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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.

技术文摘