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制造方面具有若干优势。非病毒基因传递系统是由所需的核酸（DNA或RNA）和单一或组合的生物材料（如阳离子脂质、表面活性剂、多肽、多糖、金属或合成聚合物）组成的工程复合物或纳米颗粒（NPs）。“自下而上”的设计灵活性在选择络合剂，靶向部分和其他组件提供了几个关键的优势比病毒传递载体。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传递载体更接近临床。其中，脂质/表面活性剂为基础的系统是非常有前途的，有12个主要的脂质体系统正在26个临床试验中进行研究。非病毒传递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发展需要新的和更复杂的考虑。这些系统的结构和功能都需要改进，以实现体内靶向、细胞内递送和基因编辑组件的正确路径。利用CRISPR-Cas9进行编辑的三种策略是：(1)利用基于质粒的系统编码Cas9蛋白和sgRNA；2) Cas9 mRNA与sgRNA的混合表达；或3)利用Cas9蛋白和sgRNA核糖核蛋白复合物（RNPs）可以解决与基因编辑效率、脱靶效应和靶标特异性相关的障碍。拟议的研究计划将侧重于CRISPR-Cas基因编辑系统的传递系统设计的基础和应用方面。我们建议开发体内稳定的、免疫中性的、细胞内的CRISPR-Cas基因编辑递送系统，该系统基于复合纳米颗粒，使用自组装和可靶向的生物材料、肽-gemini表面活性剂偶联物（pGs）和磷脂。该研究计划的目标将包括(1)开发中性、带正电和负电的基因编辑络合剂（肽，pG）文库，这将使合理设计在体内环境中具有适当稳定性的CRISPR- cas - np复合物成为可能；(2)研究pG纳米颗粒和CRISPR/Cas9复合物组装的新技术。(3)有或无辅助磷脂的pG-CRISPR/Cas9 NPs自组装过程和NPs形成的理化特性(4)pG-CRISPR/Cas9 NPs的传递和/或转染效率、生物相容性和功能的研究；(5)定量共聚焦荧光相互关联光谱法研究三种细胞内命运的pG-CRISPR/Cas9 NPs。

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.