Engineered Exosomes for Targeted Delivery of the CRISPR/Cas9 Genome-editor

用于 CRISPR/Cas9 基因组编辑器靶向递送的工程外泌体

• 批准号: 10383110
• 负责人: RAMESH C GUPTA
• 金额: $ 26.45万
• 依托单位: 3P BIOTECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2024-05-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10383110
• 关键词: A549; Adaptive Immune System; Address; Animal Model; Binding; Biodistribution; Biological Sciences; Cattle; Cell Culture Techniques; Cells; Chronic Obstructive Pulmonary Disease; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Colostrum; Complex; DNA; DNA Repair; DNA Sequence Alteration; Data; Detection; Development; Disease; Disease model; Drug Delivery Systems; Drug or chemical Tissue Distribution; Epithelial Cells; Fluorescence; Formulation; Genes; Genome; Genome Components; Goals; Guide RNA; Image; Immune response; In Vitro; Industry Standard; Inflammation; Inflammatory; Inflammatory Response; Inherited; Intravenous; Knock-in; Knock-out; Knowledge; Label; Laboratories; Lactoferrin; Lipofectamine; Lipopolysaccharides; Location; Lung; Mediating; Methods; Milk; Modeling; Mus; Mutation; Non-Viral Vector; Nonhomologous DNA End Joining; Nucleic Acids; Organ; Phase; Plasmids; Polyethyleneimine; Preparation; Production; Proteins; Research Personnel; Route; Source; Structure of parenchyma of lung; Surface; Symptoms; System; TP53 gene; Technology; Testing; Time; Tissues; Toxic effect; Transfection; Ultracentrifugation; Viral; Western Blotting; Wild Type Mouse; alveolar epithelium; base; biomaterial compatibility; bronchial epithelium; chemokine; cost effective; cytokine; delivery vehicle; engineered exosomes; exosome; experience; gene therapy; genome editing; in vivo; inflammatory lung disease; knock-down; lactoferrin receptors; lung cancer cell; microbial; nano; nanoformulation; new technology; novel; nuclease; nucleic acid-based therapeutics; overexpression; plasmid DNA; prevent; protein expression; public health relevance; scaffold; success; systemic toxicity; targeted delivery; tool

Technical Abstract Genetic mutations have been identified as a causative factor in numerous diseases. The genome editing system CRISPR/Cas9 is a recent development in gene therapy. Both viral and non-viral vectors have been used in attempts to direct delivery of Cas9 to specific locations with advantages and limitations similar to those known for other nucleic acid-based therapeutics. These challenges have limited the current clinical progress of this genome-editing tool. The goal of this project is to develop an effective targeted delivery system for Cas9-mediated genome editing. The investigators take advantage of a novel technology for delivery of plasmid DNA (pDNA) based on bovine milk/colostrum exosomes developed in the PI's laboratory. In this project, we will apply our knowledge and extensive experience in exosomes for efficient targeted delivery of the Cas9-mediated genome- editing tool. To establish feasibility, we have used pDNA to deliver the coding sequences for Cas9-mediated knockout of NFκB as a model gene. This single plasmid, pKO-NFκB, contains the mammalian-optimized Cas9 coding sequence, the single-guide RNA (sgRNA) specific to NFκB, as well as sequences to derive a guide RNA (gRNA) scaffold to assist in the binding of Cas9 to the target DNA. We hypothesize that pKO-NFκB, ionically entrapped in a novel exosome matrix, formulated by complexing exosomes and polycationic polyethyleneimine (PEI), will serve as an effective genome-editing tool of NFκB. Furthermore, use of engineered exosomes, prepared by loading milk lactoferrin (LF) onto exosomes, will target bronchial epithelium overexpressing LF receptors. Thus, LF-EPM-pKO-NFκB administered intranasally (i.n.) will target lung with minimal off-target effects for delivery of this genome-editing tool. Our hypothesis is supported by compelling preliminary data: high loading of nucleic acid onto EPM and protection from degradation, functionalization of exosomes by surface-bound LF loading, inhibition of NFκB expression in H2030 lung cancer cells by LF-EPM delivered pKO-NFκB, overexpression of the LF receptor intelectin (also called omentin) in the mouse lung, and predominant delivery of LF-functionalized exosomes to the mouse lung by intranasal delivery. Investigators experienced in exosomes, drug delivery, and biological sciences will pursue the following specific aims: Aim 1. Optimize targeted delivery of CRISPR/Cas9 genome-editing tool using engineered exosomes in vitro. Aim 2. Determine potential toxicity, and biodistribution and efficacy of engineered exosomes for targeted delivery of CRISPR/Cas9 genome-editing tool. If we are successful in achieving these milestones, we will move to Phase II. Results from this project will provide feasibility data for advancing this genome-editing tool delivery `platform' in a disease model. Cost-effective isolation of exosomes from a biocompatible source, combined with ultracentrifugation- independent methods currently being developed in PI's laboratory, makes the exosomes production a commercial viability as this novel delivery technology advances.

技术摘要 遗传突变已被确定为基因组编辑器 System CRISPR/CAS9是基因疗法的最新发展。已经使用了病毒和非病毒载体 试图将CAS9转移到具有类似于已知的优势和限制的特定位置 用于其他基于核酸的治疗。这些挑战限制了当前的临床进展 基因组编辑工具。该项目的目的是为Cas9介导的有效的目标交付系统开发 基因组编辑。研究人员利用一种新技术来传递质粒DNA（PDNA） 基于PI实验室中开发的牛牛奶/初乳外泌体。在这个项目中，我们将应用我们的 知识和在外泌体方面具有丰富的经验，可有效地靶向Cas9介导的基因组 - 编辑工具。为了确定可行性，我们使用pDNA提供了Cas9介导的编码序列 NFκB作为模型基因的敲除。这个单个质粒PKO-NFκB包含哺乳动物优化的Cas9 编码序列，特定于NFκB的单诱导RNA（SGRNA），以及推导引导RNA的序列 （GRNA）支架有助于CAS9与靶DNA的结合。我们假设PKO-NFκB在离子上 通过络合外泌体和聚乙烯胺配制的新型外泌体基质中 （PEI）将作为NFκB的有效基因组编辑工具。此外，使用工程外泌体， 通过将牛奶乳铁蛋白（LF）加载到外泌体上制备，将靶向支气管上皮过表达LF 接收者。那是鼻内施用（I.N。）的LF-EPM-PKO-NFκB将以最小的脱靶效应靶向肺 用于提供此基因组编辑工具。我们的假设得到了引人入胜的初步数据的支持：高负载 核酸上EPM的核酸，并免受降解，外泌体的功能化，表面结合的LF LF-EPM传递的PKO-NFκB，负载，抑制H2030肺癌细胞中NFκB表达， 小鼠肺中LF受体内染蛋白（也称为omentin）的过表达，主要递送 通过鼻内递送到小鼠肺的LF官能化外泌体。调查人员在外泌体中经历过 药物输送和生物科学将追求以下特定目的：目标1。优化目标交付 在体外使用工程外泌体的CRISPR/CAS9基因组编辑工具。目标2。确定潜力 针对CRISPR/CAS9的有针对性交付的工程外泌体的毒性，生物分布和效率 基因组编辑工具。如果我们成功地实现了这些里程碑，我们将进入第二阶段。结果 该项目将提供可行性数据，用于推进疾病中的基因组编辑工具“平台” 模型。具有成本效益的外泌体从生物相容性来源分离，并结合超速离心 - 当前在Pi的实验室开发的独立方法，使外泌体生产成为商业广告 随着这种新颖的交付技术的发展，生存能力。