Project Summary / Abstract This applications long-term objective is to develop a platform technology enabling in vivo genetic engineering technologies including DNA vaccination and precision CRISPR gene editing. A challenge in utilizing CRISPR technologies in vivo is the need to simultaneously deliver the DNA endonuclease enzyme, guide RNA, and payload DNA. Integrated nanosecond pulse intradermal reversible electroporation (INSPIRE) is a novel technique for enhancing in vivo delivery of these components. INSPIRE rapidly transports large molecules into cells by using ultrashort electrical pulses to simultaneously induce temporary nanoscale defects in the cell membrane and drive the target molecules against concentration gradients via electrokinetic drift. This proposal will demonstrate the feasibility of INSPIRE for in vivo genetic engineering in a 3D in vitro skin model and a porcine dermal model via three specific aims. 1) Development of a Hand Held System Suitable for Clinical INSPIRE Treatments, 2) Optimization of INSPIRE Protocols in a Laboratory Model, and 3) Feasibility of INSPIRE Protocols in a Large Animal Model. In Aim 1 we will construct a portable pulse generation system suitable for clinical use with the high voltage components integrated directly into a hand held applicator. In Aim 2 we will optimize protocols for delivering CRISPR cas9 plasmid, cas9 mRNA, and cas9 protein into cells using a 3D in vitro skin mimic. In Aim 3 we will demonstrate the feasibility of delivering these CRISPR components in vivo via a porcine dermal model. Following this Phase I application demonstrating in vivo feasibility, Phase II will focus on design for manufacture and initiate long-term safety/efficacy studies designed to determine serum levels of antibodies produced following transfection with DNA vaccine candidates.