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

摘要( 好了！ 我们的目标是开发新的纳米平台，用于有效地体内传递基因组编辑 用于特定细胞类型和组织的机械；无毒和可以 用于人类的治疗性基因组编辑。关于纳米载体，我们将设计出 创新的戊型肝炎病毒纳米粒子(HEVNP)由郑氏(其中一种)开发 实验室，以便细胞特异性靶向/内吞配体将显示在病毒上 纳米粒子表面，基因组编辑机和内溶肽将被 被封装在核心内部。HEVNP对胃酸环境具有抵抗力，因此可以 通过口服途径将有效载荷运送到肠道。我们假设通过引入隐形技术 病毒衣壳蛋白上的多肽元件和细胞型特异性靶向配体，如新的 纳米载体可以将基因编辑机制传递给特定类型的细胞，不仅通过口服途径， 也可以通过静脉途径。我们的目标是编辑肠腺瘤性息肉细胞的基因组， ApcMin/小鼠(家族性小鼠模型)的肠上皮细胞和肠干细胞 腺瘤性息肉病)，通过纠正突变的APC肿瘤抑制基因，导致 防止新的息肉形成和现有息肉的消退(大小和数量)。 我们将采用使能一珠一化合物(OBOC)和一珠二化合物 (OB2C)组合文库方法(由其中一位PI林博士发明)以开发D-氨基 含酸靶向/内吞配体抗肠腺瘤性息肉细胞 ApcMin/小鼠的肠上皮细胞和肠干细胞。我们将使用OBOC方法来 发现具有内溶活性的膜活性多肽。这些内溶肽 在pH=5时与内体结合，在pH=7.2时不与质膜结合。具体目标如下： UG3(阶段1) 目的1：设计和合成用于发现(A)细胞的OB2C/OBOC组合文库。 特异性靶向和内吞多肽，以及(B)内溶多肽。 目的2：(A)设计具有细胞特异性靶向配体的戊型肝炎病毒纳米粒(HEVNP)， (B)设计用于绿色荧光蛋白、荧光素酶和APC校正的基因组编辑机制；及(C) 组装一系列包裹基因组机制和内溶作用的纳米结构 多肽。 目的3：体外评价来自Aim 2的纳米结构的基因组编辑功能 小鼠肠系膜上皮细胞和腺瘤性息肉细胞及其体内(口服和静脉注射)Apcmin/ 老鼠。 UH3(第二阶段) 目标1、2和3：扩大基因组编辑纳米传输平台的生产。表演，表演 小鼠和猕猴的临床前药理学/毒理学研究。与之协作 SCGE大型动物检测中心的研究人员对输送系统进行了验证。 好了！

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. !