Delivery Technologies for In Vivo Genome Editing

项目摘要 迫切需要新的体内传递技术来实现对体细胞的选择性基因组编辑 细胞不受现有病毒传递系统或脂质纳米粒的限制。我们建议开发两个 互为补充的战略。首先，通过将Cas9和碱基编辑核糖核蛋白(RNP)捆绑在一起来定位 部分，如抗体或核酸适配子，我们将开发能够编辑 目标细胞的特定群体。作为第二种方法，我们将设计类似病毒的颗粒(VLP)来促进 高效、组织和细胞特定的基因组编辑剂递送。在此过程中，我们将发展交付 能够靶向造血干细胞和祖细胞(HSPC)以及其他组织的系统。 为了评估我们建议的交付方法的效率和细胞类型特异性，我们还将生成一个 定量和灵敏地报告来自基本编辑人员或可编程人员的基因组编辑的报告鼠标 核酸酶，不需要DNA测序。在这项建议中，我们打算： (1)设计靶向核糖核蛋白结合物，选择性结合、进入和编辑靶细胞。细胞 组织选择性Cas9和碱基编辑RNP递送系统将通过拴系基因组编辑来设计 蛋白质，直接或间接地与适体和抗体靶向部分。动力学、震级和 将确定RNP内吞、内体逃逸和核运输的特异性，并进行基因组编辑 在体外和体内测定效率和靶向特异性。 (2)用于细胞和组织靶向基因组的工程核糖核蛋白纳米粒递送系统 正在编辑。SV40衣壳蛋白将被改造成病毒样颗粒(VLP)，能够 包装核糖核蛋白，而不是DNA。VLP-RNP递送系统的化学计量比提供了 最优的细胞摄取、内体逃逸和核运输将被定义。目标特异性，如 将确定由病毒衣壳嗜性确定，并分析基因组编辑效率在体外和体内。 (3)开发一种报告小鼠，用于轻松评估靶向基因组编辑效率和细胞-和 组织类型特异性。我们将优化一个报告基因，以独立检测碱基编辑、末端连接或 同源定向修复。将该记者整合到C57BL/6小鼠的rosa26避风港基因中 胚胎干细胞以产生转基因小鼠。基因组编辑结果将通过以下方式进行评估 荧光和发光测量，并与高通量DNA测序相关。 (4)展示在非人类灵长类动物中安全有效地传递基因组编辑剂。这个 将基因组编辑传送到HSPC和其他目标组织将在恒河猴身上进行评估。两者都有 将对哺乳动物和非哺乳动物系统进行评估，以优化基因组的大规模生产 编辑器和相关的RNP交付组件。靶向特异性和基因组编辑效率，以及 安全性将在体内进行分析。我们期待找到适合临床试验的有效给药系统。

Project Summary New in vivo delivery technologies are urgently needed that enable selective genome editing of somatic cells without the limitations of existing viral delivery systems or lipid nanoparticles. We propose to develop two complementary strategies. First, by tethering Cas9 and base editor ribonucleoproteins (RNPs) to homing moieties, such as antibodies or nucleic acid aptamers, we will develop delivery systems capable of editing a specific population of target cells. As a second approach, we will engineer viral like particles (VLPs) to facilitate efficient, tissue and cell specific delivery of genome editing agents. In the process, we will develop delivery systems that are capable of targeting hematopoietic stem and progenitor cells (HSPCs), among other tissues. To evaluate the efficiency and cell-type specificity of our proposed delivery methods, we will also generate a reporter mouse that quantitatively and sensitively reports genome editing from base editors or programmable nucleases without requiring DNA sequencing. In this proposal, we intend to: (1) Design targeted ribonucleoprotein conjugates that selectively bind, enter, and edit target cells. Cell and tissue selective Cas9 and base editor RNP delivery systems will be designed by tethering genome editing proteins, directly or indirectly, to aptamer and antibody targeting moieties. The kinetics, magnitude, and specificity of RNP endocytosis, endosomal escape, and nuclear transport will be defined and genome editing efficiency and targeting specificity determined in vitro and in vivo. (2) Engineer ribonucleoprotein nanoparticle delivery systems for cell and tissue targeted genome editing. SV40 capsid proteins will be engineered to form viral like particles (VLPs) that are capable of packaging ribonucleoproteins, rather than DNA. The stoichiometry of VLP-RNP delivery systems, which affords optimal cell uptake, endosomal escape, and nuclear transport will be defined. Targeting specificity, as determined by viral capsid tropism will be defined, and genome editing efficiency analyzed in vitro and in vivo. (3) Develop a reporter mouse for facile assessment of targeted genome editing efficiency and cell- and tissue-type specificity. We will optimize a reporter gene to independently detect base editing, end-joining, or homology-directed repair. The reporter will be integrated into the Rosa26 safe harbor locus in C57BL/6 mouse embryonic stem cells to generate transgenic mice. Genome editing outcomes will be evaluated by fluorescence and luminescence measurements and correlated with high throughput DNA sequencing. (4) Demonstrate safe and effective delivery of genome editing agents in non-human primates. The delivery of genome editors to HSPCs and other target tissues will be assessed in rhesus macaques. Both mammalian and non-mammalian systems will be evaluated to optimize large scale production of the genome editor and related RNP delivery components. Targeting specificity and genome editing efficiency, as well as safety will be analyzed in vivo. We anticipate identifying effective delivery systems suitable for clinical trials.