Identifying New Therapeutics and Molecular Mechanisms in Congenital Disorders of Glycosylation.

糖基化是一种重要的生物学途径，涉及糖的翻译后或共翻译添加 部分与蛋白质结合。先天性糖基化障碍（CDG）是一种罕见的发育障碍， 糖基化途径中的先天性代谢缺陷。CDG缺乏良好的治疗选择，这是典型的 这是由于对机制知之甚少以及难以在小患者群体中建立临床试验。我 长期目标是确定CDG的机制并鉴定用于CDG患者的新疗法。 一个实例是DPAGT1-CDG-由编码第一个CDG的基因DPAGT1中的突变引起的CDG。 用于N-连接糖基化的酶。最近，我发现了许多修饰基因，它们可以被干扰， 拯救DPAGT1-CDG模型，但其机制尚不清楚。在目标1中，我建议确定 这些修饰基因的机制，使用人类细胞培养，以表征新的治疗靶点 治疗这种疾病我将使用DPAGT1-CDG细胞模型来确定这些拯救修饰基因如何影响 患者相关的增殖、压力和糖蛋白质组的健康指标。目标2：确定新药 为了拯救这种疾病，我将使用体内果蝇DPAGT1-CDG模型来进行重新利用药物 使用1，500多种小分子进行筛选（98%获得FDA/EMA批准）。使用体内模型将确保这些药物 在开发过程中是安全的，这种重新设计的药物筛选将有助于加快新的临床试验过程。 CDG疗法。在目标3中，我将描述一个新的发现，该发现表明CDG（"CDG 基因）本身代表了用于治疗CDG的修饰基因的富集组。CDG的扰动可以 DPAGT 1-CDG的拯救模型，以及最常见的CDG模型PMM2-CDG。我会用RNA 干扰干扰所有150 + CDG基因以鉴定任何能够拯救DPAGT 1-和 PMM2-CDG人细胞模型（具有与目标1中相同的健康指标）。CDG新基因的发现 能够拯救这些模型的修饰基因可能有潜力转化为未来的治疗方法， 许多其他CDG。最后，在目标4中，我将综合上述目标，以测试目标2中的治疗药物。 人细胞培养模型和果蝇模型中来自Aim 3的CDG修饰基因。我将使用高通量 细胞培养和果蝇体内应激标记的工具，以确定这些新疗法的机制。 这种多物种方法将确保从临床前模型更好地过渡到患者治疗。在 除此之外，完成这份建议书将为我提供培训，以完成我的职业目标。我会 学习细胞培养和小分子筛选的新技术，同时参加正式的导师课程 和写作我有一个杰出的导师，共同导师，和咨询委员会组成的教师与专业知识 CDG、细胞培养、药物筛选、遗传学和分子生物学。我还有最先进的设备 犹他州大学的教职员工。有了我的计划，忠诚的教职员工和优秀的机构， 完成这份建议书将帮助我成功地过渡到一个独立的研究生涯。

Glycosylation is an essential biological pathway that involves the post- or co-translational addition of sugar moieties to proteins. Congenital Disorders of Glycosylation (CDGs) are rare developmental disorders caused by inborn errors of metabolism in glycosylation pathways. CDGs lack good treatment options, and this is typically due to poorly understood mechanisms and difficulty in establishing clinical trials in small patient populations. My long-term objectives are to determine mechanisms of CDGs and identify new therapeutics for CDG patients. One example is DPAGT1-CDG - a CDG caused by mutations in the gene DPAGT1 which encodes for the first enzyme used in N-linked glycosylation. Recently, I identified many modifier genes which can be perturbed to rescue a model of DPAGT1-CDG, but their mechanisms are not yet known. In Aim 1, I propose to determine the mechanisms of these modifier genes using human cell culture in order to characterize new therapeutic targets for this disorder. I will use a DPAGT1-CDG cell model to determine how these rescuing modifier genes affect patient-related health metrics of proliferation, stress, and their glycoproteome. In Aim 2, to identify new drugs that can rescue this disorder, I will use an in vivo Drosophila DPAGT1-CDG model to perform a repurposed drug screen using 1,500+ small molecules (98% FDA/EMA-approved). Using an in vivo model will ensure these drugs are safe during development, and this repurposed drug screen will help expedite the clinical trial process for new CDG therapies. In Aim 3, I will characterize a new finding that suggests that genes underlying CDGs ("CDG genes") themselves represent an enriched set of modifier genes for treating CDGs. Perturbation of CDGs can rescue models of DPAGT1-CDG, as well as a model of the most common CDG, PMM2-CDG. I will use RNA interference to perturb all 150+ CDG genes to identify any that are capable of rescuing both DPAGT1- and PMM2-CDG human cell models (with the same health metrics as in Aim 1). The discovery of new CDG gene modifier genes capable of rescuing these models could have the potential to translate into future therapies for many other CDGs. Finally, in Aim 4, I will synthesize the above Aims to test therapeutic drugs from Aim 2 in human cell culture models and CDG modifier genes from Aim 3 in Drosophila models. I will use high-throughput tools in cell culture and in vivo stress markers in Drosophila to determine the mechanisms of these new therapies. This multi-species approach will ensure a better transition from preclinical models into therapies for patients. In addition to the above, completion of this proposal will provide me with training to complete my career goals. I will learn new techniques in cell culture and small molecule screens while also taking formal courses in mentorship and writing. I have an outstanding mentor, co-mentor, and advisory committee consisting of faculty with expertise in CDGs, cell culture, drug screening, genetics, and molecular biology. I also have state-of-the-art facilities and staff at the University of Utah available to me. With my plan, committed faculty members, and excellent institution, completing this proposal will help me successfully transition to an independent research career.