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. Public Health Relevance Statement Relevance to Public Health CRISPR based technologies have the potential to address a multitude of human malignancies provided that safe and effective mechanisms for precisely delivering the endonuclease, guide RNA, and donor DNA in vivo can be developed. This proposal aims to significantly enhance the size and type of genome editing machinery that can be utilized by developing a minimally invasive pulsed electric field technique for in vivo transmembrane transport of large genome editing molecules.
Project Terms: Animals ; Antibodies ; Malignant Neoplasms ; Cancers ; Malignant Tumor ; malignancy ; neoplasm/cancer ; Cell membrane ; Cytoplasmic Membrane ; Plasma Membrane ; plasmalemma ; Cell Nucleus ; Nucleus ; Cells ; Cell Body ; Charge ; Collagen ; Deoxyribonuclease I ; DNA Endonuclease ; DNase I ; Pancreatic DNase ; Thymonuclease ; DNA ; Deoxyribonucleic Acid ; endonuclease ; Enzymes ; Enzyme Gene ; Epidermis ; Feasibility Studies ; Fibroblasts ; Genes ; Genetic Engineering ; Genetic Engineering Biotechnology ; Genetic Engineering Molecular Biology ; Recombinant DNA Technology ; genetically engineered ; Genome ; Hand ; Human ; Modern Man ; In Vitro ; Laboratories ; Methods ; Muscle Contraction ; Muscle Cell Contraction ; Muscular Contraction ; Muscle ; Muscle Tissue ; muscular ; Pain ; Painful ; Plasmids ; Probability ; Proteins ; Public Health ; Reagent ; Research Design ; Study Type ; study design ; Messenger RNA ; mRNA ; Safety ; Computer software ; Software ; Family suidae ; Pigs ; Suidae ; Swine ; porcine ; suid ; Technology ; Testing ; Time ; Tissues ; Body Tissues ; Transfection ; Transistors ; Transportation ; Vaccination ; silicon carbide ; Generations ; Guide RNA ; gRNA ; Mediating ; Custom ; base ; Label ; improved ; Clinical ; Phase ; Series ; Electroporation ; electroporative delivery ; gene electrotransfer ; Evaluation ; Dermal ; Serum ; Blood Serum ; Subcutaneous Tissue ; Hypodermis ; Subcutis ; Superficial Fascia ; Tela Subcutanea ; subdermal tissue ; electric field ; Naked DNA Vaccines ; Recombinant DNA Vaccines ; DNA Vaccines ; Pulse ; Physiologic pulse ; Msec ; millisecond ; nano second ; nanosecond ; Event ; Protocol ; Protocols documentation ; Techniques ; System ; 3-D ; 3D ; three dimensional ; 3-Dimensional ; Width ; voltage ; Animal Models and Related Studies ; model of animal ; model organism ; Animal Model ; simulation ; novel ; Devices ; Modeling ; portability ; Skin ; preventing ; prevent ; deliver vaccines ; vaccine delivery ; Address ; Dose ; Tissue Viability ; Defect ; Data ; in vivo ; in vivo Model ; Transmembrane Transport ; Membrane Transport ; Process ; Gene Delivery ; Development ; developmental ; design ; designing ; nanoscale ; nano meter scale ; nano meter sized ; nano scale ; nanometer scale ; nanometer sized ; prototype ; minimally invasive ; vaccine candidate ; 3D Print ; 3-D print ; 3-D printer ; 3D printer ; 3D printing ; three dimensional printing ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR ; CRISPR/Cas system ; genome editing ; genomic editing ; CRISPR/Cas technology ; CRISPR method ; CRISPR methodology ; CRISPR technique ; CRISPR technology ; CRISPR-CAS-9 ; CRISPR-based method ; CRISPR-based technique ; CRISPR-based technology ; CRISPR-based tool ; CRISPR/Cas method ; CRISPR/Cas9 ; CRISPR/Cas9 technology ; Cas nuclease technology ; Clustered Regularly Interspaced Short Palindromic Repeats method ; Clustered Regularly Interspaced Short Palindromic Repeats methodology ; Clustered Regularly Interspaced Short Palindromic Repeats technique ; Clustered Regularly Interspaced Short Palindromic Repeats technology ; histological specimens ; histological samples ; histology samples ; histology specimens ; experimental study ; experiment ; experimental research ; efficacy study ; Injections ;
