Cell-specific nanocarrier with endocytic and endosomolytic activities for therapeutic genome editing

具有内吞和内体溶解活性的细胞特异性纳米载体，用于治疗性基因组编辑

基本信息

批准号：
10227681
负责人：
R.Holland Cheng
金额：
$ 78.48万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
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 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10227681
关键词：
APC gene Adenomatous Polyps Amino Acids Animal Model Animal Testing ApcMin/+ mice Binding Biological Assay Blood Circulation Capsid Proteins Cell membrane Cells Chemical Structure Computer software Cryoelectron Microscopy Cytoplasm DNA Vaccines DNA delivery Elements Encapsulated Endocytosis Endosomes Engineering Enterocytes Environment Epithelial Cells Exposure to Familial Adenomatous Polyposis Syndrome Funding Galectin 1 Gastric Acid Genes Genome Goals Guide RNA Hepatitis E Human Image Immobilization In Vitro Incubated Intestines Intravenous Label Laboratories Libraries Ligands Lipids Liposomes Luciferases Macaca mulatta Medical Oncology Membrane Methods Modeling Mus Nano delivery Oral Peptide Library Peptides Periodicity Pharmacology Pharmacology and Toxicology Phase Polyps Polystyrenes Prevention Production Random Peptide Libraries Reporting Research Personnel Resistance Rhodamine Route Series Site Specificity Surface System Technology Testing Therapeutic Uses Tissues Tumor Suppressor Genes Validation Viral Xenograft Model biological systems brain endothelial cell cell type cellular targeting combinatorial combinatorial chemistry design genome editing high throughput screening in vivo inhibitor/antagonist innovation intestinal epithelium macrophage mouse model mutant nanocarrier nanotechnology platform nanotheranostics novel particle peptide B pre-clinical scale up screening skills small molecule stem cells structural biology therapeutic genome editing tumor tumor xenograft uptake viral nanoparticle

项目摘要

Abstract( ! Our goal is to develop novel nanoplatforms for efficient in vivo delivery of genome editing machinery into specific cell types and tissues; nanodelivery systems that are non-toxic and can be used for therapeutic genome editing in human. Regarding nanocarrier, we will engineer the innovative hepatitis E viral nanoparticle (HEVNP) developed by the Cheng (one of the PIs) laboratory, such that cell specific targeting/endocytic ligand will be displayed on the viral nanoparticle surface, and genome editing machinery and endosomolytic peptides will be encapsulated inside the core. HEVNP is resistant to gastric acid environment and therefore can deliver payload to intestine via oral route. We hypothesize that through introduction of stealth peptide elements and cell-type specific targeting ligand on the viral capsid proteins, such novel nanocarrier can deliver gene editing machinery to specific cell type, not only via the oral route, but also via the intravenous route. Our goal is to genome edit intestinal adenomatous polyp cells, intestinal epithelial cells, and intestinal stem cells of ApcMin/+ mice (a mouse model of familial adenomatous polyposis) by correcting a mutant Apc tumor suppressor gene, leading to prevention of new polyp formation and regression of existing polyps (size & number). We will employ the enabling one-bead one-compound (OBOC) and one-bead two-compound (OB2C) combinatorial library methods (invented by Dr. Lam, one of the PIs) to develop D-amino acid containing targeting/endocytic ligands against intestinal adenomatous polyp cells, intestinal epithelial cells, and intestinal stem cells of ApcMin/+ mice. We will use the OBOC method to discover membrane active peptides with endosomolytic activities. These endosomolytic peptides will bind to endosome at pH5 but not to plasma membrane at pH7.2. Specific aims are as follows: UG3 (Phase 1) Aim 1: To design and synthesize OB2C/OBOC combinatorial libraries for the discovery of (a) cell- specific targeting and endocytic peptides, and (b) endosomolytic peptides. Aim 2: (a) To engineer hepatitis E viral nanoparticle (HEVNP) with cell-specific targeting ligands, (b) to design genome editing machinery for GFP, luciferase and Apc correction, and (c) to assemble a series of nanoconstructs encapsulating the genome machinery and endosomolytic peptides. Aim 3: To evaluate the genome editing functions of the nanoconstructs from aim 2, in vitro with murine intestinal enterocytes and adenomatous polyp cells, and in vivo (oral & iv) with Apcmin/+ mice. UH3 (Phase 2) Aim 1, 2 & 3: To scale up the production of the genome editing nanodelivery platform. To perform preclinical pharmacology/toxicology studies in mouse and Rhesus macaques. To collaborate with investigator from SCGE Large Animal Testing Centers on validation of the delivery system. !

抽象的（呢我们的目标是开发新型的纳米植物，以在基因组编辑的体内有效递送机械成特定的细胞类型和组织；无毒的纳米传递系统用于人类的治疗基因组编辑。关于纳米载体，我们将设计创新的肝炎病毒纳米颗粒（HEVNP）由Cheng（PIS之一）开发实验室，使细胞特异性靶向/内吞配体将显示在病毒上纳米颗粒表面以及基因组编辑机械和内溶胶肽将是封装在核心内。 HEVNP对胃酸环境具有抵抗力，因此可以通过口头途径将有效载荷传递到肠道。我们假设通过引入隐形肽元素和细胞类型特异性靶向配体在病毒式衣壳蛋白上，这种新型纳米载体可以将基因编辑机械传递到特定的细胞类型，不仅是通过口服途径，而且还通过静脉路线。我们的目标是基因组编辑肠道腺瘤细胞，肠上皮细胞和apcmin/+小鼠的肠道干细胞（家族性的小鼠模型腺瘤性息肉病）通过校正突变的APC抑制基因，导致预防新的息肉形成和现有息肉的回归（尺寸和数字）。我们将采用启用的单珠一体化合物（OBOC）和一孔的两种化合物（OB2C）组合图书馆方法（由PI的Lam博士发明）来开发D-Amino 含有靶向/内吞配体的酸肠腺瘤细胞，肠道上皮细胞和Apcmin/+小鼠的肠干细胞。我们将使用OBOC方法发现具有内溶液活性的膜活性肽。这些内溶胶肽将在pH5时与内体结合，但不与pH7.2的质膜结合。具体目的如下： UG3（阶段1）目标1：设计和合成OB2C/OBOC组合库以发现（a）细胞 - 特定的靶向和内吞肽，以及（b）内溶胶肽。目标2：（a）用特异性靶向配体来设计乙型肝炎病毒纳米颗粒（HEVNP），（b）设计用于GFP，荧光素酶和APC校正的基因组编辑机械，以及（c）组装一系列封装基因组机制和内溶血的纳米结构肽。目标3：评估AIM 2的纳米构造的基因组编辑功能，并在体外与鼠肠肠上皮细胞和腺瘤息肉细胞，以及带有apcmin/+的体内（口腔和IV）老鼠。 UH3（第2阶段）目标1、2和3：扩展基因组编辑纳米传递平台的生产。执行小鼠和恒河猕猴中的临床前药理学/毒理学研究。与之合作 SCGE大型动物测试的研究人员关于验证系统验证的中心。呢