Harnessing the biology of glycosphingolipid trafficking for biologic drug delivery

利用鞘糖脂运输的生物学特性进行生物药物输送

• 批准号: 9174596
• 负责人: Daniel Jean-Francois Chinnapen
• 金额: $ 44.25万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-04 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9174596
• 关键词: Address; Area; Back; Biology; Blood; Blood Vessels; Brain; Cell membrane; Cells; Ceramides; Complex; Coupled; Couples; Disease; Drug Delivery Systems; Endocytosis; Endothelial Cells; Endothelium; Environment; Epithelial; Epithelial Cells; Esters; Figs - dietary; Glycosphingolipids; Goals; Head; Heart; Image; In Vitro; Inflammatory; Intestines; Link; Lipids; Liver; Lysosomal Storage Diseases; Lysosomes; Mediating; Membrane; Membrane Microdomains; Membrane Protein Traffic; Methods; Modeling; Molecular; Mus; Non-Insulin-Dependent Diabetes Mellitus; Oligosaccharides; Outcome; Pathway interactions; Peptide Hydrolases; Peptide Transport; Peptides; Permeability; Physiological; Process; Proteins; Reporter; Resistance; Shapes; Signal Transduction; Sorting - Cell Movement; Sphingolipids; Stomach; Stream; Structure; Surface; Technology; Testing; Therapeutic; Thick; Tight Junctions; Tissues; Translating; Volatile Fatty Acids; base; clinical application; design; enzyme replacement therapy; extracellular; flotillin; glucagon-like peptide 1; glucose metabolism; in vivo; incretin hormone; intestinal epithelium; molecular carrier; molecular shape; monolayer; mucosal vaccine; non-Native; novel; peptide drug; polarized cell; prevent; solute; sugar; technology development; therapeutic protein; trafficking; transcytosis; uptake

PROJECT SUMMARY The goal of this proposal is to test if a discovery we recently made on glycosphingolipid trafficking in epithelial cells can be translated to clinical application as a platform for drug delivery of biologics. Mucosal surfaces represent vast areas where host tissues are separated from the environment only by a delicate but highly effective single layer of columnar epithelial cells, joined by tight junctions that are impermeable to proteins and even small peptides. So far, the lack of rational and efficient methods to circumvent this barrier has prevented the application of most therapeutic proteins and peptides for mucosal drug delivery. Endothelial cells also form vast and highly restrictive single cell thick barriers that separate most tissues from the blood stream. Most healthy non-inflamed endothelial barriers strongly limit the permeability of large molecules; thus preventing access of many protein-based biologics to cells of many tissues - even when the therapeutic proteins are applied intravenously. Here, we address these problems by testing whether non-native “short-chain” GM1 glycosphingolipids can serve as molecular carriers for drug delivery of peptide and protein biologics. Substantial progress was made since the original submission of this proposal in 2014. Structure-function studies on transepithelial transport of the ceramide domain identified non-native GM1-species that have the combined features of enhanced uptake, transcytosis across epithelial barriers, and efficient release from cell membranes after transport. These GM1 species are studied as vehicles for cargo transport. In Aim 1, we will conclude and expand our preliminary studies testing for GM1-mediated transport of peptide and protein cargoes across mouse epithelial and endothelial barriers in vivo. The GM1 lipids are fused via their extracellular oligosaccharide domain to reporter peptides yielding robust signals for tracking the molecules by imaging and biochemically. We will test for transport of larger cargoes, and for proof of principle by mucosal administration of the non-native GM1 species fused to the incretin hormone GLP-1, as model for treatment of Type II Diabetes. Transport of peptides across tight endothelial barriers (heart and brain) will be tested in vivo. Aim 2 will test for mechanism of transcytosis for the non-native GM1 species. The major hypotheses for sorting by molecular shape or by association with membrane microdomains will be examined. Aim 3 will test if the extracellular oligosaccharide domain of GM1 can be truncated or eliminated while still maintaining functionality of the GM1 ceramide domain in trafficking. We will explore novel structures of the linker peptide to replace functionalities of the oligosaccharide head groups if necessary. In Aim 4, we will test if the linker between GM1 and peptide can be designed to release the cargo after or during transcytosis. We will test if the incorporation of a cleavable ester bond, or a motif for the endosomal- protease furin, can achieve this goal.

项目摘要 该提案的目的是测试我们最近对上皮糖脂贩运的发现是否 可以将细胞转化为临床应用，作为生物制剂药物输送的平台。 粘膜表面代表了广阔的区域，其中宿主组织仅与环境分开 精致但高效的柱状上皮细胞的单层，由紧密连接连接 不受蛋白质甚至小胡椒的不渗透。到目前为止，缺乏理性和高效的方法 绕过这种障碍阻止了大多数治疗蛋白和胡椒粉用于粘膜的应用 药物输送。内皮细胞还形成巨大且高度限制的单细胞厚屏障，大多数分离 血液中的组织。大多数健康的非影响内皮屏障极大地限制了 大分子；从而阻止许多基于蛋白质的生物制剂进入许多组织的细胞 - 即使 静脉注射治疗蛋白。 在这里，我们通过测试非本地“短链” GM1糖脂果脂能够解决这些问题 用作药物输送胡椒和蛋白质生物制剂的分子载体。 自2014年本提案的最初提交以来，取得了重大进展。结构功能 关于神经酰胺结构域的跨多运输的研究确定了具有的非本地GM1种类 增强摄取，上皮屏障的跨胞菌病的结合特征以及从细胞中有效释放 运输后的膜。这些GM1物种是研究货物运输的车辆。 在AIM 1中，我们将包括并扩展我们的初步研究测试，用于GM1介导的胡椒的运输 蛋白质在体内跨小鼠上皮和内皮屏障。 GM1脂质通过它们的 细胞外寡糖结构域与报告基因肽产生可靠的信号，以跟踪分子通过 成像和生化。我们将测试较大货物的运输，并通过粘膜证明原理证明 给予与激素GLP-1融合的非本地GM1物种，作为治疗的模型 II型糖尿病。将在体内测试肽在紧密的内皮屏障（心脏和大脑）上的运输。 AIM 2将测试非本地GM1物种的转胞胞菌病机理。分类的主要假设 将检查通过分子形状或与膜微区的关联。 AIM 3将测试GM1的细胞外寡糖结构域是否可以被截短或消除 在运输中维持GM1神经酰胺域的功能。我们将探索新颖的结构 如有必要，接头肽可以替代寡糖头组的功能。 在AIM 4中，我们将测试GM1和Pepper之间的连接器是否可以设计为释放货物之后或 在跨胞菌病期间。我们将测试是否掺入可裂解的酯键，还是内体 - 蛋白酶肉手可以实现这一目标。