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

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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.