/或蛋白质的 Prime 编辑组件。 LNP 是非病毒、无毒的，并且在临床上经过验证的交付工具。然而，可能的 LNP 空间极其多样化，其中数以万计的潜在脂质结构可能对 LNP 递送有用。选择因此，对于prime编辑应用来说，最佳的LNP是具有挑战性的，因为在体外测试通常不可靠，并且一次对一种 LNP 进行体内测试的通量极低。在这里，我们建议结合两种可扩展的技术来生成和测试安全、有效的 LNP 用于执行主要编辑的公式。首先，我们将采用组合化学生成大型可生物降解脂质库以纳入 LNP 的技术。第二，我们将引入一种新技术，我们称之为 pegRNA 条形码来筛选数十个数百个 LNP，可在单只小鼠中成功进行 Prime 编辑。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。确定最佳候选人后，我们将继续使用我们的 LNP 来应用prime编辑治疗两种不同遗传性疾病的小鼠模型：遗传性 I 型酪氨酸血症 (HTI)（一种肝脏疾病）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的效率、不良编辑事件的水平，以及这些小鼠的表型校正。结果可能会确定有前途的临床前候选药物用于治疗 HTI、CF 和许多其他肺和肝疾病。