Nanoparticle-based gene editing delivery systems

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Non-viral gene therapy strategies provide several advantages with regards to safety profile, localized gene expression and cost-effective manufacturing compared to viral systems. Non-viral gene delivery systems are engineered complexes or nanoparticles (NPs) composed of the required nucleic acid (DNA or RNA) and a single or a combination of biomaterials such as cationic lipids, surfactants, peptides, polysaccharides, metals or synthetic polymers. The "bottom-up" design flexibility in selecting complexing agents, targeting moieties and other components provides several key advantages over viral delivery vectors. Recent advancements, including results from our laboratory, have brought nanoparticle-based non-viral gene delivery vectors one step closer to the clinic. Among these, lipid/surfactant-based systems are highly promising with 12 major liposome systems being investigated in 26 clinical trials. Non-viral delivery systems offer unique advantages for gene editing as well. However, the development of the delivery systems for the components of gene editing tools require new and more complex considerations. Improvements in both the structure and function of these systems are required for in vivo targeting, intracellular delivery and proper routing of the gene editing components. The three strategies to edit with CRISPR-Cas9 , i.e. (1) using plasmid based system encoding Cas9 protein and sgRNA; 2) using mixture of Cas9 mRNA and sgRNA; or 3) using Cas9 protein and sgRNA ribonucleoprotein complexes (RNPs)) may work provided the obstacles related to gene-editing efficiency, off-target effects and target specificity can be solved. The proposed research program will focus on both fundamental and applied aspects of delivery system design for CRISPR-Cas gene editing systems. We propose developing in vivo-stable, immune neutral, intracellular delivery systems for CRISPR-Cas gene editing based on composite nanoparticles using self-assembling and targetable biomaterials, peptide-gemini surfactant conjugates (pGs) and phospholipids. The objectives of the research program will include (1) the development of libraries of neutral, positively and negatively charged gene editing complexing agents (peptides, pGs) that will enable the rational design of CRISPR-Cas-NP complexes with suitable stability in in vivo environments, (2) the investigation of novel techniques for assembly of pG nanoparticles and CRISPR/Cas9 complexes, (3) physicochemical characterization of the pG-CRISPR/Cas9 NPs self-assembly process and formation of NPs with or without helper phospholipids (4) investigation of delivery and/or transfection efficiency, biocompatibility and functionality of pG-CRISPR/Cas9 NPs ; (5) the investigation of thee intracellular fate pG-CRISPR/Cas9 NPs by quantitative confocal fluorescence cross-correlation spectroscopy.