Nanoparticle-based gene editing delivery systems

与病毒系统相比，非病毒基因治疗策略在安全性、局部基因表达和成本效益方面提供了几个优势。非病毒基因递送系统是由所需的核酸（DNA或RNA）和单一或组合的生物材料（例如阳离子脂质、表面活性剂、肽、多糖、金属或合成聚合物）组成的工程化复合物或纳米颗粒（NP）。在选择络合剂、靶向部分和其他组分方面的“自下而上”设计灵活性提供了优于病毒递送载体的几个关键优点。最近的进展，包括我们实验室的结果，使基于纳米颗粒的非病毒基因递送载体更接近临床。其中，脂质/表面活性剂为基础的系统是非常有前途的12个主要的脂质体系统正在研究中的26个临床试验。 非病毒递送系统也为基因编辑提供了独特的优势。然而，基因编辑工具组件的递送系统的开发需要新的和更复杂的考虑。这些系统的结构和功能的改进对于基因编辑组分的体内靶向、细胞内递送和适当路由是必需的。用CRISPR-Cas9编辑的三种策略，即（1）使用编码Cas9蛋白和sgRNA的基于质粒的系统; 2）使用Cas9 mRNA和sgRNA的混合物;或3）使用Cas9蛋白和sgRNA核糖核蛋白复合物（RNP））可以起作用，只要可以解决与基因编辑效率、脱靶效应和靶特异性相关的障碍。 拟议的研究计划将侧重于CRISPR-Cas基因编辑系统的基础和应用方面的递送系统设计。我们建议开发体内稳定的，免疫中性的，基于复合纳米颗粒的CRISPR-Cas基因编辑细胞内递送系统，使用自组装和靶向生物材料，肽-双子表面活性剂缀合物（PG）和磷脂。 该研究计划的目标将包括（1）开发中性、带正电荷和带负电荷的基因编辑络合剂库（2）研究用于组装pG纳米颗粒和CRISPR/Cas9复合物的新技术，（3）pG-CRISPR/Cas9 NP自组装过程的物理化学表征和具有或不具有辅助磷脂的NP的形成（5）通过定量共聚焦荧光互相关光谱研究pG-CRISPR/Cas9 NP的细胞内命运。

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-CasNP 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.