Delivery Technologies for In Vivo Genome Editing

体内基因组编辑的传递技术

• 批准号: 10222522
• 负责人: Elliot Chaikof
• 金额: $ 71.1万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-22 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10222522
• 关键词: 3T3 Cells; Animal Testing; Animals; Antibodies; Base Pairing; Binding; C57BL/6 Mouse; Capsid; Capsid Proteins; Cells; Clinical Trials; Collaborations; DNA; DNA Double Strand Break; DNA delivery; DNA sequencing; Development; Endocytosis; Engineering; Exposure to; Flow Cytometry; Fluorescence; Genetic Diseases; Genome; Hematopoietic stem cells; High-Throughput DNA Sequencing; High-Throughput Nucleotide Sequencing; Homing; In Vitro; Individual; Injections; Kinetics; Lipids; Liver; Luminescent Measurements; Macaca mulatta; Mediating; Methods; Mitotic; Modification; Mus; Mutate; Nucleic Acids; Oncogenic; Outcome; Patients; Point Mutation; Process; Proteins; RNA; Reagent; Reporter; Reporter Genes; Reporting; Ribonucleoproteins; Risk; Safety; Simian virus 40; Site; Somatic Cell; Specificity; System; Target Populations; Technology; Tissues; Transgenic Mice; Tropism; Viral; aptamer; base; cell type; clinical development; clinical translation; clinically relevant; design; embryonic stem cell; genome editing; immunogenicity; improved; in vivo; insertion/deletion mutation; large scale production; lipid nanoparticle; luminescence; nanoparticle delivery; nonhuman primate; novel; nuclease; nucleocytoplasmic transport; particle; programs; protective allele; repaired; somatic cell gene editing; stoichiometry; tool; uptake; whole body imaging

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.

项目摘要 迫切需要新的体内递送技术，其使得能够对体细胞的基因组进行选择性基因组编辑。 细胞，而不受现有病毒递送系统或脂质纳米颗粒的限制。我们建议发展两个 互补战略。首先，通过将Cas9和碱基编辑器核糖核蛋白（RNP）连接到归巢， 对于诸如抗体或核酸适体的部分，我们将开发能够编辑核酸适体的递送系统。 靶细胞的特定群体。作为第二种方法，我们将设计病毒样颗粒（VLP）， 高效、组织和细胞特异性递送基因组编辑剂。在此过程中，我们将开发交付 能够靶向造血干细胞和祖细胞（HSPC）以及其他组织的系统。 为了评估我们提出的递送方法的效率和细胞类型特异性，我们还将生成一个 定量且灵敏地报告来自碱基编辑器的基因组编辑的报告小鼠或可编程的 不需要DNA测序。在本提案中，我们打算： (1)设计靶向核糖核蛋白缀合物，选择性地结合，进入和编辑靶细胞。细胞 组织选择性Cas9和碱基编辑器RNP递送系统将通过系留基因组编辑来设计 蛋白质直接或间接地与适体和抗体靶向部分连接。动力学、强度和 将定义RNP内吞、内体逃逸和核转运的特异性，并进行基因组编辑。 在体外和体内测定的效率和靶向特异性。 (2)用于细胞和组织靶向基因组的工程核糖核蛋白纳米颗粒递送系统 编辑. SV 40衣壳蛋白将被工程化以形成病毒样颗粒（VLP），其能够 包装核糖核蛋白，而不是DNA。VLP-RNP递送系统的化学计量，其提供了 将定义最佳细胞摄取、内体逃逸和核转运。靶向特异性，如 将定义通过病毒衣壳向性确定的基因组编辑效率，并在体外和体内分析基因组编辑效率。 (3)开发报告小鼠，用于靶向基因组编辑效率和细胞-和 组织类型特异性。我们将优化报告基因，以独立检测碱基编辑，末端连接或 同源定向修复报告基因将整合到C57 BL/6小鼠的Rosa 26安全港基因座中 胚胎干细胞来产生转基因小鼠。基因组编辑结果将由以下人员进行评估： 荧光和发光测量并与高通量DNA测序相关。 (4)证明基因组编辑剂在非人类灵长类动物中的安全有效递送。的 将在恒河猴中评估基因组编辑器向HSPC和其它靶组织的递送。两 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 将在体内分析安全性。我们期望确定适用于临床试验的有效输送系统。