Engineered exosomes for targeted delivery of the crispr/CAS9 genome-editor - 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 NFB as a model gene. This single plasmid, PKO-NFB, contains the mammalian-optimized CAS9 coding sequence, the single-guide RNA (SGRNA) specific to NFB, 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-NFB, ionically entrapped in a novel exosome matrix, formulated by complexing exosomes and polycationic polyethyleneimine (PEI), will serve as an effective genome-editing tool of NFB. Furthermore, use of engineered exosomes, prepared by loading milk lactoferrin (LF) onto exosomes, will target bronchial epithelium overexpressing LF receptors. Thus, LF-EPM-PKO-NFB 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 NFB expression in H2030 lung cancer cells by LF-EPM delivered PKO-NFB, 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.