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

糖基化是一种基本的生物途径，涉及糖的翻译后或共翻译后加成。 蛋白质的一部分。先天性糖基化紊乱(CDGs)是一种罕见的发育障碍，由 糖基化途径中的先天代谢错误。CDG缺乏良好的治疗选择，这是典型的 由于对机制知之甚少以及在小患者群体中建立临床试验的困难。我的 长期目标是确定CDG的机制，并为CDG患者寻找新的治疗方法。 一个例子是DPAGT1-CDG-一种由DPAGT1基因突变引起的CDG，它编码第一个 用于N-连接的糖基化的酶。最近，我发现了许多可以被干扰的修饰基因。 拯救DPAGT1-CDG模型，但其作用机制尚不清楚。在目标1中，我建议确定 利用人类细胞培养研究这些修饰基因的作用机制以确定新的治疗靶点 来治疗这种疾病。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 与患者相关的增殖、应激及其糖蛋白组的健康指标。在目标2中，确定新药 为了挽救这种疾病，我将使用体内的果蝇DPAGT1-CDG模型来执行一种改变用途的药物 使用1500+小分子进行筛查(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，这种重新调整用途的药物筛选将有助于加快新的临床试验进程 CDG疗法。在目标3中，我将描述一项新发现，该发现表明CDG(CDG)背后的基因 基因“)本身代表了用于治疗CDG的一组丰富的修饰基因。CDG的扰动可以 救援模型DPAGT1-CDG，以及最常见的CDG模型PMM2-CDG。我将使用RNA 干扰所有150+CDG基因以识别能够同时拯救DPAGT1和 PMM2-CDG人体细胞模型(具有与目标1相同的健康指标)。新CDG基因的发现 能够挽救这些模型的修饰基因可能有可能转化为未来的治疗方法 很多其他的CDG。最后，在目标4中，我将合成上述目标，以测试来自目标2的治疗药物。 果蝇模型中人细胞培养模型和AIM 3中的CDG修饰基因。我将使用高吞吐量 在果蝇细胞培养和体内应激标志物中的工具，以确定这些新疗法的机制。 这种多物种方法将确保更好地从临床前模型过渡到患者的治疗方法。在……里面 除此之外，完成这份提案将为我提供完成职业目标的培训。这就做 学习细胞培养和小分子筛选的新技术，同时参加正规的辅导课程 还有写作。我有一个优秀的导师、共同导师和咨询委员会，由具有专业知识的教师组成。 CDGs、细胞培养、药物筛选、遗传学和分子生物学。我也有最先进的设施和 犹他大学的工作人员对我来说是有空的。有了我的计划，尽职尽责的教职员工和优秀的机构， 完成这项建议将帮助我成功地过渡到独立的研究生涯。

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.