Technical Genetic mutations have been identified as a causative factor in numerous diseases. The genome editingsystem CRISPR/Cas9 is a recent development in gene therapy. Both viral and non-viral vectors have been usedin attempts to direct delivery of Cas9 to specific locations with advantages and limitations similar to those knownfor other nucleic acid-based therapeutics. These challenges have limited the current clinical progress of thisgenome-editing tool. The goal of this project is to develop an effective targeted delivery system for Cas9-mediatedgenome 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 ourknowledge 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-mediatedknockout of NFκB as a model gene. This single plasmid, pKO-NFκB, contains the mammalian-optimized Cas9coding 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, ionicallyentrapped 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 LFreceptors. Thus, LF-EPM-pKO-NFκB administered intranasally (i.n.) will target lung with minimal off-target effectsfor delivery of this genome-editing tool. Our hypothesis is supported by compelling preliminary data: high loadingof nucleic acid onto EPM and protection from degradation, functionalization of exosomes by surface-bound LFloading, 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 ofLF-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 deliveryof CRISPR/Cas9 genome-editing tool using engineered exosomes in vitro. Aim 2. Determine potentialtoxicity, and biodistribution and efficacy of engineered exosomes for targeted delivery of CRISPR/Cas9genome-editing tool. If we are successful in achieving these milestones, we will move to Phase II. Results fromthis project will provide feasibility data for advancing this genome-editing tool delivery `platform' in a diseasemodel. 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 commercialviability as this novel delivery technology advances.
Public Health Relevance Statement: Narrative (Public Health Relevance Statement)
In this project, we will develop novel nano formulations of the genome-editing machinery (CRISPR Cas9)
using bovine colostrum exosomes complexed with a polycationic matrix. Exosomes will be attached to milk-
derived lactoferrin to target bronchial epithelial cells minimizing off-target effects. This novel and efficient
delivery technology will be developed using plasmid DNA containing all necessary components of this genome
editing tool targeting NFκB, a hallmark of inflammation and disease development in the lung and other organs,
using established cell culture and animal models.
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