背景：线粒体肌病、乳酸酸中毒和铁粒幼细胞性贫血综合征（MLASA）是遗传性铁粒幼细胞性贫血（CSA）中的一类，根据患者携带的突变基因可以将其分为三种亚型。目前对于MLASA患者，除输血、祛铁及其他系统的对症治疗外，暂无有效的干预策略。I型MLASA（MLASA1）是由PUS1基因纯合突变引起的，PUS1作为一种RNA假尿苷合酶，可以通过催化尿苷生成假尿苷来修饰多种RNA，进而影响其修饰的RNA稳定性或功能。我院发现一例携带未报道过的PUS1基因纯合突变（c.523delC, p.P175fs）的MLASA1患者。由于患者细胞来源有限，不便于研究。而诱导性多能干细胞（iPSCs）的出现实现了人疾病模型的体外模拟，为疾病的病理机制研究以及后续的药物筛选提供了基础平台。本研究首次应用iPSCs模型研究PUS1基因突变导致的MLASA患者表型及致病机制，以期找到可行的MLASA疾病干预策略。

目的：建立患者特异性的MLASA-iPSCs及其修复细胞株，在此基础上，确定MLASA表型特征、解析疾病机制、探究疾病干预策略，以期缓解患者的疾病表型。

方法：通过电转非整合型Yamanaka因子质粒的方式，将携带PUS1纯合突变（c.523delC, p.P175fs）的患者骨髓来源的单个核细胞重编程为诱导性多能干细胞。同时，针对该突变位点设计定点sgRNA和修复模板，利用CRISPR/Cas9技术构建PUS1突变位点修复的多能干细胞株。对正常人来源的iPSCs（Normal-iPSCs）、患者特异性iPSCs（MLASA-iPSCs）及其修复株（MLASA-Res-iPSCs）进行红系定向分化来评估其分化能力。通过Seahorse、ELASA和流式细胞术等方法对线粒体功能的相关指标进行检测。联合tRNA PCR array、RNA-seq和Ribo-seq等技术确定疾病的差异表达基因及通路，深入探究PUS1突变的致病机制。基于机制探究结果，选择合适的药物对患者特异性的iPSCs进行处理，并通过对比药物处理前后的效果，筛选出有效的药物，为临床用药提供理论依据。

结果：

1.     成功获得由患者骨髓单个核细胞来源的iPSCs和PUS1突变原位修复细胞株，经过PCR和Sanger测序证实MLASA-iPSCs携带了源自患者的突变，而MLASA-Res-iPSCs实现了基因校正。免疫荧光和畸胎瘤形成等实验证明了iPSCs的多能性。

2.     RT-qPCR和Western blot实验结果证实PUS1基因的P175fs突变导致其mRNA表达水平下降以及蛋白水平缺失。

3.     通过两种不同的方法对Normal-iPSCs、MLASA-iPSCs及MLASA-Res-iPSCs进行红系分化诱导，发现这三种细胞均可产生相似比例的生血内皮（hemogenic endothelium，HE）细胞（CD34⁺CD31⁺），但MLASA-iPSCs的有核红细胞（CD71⁺CD235a⁺）生成比例显著低于Normal-iPSCs和MLASA-Res-iPSCs，存在红系分化阻滞，很好地模拟了患者的贫血表型。

4.     Seahorse、ELASA和流式细胞术等实验结果表明MLASA-iPSCs线粒体功能缺陷。我们在MLASA-iPSCs中观察到细胞耗氧量减少，线粒体氧化呼吸链复合物I和III活性显著下降，线粒体膜电位明显降低，线粒体生物量和ROS水平异常升高，而这些表型在MLASA-Res-iPSC均能实现修复。

5.     通过联合分析tRNA PCR array、RNA-seq和Ribo-seq的结果，我们发现PUS1通过调控其修饰的mt-tRNA的表达丰度，从而影响线粒体和胞质的翻译，导致线粒体功能缺陷和mTOR信号通路的异常激活。

6.     经线粒体补充剂烟酰胺腺嘌呤二核苷酸（NAD⁺）的前体物质烟酰胺核苷（NR）和CoQ10/MitoQ处理后，MLASA-iPSCs的线粒体功能实现部分恢复，尽管阻滞的红系分化没有明显改善。PUS1突变导致的红系分化缺陷经mTOR抑制剂雷帕霉素（Rapamycin）处理后得以改善，提示抑制mTOR信号通路可能是治疗该贫血的途径。

7.     在患者尝试服用西罗莫司（Rapamycin商品名）后，患者血红蛋白和红细胞含量均显著升高，有效地缓解了贫血症状，初步揭示了该贫血的有效治疗途径。

结论：MLASA患者特异性iPSCs和修复细胞系成功建立后，经表型及功能性实验的验证，证实PUS1基因纯合突变（c.523delC, p.P175fs）直接导致了 MLASA疾病发生。该突变造成PUS1蛋白丢失，导致其对tRNA表达的调控异常，从而影响线粒体和胞质的翻译，最终造成线粒体功能缺陷和红系分化障碍。由MLASA-iPSCs及其分化而来的生血内皮细胞出现mTOR信号通路的异常活化，使用mTOR抑制剂Rapamycin处理可部分恢复因PUS1突变导致的红系分化障碍。根据此实验结果，我们尝试让患者按照医嘱服用西罗莫司，在服用一个月以后，血红蛋白水平达到80 g/L以上，且持续用药可维持疗效。我们在疾病模型的实验研究和临床初始研究数据都表明适当抑制mTOR信号通路可以有效缓解PUS1基因突变导致的贫血。

