Delivery Technologies for In Vivo Genome Editing

体内基因组编辑的传递技术

基本信息

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
项目类别：
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

项目摘要

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.

项目概要迫切需要新的体内递送技术来实现体细胞的选择性基因组编辑细胞不受现有病毒递送系统或脂质纳米粒子的限制。我们建议开发两个互补策略。首先，通过将 Cas9 和碱基编辑核糖核蛋白 (RNP) 束缚到归巢部分，例如抗体或核酸适体，我们将开发能够编辑的递送系统特定的靶细胞群。作为第二种方法，我们将设计病毒样颗粒（VLP）以促进基因组编辑剂的高效、组织和细胞特异性递送。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）等组织的系统。为了评估我们提出的递送方法的效率和细胞类型特异性，我们还将生成报告鼠标，可定量、灵敏地报告碱基编辑器或可编程的基因组编辑核酸酶，无需 DNA 测序。在本提案中，我们打算： (1) 设计选择性结合、进入和编辑靶细胞的靶向核糖核蛋白缀合物。细胞组织选择性 Cas9 和碱基编辑器 RNP 传递系统将通过束缚基因组编辑来设计蛋白质直接或间接地结合到适体和抗体靶向部分。动力学、幅度和 RNP 内吞作用、内体逃逸和核运输的特异性将被定义并进行基因组编辑体外和体内确定的效率和靶向特异性。 (2) 设计用于细胞和组织靶向基因组的核糖核蛋白纳米粒子递送系统编辑。 SV40衣壳蛋白将被设计成病毒样颗粒（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，可提供将定义最佳细胞摄取、内体逃逸和核运输。目标特异性，如将定义由病毒衣壳向性决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。将报告基因整合到C57BL/6小鼠中的Rosa26安全港位点胚胎干细胞产生转基因小鼠。基因组编辑结果将由以下机构评估荧光和发光测量并与高通量 DNA 测序相关。 (4) 证明在非人类灵长类动物中安全有效地递送基因组编辑剂。这将在恒河猴中评估基因组编辑器向 HSPC 和其他靶组织的传递。两个都将评估哺乳动物和非哺乳动物系统，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。我们期望找到适合临床试验的有效输送系统。