Harnessing the biology of glycosphingolipid trafficking for biologic drug delivery

项目总结 这项提议的目标是测试我们最近在上皮细胞鞘糖脂运输方面的一项发现 细胞作为生物制剂给药的平台可以转化为临床应用。 粘膜表面代表宿主组织与环境仅有一段距离的广阔区域。 精致但高效的单层柱状上皮细胞，由紧密的连接连接在一起 对蛋白质甚至小肽都是不透水的。到目前为止，缺乏合理有效的方法来 绕过这一障碍阻止了大多数治疗性蛋白质和多肽在粘膜中的应用 药物递送。内皮细胞也会形成巨大的高度限制性的单细胞厚屏障，将大多数 血液中的组织。大多数健康的非炎症内皮屏障强烈限制血管内皮细胞的通透性 大分子；从而阻止许多基于蛋白质的生物制品进入许多组织的细胞-即使在 治疗性蛋白质被静脉注射。 在这里，我们通过测试非天然的短链GM1糖鞘糖脂是否可以 作为多肽和蛋白质生物制品的分子载体。 自2014年首次提交该提案以来，取得了实质性进展。结构-功能 神经酰胺结构域跨皮转运的研究--鉴定具有 增强摄取、跨上皮屏障和有效释放细胞的综合特征 转运后的膜。这些GM1物种被作为货物运输工具进行研究。 在目标1中，我们将总结和扩展我们对GM1介导的多肽转运的初步研究测试 在体内，蛋白质可以穿过小鼠的上皮和内皮屏障。GM1类脂通过它们的 胞外寡糖结构域报告多肽产生强大的信号用于跟踪分子通过 在成像和生化方面。我们将测试较大货物的运输，并通过粘膜进行原则证明 给予非本地GM1物种与胰岛素激素GLP-1融合的药物作为治疗糖尿病的模型 II型糖尿病。将在体内测试通过紧密的内皮屏障(心脏和大脑)的多肽运输。 目标2将测试非本地GM1物种的细胞转运机制。关于排序的主要假设 通过分子形状或通过与膜微域的结合来检查。 目标3将测试GM1的胞外寡糖结构域是否可以在静止的情况下被截断或消除 维持GM1神经酰胺结构域在贩运中的功能。我们将探索新的结构 如有必要，用连接肽取代低聚糖头基团的官能团。 在目标4中，我们将测试GM1和多肽之间的连接物是否可以设计为在或 在细胞穿透过程中。我们将测试是否加入了可切割的酯键，或内体的基序- 蛋白水解酶，可以达到这一目的。

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.