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

摘要（ ! 我们的目标是开发新型纳米平台，用于基因组编辑的有效体内递送 纳米递送系统是无毒的，并且可以 用于人类的治疗性基因组编辑。关于纳米载体，我们将设计 由Cheng（PI之一）开发的创新戊型肝炎病毒纳米颗粒（HEVNP） 实验室，使得细胞特异性靶向/内吞配体将展示在病毒载体上。 纳米颗粒表面，基因组编辑机制和内体溶解肽将被 包裹在核心里HEVNP对胃酸环境具有抗性，因此可以 通过口服途径将有效载荷递送至肠道。我们假设通过引入隐形技术 肽元件和细胞类型特异性靶向配体，这种新的 纳米载体可以将基因编辑机制递送到特定细胞类型，不仅通过口服途径， 也可以通过静脉途径。我们的目标是对肠腺瘤性息肉细胞进行基因组编辑， 肠上皮细胞和肠干细胞的ApcMin/+小鼠（家族性肠上皮细胞瘤的小鼠模型）， 腺瘤性息肉病）通过纠正突变的Apc肿瘤抑制基因，导致 防止新息肉形成和现有息肉的消退（大小和数量）。 我们将采用使能的一珠一化合物（OBOC）和一珠两化合物 （OB 2C）组合文库方法（由PI之一的Dr. Lam发明）开发D-氨基 针对肠腺瘤性息肉细胞的含酸靶向/内吞配体，肠 上皮细胞和肠干细胞。我们将使用OBOC方法， 发现具有内体溶解活性的膜活性肽。这些内体溶解肽 在pH 5时与内体结合，在pH7.2时不与质膜结合。具体目标如下： UG 3（第1阶段） 目的1：设计和合成OB 2C/OBOC组合文库，用于发现（a）细胞- 特异性靶向和内吞肽，和（B）内体溶解肽。 目的2：（a）构建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GFP、荧光素酶和Apc校正的基因组编辑机器，以及（c） 组装一系列封装基因组机器和内体溶解的纳米结构， 缩氨酸 目的3：在体外用DNA测序技术评估来自目的2的纳米构建体的基因组编辑功能。 小鼠肠上皮细胞和腺瘤性息肉细胞，以及体内（口服和静脉注射）Apcmin/+ 小鼠 UH 3（第2阶段） 目标1、2和3：扩大基因组编辑纳米递送平台的生产规模。执行 小鼠和恒河猴的临床前药理学/毒理学研究。协作 来自SCGE大型动物试验中心的研究者对输送系统进行了验证。 !

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. !