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 这项提案的目的是测试我们最近在上皮细胞中鞘糖脂运输方面的发现， 细胞可以转化为临床应用，作为生物制剂的药物递送平台。 粘膜表面代表了宿主组织仅通过膜与环境分离的广阔区域。 精细但高效的单层柱状上皮细胞，由紧密连接连接， 蛋白质甚至小肽都无法渗透。到目前为止，缺乏合理有效的方法， 绕过这一屏障已经阻止了大多数治疗性蛋白质和肽用于粘膜炎的应用。 药物输送内皮细胞也形成巨大的和高度限制性的单细胞厚屏障， 血液中的组织大多数健康的非炎症内皮屏障强烈限制了细胞的渗透性。 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-即使当 治疗性蛋白质通过静脉内施用。 在这里，我们通过测试非天然的“短链”GM 1鞘糖脂是否可以 用作肽和蛋白质生物制剂的药物递送的分子载体。 自2014年最初提交该提案以来，已取得实质性进展。结构功能 对神经酰胺结构域的跨上皮转运的研究鉴定了具有神经酰胺结构域的非天然GM 1-种类。 增强的摄取、穿过上皮屏障的转胞吞作用和从细胞中的有效释放的组合特征 运输后的膜。这些GM 1物种被研究作为货物运输的载体。 在目标1中，我们将总结和扩展我们的初步研究测试GM 1介导的肽转运 以及体内穿过小鼠上皮和内皮屏障的蛋白质货物。GM 1脂质通过它们的 细胞外寡糖结构域与报告肽结合，产生用于跟踪分子的稳健信号， 成像和生物化学。我们将测试较大货物的运输，并通过粘膜试验验证原理。 施用与肠降血糖素激素GLP-1融合的非天然GM 1种类，作为治疗糖尿病的模型。 2型糖尿病将在体内测试肽穿过紧密内皮屏障（心脏和脑）的转运。 目的2将测试非天然GM 1物质的转胞吞作用机制。分类的主要假设 通过分子形状或通过与膜微区的关联来进行检查。 目的3将测试GM 1的胞外寡糖结构域是否可以被截短或消除，同时仍然 维持GM 1神经酰胺结构域在运输中的功能。我们将探索新的结构， 如果需要，连接肽取代寡糖头基的官能团。 在目标4中，我们将测试GM 1和肽之间的接头是否可以被设计为在或之后释放货物。 在转胞吞作用中。我们将测试是否掺入可裂解的酯键，或内体的基序- 蛋白酶弗林蛋白酶可以实现这一目标。