Nanoparticle-based gene editing delivery systems

基于纳米颗粒的基因编辑传递系统

• 批准号: RGPIN-2020-06002
• 负责人: Foldvari, Marianna
• 金额: $ 4.01万
• 依托单位: University of Waterloo
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740968
• 关键词: Nanoparticle; based; gene; editing; delivery

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.

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多种优势。非病毒基因传递系统是由所需的核酸（DNA或RNA）和单个或多种生物材料（例如阳离子脂质、表面活性剂、肽、多糖、金属或合成聚合物）组成的工程复合物或纳米颗粒（NP）。在选择络合剂、靶向部分和其他成分方面的“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势。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，基于脂质/表面活性剂的系统非常有前景，26 项临床试验正在研究 12 种主要脂质体系统。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传输系统的开发需要新的、更复杂的考虑。基因编辑组件的体内靶向、细胞内递送和正确路由需要改进这些系统的结构和功能。 CRISPR-Cas9编辑的三种策略，即（1）使用基于质粒的系统编码Cas9蛋白和sgRNA； 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）可能会起作用，前提是与基因编辑效率、脱靶效应和靶标特异性相关的障碍能够得到解决。拟议的研究计划将重点关注 CRISPR-Cas 基因编辑系统的传递系统设计的基础和应用方面。我们建议使用自组装和靶向生物材料、肽-双子表面活性剂缀合物（pGs）和磷脂，基于复合纳米粒子开发用于CRISPR-Cas基因编辑的体内稳定、免疫中性、细胞内递送系统。该研究计划的目标将包括（1）开发中性、带正电和带负电的基因编辑复合物（肽，pG）文库，从而能够合理设计在体内环境中具有适当稳定性的 CRISPR-Cas-NP 复合物，（2）研究 pG 纳米颗粒和 CRISPR/Cas9 复合物组装的新技术，（3） pG-CRISPR/Cas9 NPs 自组装过程以及有或没有辅助磷脂的 NPs 形成 (4) pG-CRISPR/Cas9 NPs 的递送和/或转染效率、生物相容性和功能的研究； (5)通过定量共焦荧光互相关光谱研究pG-CRISPR/Cas9 NPs的细胞内命运。