Develop a human liver system to study SLC25A13 mutations in citrin deficiency

项目摘要 Citrin缺乏症（CD）是一种由SLC 25 A13基因突变引起的常染色体隐性遗传病，是一种泛种族性疾病， 罕见病目前尚无CD的特效药。饮食管理被认为是最有效的 CD的治疗，以及患者临床结局的决定性因素。然而，饮食管理 仅基于小病例报告，CD儿童的饮食管理通常很差， 特别是在以碳水化合物为主的饮食的地区。因此，需要进一步研究， 了解疾病的发病机制，促进更具体的治疗发展。 CD表现为年龄依赖性表型，可能具有严重的临床结局，包括无法茁壮成长 和由瓜氨酸缺乏症（FTTDCD）和瓜氨酸血症2型（CTLN 2）引起的血脂异常， 高氨性脑病的发展以及其他临床表现。但 SLC 25 A13功能丧失如何导致上述疾病表型的机制仍然存在 基本上不清楚。CD患者的发病年龄和不完全缓解率与CD的发病年龄和不完全缓解率之间的关系仍知之甚少。 病人是有决心的。 CD研究中的一个主要未满足的需求是缺乏一个可靠的与人类相关的模型， 这种疾病在人类中的传播范围。到目前为止，研究CD的唯一可用模型是基因敲除（KO）小鼠 (i.e., Slc 25 a13-KO和Slc 25 a13和Gpd 2-双KO）。由于在基因上存在明显的物种特异性差异， 特别是在糖酵解中的调节，这些模型的实用性和临床相关性是有争议的。需要 人类相关的系统进一步强调的意见，显示变量的发病年龄和不完整的 在CTLN 2患者中观察到的突变，其中怀疑有额外的环境和/或遗传触发因素。 本申请的主要研究目标是利用人类诱导多能干细胞（iPSC）- 我们实验室开发的衍生多细胞体外肝脏模型，以创建人类相关模型， 研究SLC 25 A13的CD。在这两个具体目标中，我们计划产生含有野生型的肝培养物， 或SLC 25 A13 KO细胞，并表征与人CD相关的可能的疾病表型。 开发iPSC衍生的可再生和可遗传操纵的人类系统用于研究SLC 25 A13 将有助于详细解剖疾病的发病机制。此外，由于缺乏合适的人- 相关系统的研究光盘，我们的iPSC衍生平台将提供一个迫切需要的新平台来测试 干预措施，并促进特定疗法的发展。最后，这种初步支持将使我们能够 进行试点分析和表征，并为更大的赠款申请获得关键的初步数据 这将有助于这些努力。

PROJECT SUMMARY Citrin deficiency (CD), an autosomal recessive disorder caused by mutations in SLC25A13 gene, is a pan-ethnic rare disease. Currently there is no specific drug for CD. Dietary management is regarded as the most effective treatment for CD, as well as a decisive factor in the clinical outcome of patients. However, dietary management has only been based on small case reports and dietary management for children with CD is generally poor, especially in areas where carbohydrate-based diets are predominant. Thus, further research is needed to understand disease pathogenesis and facilitate more specific therapeutic development. CD manifests as age-dependent phenotypes and can have severe clinical outcome including failure to thrive and dyslipidemia caused by citrin deficiency (FTTDCD) and citrullinemia type 2 (CTLN2) with a sudden development of hyperammonemic encephalopathy, as well as other clinical manifestations. However, the mechanisms by which how SLC25A13 loss-of-function leads to the abovementioned disease phenotypes remain largely unclear. It also remains poorly understood how the age of onset and incomplete penetrance seen in CD patients are determined. A major unmet need in the study of CD is the lack of a reliable human-relevant model that mimics the entire spectrum of this disease in humans. So far, the only available models to study CD are the knockout (KO) mice (i.e., Slc25a13-KO and Slc25a13 and Gpd2-double KO). Owing to marked species-specific differences in gene regulation especially in glycolysis, the utility and clinical relevance of these models are debated. The need for human-relevant system is further underscored by the observations showing variable age of onset and incomplete penetrance seen in CTLN2 patients where additional environmental and/or genetic triggers are suspected. The primary research goal of this application is to leverage a human induced pluripotent stem cell (iPSC)- derived multicellular in vitro liver model developed in our laboratory to create a human-relevant model for studying SLC25A13 in CD. Across the two specific aims, we plan to generate liver cultures harboring wild-type or SLC25A13 KO cells and to characterize the possible disease phenotypes that are relevant to human CD. Developing an iPSC-derived renewable and genetically manipulatable human system for the study of SLC25A13 in CD will facilitate the detailed dissection of disease pathogenesis. Moreover, given the lack of suitable human- relevant systems to study CD, our iPSC-derived platform will provide an urgently needed new platform to test interventions and promote the development of specific therapeutics. Finally, this initial support will allow us to perform pilot analysis and characterization and to acquire critical preliminary data for a bigger grant application that will facilitate these efforts.