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

技术摘要 基因突变已被确认为多种疾病的致病因素。基因组编辑 CRISPR/Cas9系统是基因治疗领域的最新进展。病毒载体和非病毒载体都已被使用 在试图将Cas9定向递送到具有类似于已知优点和限制的特定位置中 用于其他基于核酸的疗法。这些挑战限制了本病目前的临床进展。 基因组编辑工具。该项目的目标是开发一种有效的针对Cas9介导的靶向递送系统 基因组编辑。研究人员利用一种新的技术来传递质粒DNA(PDNA) 基于PI实验室开发的牛乳/初乳外显体。在这个项目中，我们将应用我们的 在外体方面的知识和丰富的经验，以有效地靶向传递Cas9介导的基因组- 编辑工具。为了确定可行性，我们使用PDNA来传递Cas9介导的编码序列 以敲除核因子κB为模型基因。这个单质粒pKO-NFCAS9 B含有哺乳动物优化的κ 编码序列、针对NFκB的单导引核糖核酸(SGRNA)以及衍生引导核糖核酸的序列 (GRNA)辅助Cas9与靶DNA结合的支架。我们假设PKO-NFκB，离子上 包裹在一种新型的外切体基质中，由外切体和聚阳离子聚乙烯亚胺络合而成 (PEI)，将作为核因子κB的一种有效的基因组编辑工具。此外，利用工程外切体， 通过将牛奶乳铁蛋白(LF)负载到外切体上而制备的，将靶向过度表达LF的支气管上皮 感受器。因此，LF-EPM-PKO-NFκB鼻腔给药(i.n.)将以肺部为目标，最大限度地减少偏离目标的影响 为这个基因组编辑工具的交付。我们的假设有令人信服的初步数据支持：高负荷 核酸在EPM上的结合和表面结合的LF对外切体的降解和功能化的保护 LF-κ-NF-κB对H2030肺癌细胞株表达的抑制作用 LF型受体内毒素(也称为omentin)在小鼠肺组织中的过度表达，以及主要的 LF功能化外切体经鼻腔给药至小鼠肺内。在外显体方面经验丰富的研究人员， 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 使用体外工程外切体的CRISPR/Cas9基因组编辑工具。目标2.确定潜力 靶向递送CRISPR/Cas9的工程外切体的毒性、生物分布和有效性 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。 该项目将为推进这一基因组编辑工具在疾病中的交付“平台”提供可行性数据 模特。从生物相容的来源中经济高效地分离外切体，结合超速离心法- 目前，Pi的实验室正在开发独立的方法，使Exosome的生产成为商业 随着这一新的递送技术的进步，它的可行性。

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.