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

基本信息

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
项目类别：
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

项目摘要

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.

技术摘要基因突变已被确定为许多疾病的致病因素。 CRISPR/Cas9系统是基因治疗领域的最新发展，病毒和非病毒载体均已被使用。尝试将 Cas9 直接递送到特定位置，具有与已知的优点和类似的局限性对于其他基于核酸的疗法来说，这些挑战限制了其当前的临床进展。该项目的目标是开发一种有效的 Cas9 介导的靶向递送系统。研究人员利用一种新技术来传递质粒 DNA (pDNA)。基于 PI 实验室开发的牛乳/初乳外泌体，我们将在这个项目中应用我们的技术。外泌体方面的知识和丰富的经验，可有效靶向递送 Cas9 介导的基因组为了建立可行性，我们使用 pDNA 来提供 Cas9 介导的编码序列。敲除 NFκB 作为模型基因。这个单一质粒 pKO-NFκB 包含哺乳动物优化的 Cas9。编码序列、NFκB 特异性的单向导 RNA (sgRNA) 以及衍生向导 RNA 的序列 (gRNA) 支架协助 Cas9 与靶 DNA 的结合我们以离子方式追踪 pKO-NFκB。包裹在一种新型外泌体基质中，该基质由外泌体和聚阳离子聚乙烯亚胺复合而成 (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，将靶向过度表达 LF 的支气管上皮因此，鼻内 (i.n.) 施用的 LF-EPM-pKO-NFκB 将靶向肺部，且脱靶效应最小。我们的假设得到了令人信服的初步数据的支持：高负载。将核酸转移到 EPM 上并防止外泌体降解，通过表面结合 LF 实现外泌体功能化 LF-EPM 递送的 pKO-NFκB 负载、抑制 H2030 肺癌细胞中 NFκB 的表达，小鼠肺中 LF 受体肠蛋白（也称为网膜蛋白）的过度表达，以及主要的递送通过鼻内递送将 LF 功能化的外泌体输送至小鼠肺部，具有外泌体经验的研究人员，药物递送和生物科学将追求以下具体目标：目标 1. 优化靶向递送使用体外工程外泌体开发 CRISPR/Cas9 基因组编辑工具。目标 2. 确定潜力。用于 CRISPR/Cas9 靶向递送的工程化外泌体的毒性、生物分布和功效如果我们成功实现这些里程碑，我们将进入第二阶段的结果。该项目将为在疾病中推进基因组编辑工具交付“平台”提供可行性数据结合超速离心从生物相容性来源中经济高效地分离外泌体 PI 实验室目前正在开发的独立方法使外泌体生产商业化随着这种新颖的递送技术的进步，它的可行性。