3P Biotechnologies, Inc.

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.