Background：Mitochondrial myopathy, lactic acidosis and sideroblastic anemia syndrome (MLASA) is a type of congenital sideroblastic anemia (CSA), which can be divided into three subtypes based on the mutation genes. Currently, there is no effective intervention strategy for MLASA, only blood transfusion, iron removal and other systematic symptomatic treatment were used but with minute effects. As a RNA pseudouridine synthase, PUS1 is one of the mutated gene reported in MLASA. It converts the uridines on various RNAs into pseudouridines, thereby affecting the stability or function of the targeted RNAs. A MLASA patient carrying an unreported homozygous PUS1 mutation (c.523delC, p.P175fs) was admitted to our hospital, however the limited cells from patient restricted the deep investigation. The emergence of induced pluripotent stem cells (iPSCs) has greatly facilitated in vitro study models of human diseases that provide a platform for the investigation of disease pathogenesis and subsequent drug screening. In this study, the MLASA patient-specific iPSCs (named as MLASA-iPSCs) with the homozygous PUS1 mutation was established for the first time. The phenotype and pathogenesis of MLASA caused by PUS1 mutation was explored to find feasible intervention strategies to treat and relieve the disease.

Objective: To generate MLASA patient-specific iPSCs and its corrected cell line (named as MLASA-Res-iPSCs) to identity the characteristics of MLASA, to define the pathological mechanism, and to explore intervention strategies in order to treat the patient with MLASA.

Methods: BM-MNCs (bone marrow mononuclear cells) of the patient carrying a homozygous PUS1 mutation (c.523delC, p.P175fs) were reprogrammed into patient-specific iPSCs through an integration-free delivery of epitomal plasmid vectors expressing Yamanaka factors with electroporation. In addition, targeted sgRNA and template were designed to correct the mutation and generate repaired iPS cell line by CRISPR/Cas9 technology. Normal-, MLASA- and MLASA-Res-iPSCs were induced into erythroblasts by two methods to evaluate their capacity of erythroid differentiation. The function of mitochondrial were measured by Seahorse, ELASA, flow cytometry and other methods. Combined analysis of tRNA PCR array, RNA-seq, and Ribo-seq were used to investigate differentially expressed genes and pathways in  MLASA- and MLASA-Res-iPSCs, and to further explore the pathogenesis of MLASA harboring PUS1 mutations. Based on the above findings, effective drugs were screened out to treat iPSCs, and provide a theoretical guidance for clinical medication.

Results:

1.     The successful establishment of patient-specific iPSCs and its repaired iPS cell line. MLASA-iPSCs did carry patient-derived mutation, which was corrected in MLASA-Res-iPSCs, were confirmed by PCR and Sanger sequencing. Immunofluorescence and teratoma formation assay were used to validate the pluripotency of iPSCs.

2.     P175fs mutation in PUS1 gene caused its mRNA reduction and protein deletion were measured by RT-qPCR and Western blot.

3.     Normal-, MLASA- and MLASA-Res-iPSCs were induced to erythroblast using two methods. It was found that all of three iPS cell lines could produce similar proportions of hematopoietic endothelial cells (CD34⁺CD31⁺). However, the percentage of generated erythroblasts (CD71⁺CD235a⁺) from MLASA-iPSCs was significantly lower than that of Normal-iPSCs and MLASA-Res-iPSCs, indicating that PUS1 mutation led to the erythroid differentiation blockage, which mimics the anemic phenotype of patient.

4.     The mitochondrial function was defective in MLASA-iPSCs, which detected by Seahorse, ELASA and flow cytometry. We observed decreased cellular oxygen consumption, attenuated enzyme activities of complex I and III of mitochondrial oxidative respiratory chain, reduced mitochondrial membrane potential, elevated mitochondrial biomass and ROS levels in MLASA-iPSCs, and these phenotypes could be rescued in MLASA-Res-iPSCs.

5.     The combination analysis of tRNA PCR array, RNA-seq, and Ribo-seq showed that PUS1 affected the abundance of targeted mt-tRNA, and thus controlled mitochondrial and cytoplasmic translation, resulting in mitochondrial dysfunction and aberrant activation of mTOR signaling pathway.

6.     Treatment with nicotinamide riboside (NR), the precursor of nicotinamide adenine dinucleotide (NAD⁺), and CoQ10/MitoQ could partially restore the mitochondrial dysfunction of MLASA-iPSCs, which did not improve the capacity of erythroid differentiation. While treatment with mTOR inhibitor Rapamycin could mitigate the erythroid differentiation disorder of iPSCs caused by PUS1 mutation.

7.     Encouragingly, both hemoglobin level and red blood cell count of patient were significantly increased after taking Sirolimus (Rapamycin), and showed an effective relief of anemia symptoms.

Conclusion: The MLASA patient-specific iPSCs and its repaired cell line were successfully generated, and the phenotypic and functional experiments confirmed that the homozygous P175fs mutation in PUS1gene directly caused MLASA. P175fs mutation of PUS1 resulted in its protein loss, which affecting mitochondrial and cytoplasmic translation through aberrant regulation of mt-tRNA expression, and ultimately impaired mitochondrial function and erythroid differentiation. Abnormal activation of mTOR was observed in MLASA-iPSCs and their differentiated hematopoietic endothelial cells, and treatment with mTOR inhibitor Rapamycin could partially restore the disorder of erythroid differentiation caused by PUS1 mutation. Based on the above results, the patient was given Sirolimus under the guidance of doctor for one month. The hemoglobin level of patient reaches up to 80 g/L, and continued medication can maintain the curative efficacy. The experimental results of iPSCs and the clinical data of patient indicate that the inhibition of mTOR signaling pathway can effectively alleviate the anemia symptoms caused by PUS1 mutation.