Unsaturated glycoceramide as novel platform for mucosal vaccine and drug delivery

描述(由申请人提供)：这项提案的目标是测试我们最近关于上皮细胞脂质运输的发现是否可以转化为临床应用。具体地说，我们将测试使用“短的或不饱和的”神经酰胺基脂作为分子载体的效用，以跨越粘膜上皮屏障输送治疗性多肽或疫苗佐剂。粘膜表面代表着广阔的区域，宿主组织与环境之间只有一层微妙但高效的柱状上皮细胞，由不能渗透到蛋白质甚至小肽的紧密连接连接在一起。到目前为止，缺乏合理和有效的方法来绕过这一障碍，阻碍了大多数治疗性蛋白质在口腔或鼻腔给药和粘膜疫苗中的应用。在我们对霍乱毒素糖脂受体GM1生物学的研究过程中，我们最近发现神经酰胺(脂)结构域的结构决定了GM1在上皮细胞中的运输。当顶端使用时，含有“短”C12：0或“扭链”不饱和C16：1脂肪酸(GM1短/不饱和)的GM1-神经酰胺进入公共/循环内体。在这里，它们被分拣，运输到不同的细胞内目的地，并进入到基侧细胞表面的“跨细胞”途径。相反，具有完全饱和脂肪酸链(C16：0或更长)的GM1-神经酰胺(GM1长/饱和)被运输到晚期内体和溶酶体进行降解。在这个探索性项目中，我们将测试这一基本发现是否可以用于生物活性多肽或蛋白质佐剂的经皮递送，这两种药物都有临床应用。我们将把含有不饱和“扭结”或饱和脂肪酸的GM1分子与作用于调节血糖的治疗性多肽激素类高血糖素样多肽-1(GLP1)以及TLR5激动剂Flc(鞭毛沙门氏菌)联系起来，我们在这里使用它来模拟一种粘膜疫苗佐剂(目标2)。这些研究将测试一种通过粘膜表面运输治疗/疫苗分子的新平台。对治疗性多肽和蛋白质的非亲本递送的需求很大。迫切需要改进的粘膜疫苗策略来保护免受病原体的侵袭，因为绝大多数病原体是通过粘膜表面入侵的。 公共卫生相关性：该应用程序的目标是开发一种允许口服或经鼻给药治疗性蛋白质和疫苗的方法。正常情况下，这种蛋白质不会被吸收，必须通过注射输送；这严重限制了治疗应用。

DESCRIPTION (provided by applicant): The goal of this proposal is to test if our recent discovery on lipid trafficking in epithelial cells can be translated to clinical applications. Specifically, we will test the utility of using "short or unsaturated" ceramide-based lipids as molecular carriers to deliver therapeutic peptides or vaccine adjuvants across mucosal epithelial barriers. 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 for oral or nasal drug delivery and for mucosal vaccines. In the course of our studies on the biology of the glycolipid receptor for cholera toxin, ganglioside GM1, we recently discovered that the structure of the ceramide (lipid) domain dictates GM1 trafficking in epithelial cells. When applied apically, GM1-ceramides containing "short" C12:0 or "kinked chain" unsaturated C16:1 fatty acids (GM1short/unsat) enter the common/recycling endosome. Here, they are sorted for transport to various intracellular destinations and into the "transcytotic" pathway to the basolateral cell surface. In contrast, the GM1-ceramides with fully saturated fatty acid chains (C16:0 or longer) (GM1 long/sat) are instead transported to the late endosome and lysosome for degradation. In this exploratory project, we will test whether this basic discovery can be harnessed for transepithelial delivery of a bioactive peptide or protein adjuvant, both of which have clinical applications. We will link GM1 molecules containing unsaturated "kinked" or saturated fatty acids to the therapeutic peptide hormone glucagon-like peptide-1 (GLP1), which acts to regulate blood sugar (Aim 1); and to the TLR5-agonist FliC (Salmonella flagellin), which we use here to model a mucosal vaccine adjuvant (Aim 2). These studies will test a novel platform for transport of therapeutic/vaccine molecules across mucosal surfaces. There is great need for non-parental delivery of therapeutic peptides and proteins. Improved mucosal vaccine strategies are greatly needed for protection against pathogens, the vast majority of which invade via mucosal surfaces. PUBLIC HEALTH RELEVANCE: The goal of this application is to develop a way to allow for oral or nasal administration of therapeutic proteins and vaccines. Normally such proteins are not absorbed and must be delivered by injection; severely limiting therapeutic applications.