Mesenchymal stem cells (MSCs) serve as an attractive vehicle for cell-directed enzyme prodrug therapy (CDEPT) due to their unique tumour-nesting ability. Such approach holds high therapeutic potential for treating solid tumours including glioblastoma multiforme (GBM), a devastating disease with limited effective treatment options. Currently, it is a common practice in research and clinical manufacturing to use viruses to deliver therapeutic genes into MSCs. However, this is limited by the inherent issues of safety, high cost and demanding manufacturing processes. The aim of this study is to identify a facile, scalable in production and highly efficient non-viral method to transiently engineer MSCs for prolonged and exceptionally high expression of a fused transgene: yeast cytosine deaminase::uracil phosphoribosyl-transferase::green fluorescent protein (CD::UPRT::GFP). MSCs were transfected with linear polyethylenimine using a cpg-free plasmid encoding the transgene in the presence of a combination of fusogenic lipids and β tubulin deacetylase inhibitor (Enhancer). Process scalability was evaluated in various planar vessels and microcarrier-based bioreactor. The transfection efficiency was determined with flow cytometry, and the therapeutic efficacy of CD::UPRT::GFP expressing MSCs was evaluated in cocultures with temozolomide (TMZ)-sensitive or TMZ-resistant human glioblastoma cell lines. In the presence of 5-fluorocytosine (5FC), the 5-fluorouracil-mediated cytotoxicity was determined by performing colometric MTS assay. In vivo antitumor effects were examined by local injection into subcutaneous TMZ-resistant tumors implanted in the athymic nude mice. At > 90% transfection efficiency, the phenotype, differentiation potential and tumour tropism of MSCs were unaltered. High reproducibility was observed in all scales of transfection. The therapeutically modified MSCs displayed strong cytotoxicity towards both TMZ-sensitive and TMZ-resistant U251-MG and U87-MG cell lines only in the presence of 5FC. The effectiveness of this approach was further validated with other well-characterized and clinically annotated patient-derived GBM cells. Additionally, a long-term suppression (> 30 days) of the growth of a subcutaneous TMZ-resistant U-251MG tumour was demonstrated. Collectively, this highly efficient non-viral workflow could potentially enable the scalable translation of therapeutically engineered MSC for the treatment of TMZ-resistant GBM and other applications beyond the scope of this study.

中文翻译：

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一种易于生产且可扩展的非病毒基因工程化间充质干细胞，可有效抑制耐替莫唑胺（TMZR）胶质母细胞瘤的生长。

间充质干细胞（MSC）由于其独特的肿瘤嵌套能力，可作为细胞定向酶前药治疗（CDEPT）的诱人载体。这种方法在治疗包括多形性胶质母细胞瘤（GBM）在内的实体瘤方面具有很高的治疗潜力，这是一种毁灭性疾病，有效治疗选择有限。当前，使用病毒将治疗性基因传递到MSC中是研究和临床生产中的普遍做法。但是，这受到安全性，高成本和苛刻的制造过程等固有问题的限制。这项研究的目的是确定一种简便，可扩展的生产方法和高效的非病毒方法，以瞬时改造MSC，以实现融合转基因的延长和异常高表达：酵母胞嘧啶脱氨酶：：尿嘧啶磷酸核糖基转移酶：：绿色荧光蛋白（CD :: UPRT :: GFP）。在融合脂质和β微管蛋白脱乙酰基酶抑制剂（Enhancer）结合存在的情况下，使用无cpg的编码转基因的质粒，用线性聚乙烯亚胺转染MSC。在各种平面容器和基于微载体的生物反应器中评估了工艺可扩展性。用流式细胞术确定转染效率，并在与替莫唑胺（TMZ）敏感或TMZ耐药的人胶质母细胞瘤细胞系共培养物中评估表达CD :: UPRT :: GFP的MSC的治疗效果。在存在5-氟胞嘧啶（5FC）的情况下，通过进行MTS计量法测定5-氟尿嘧啶介导的细胞毒性。通过局部注入植入无胸腺裸鼠的皮下TMZ耐药性肿瘤中检查体内抗肿瘤作用。在> MSCs的90％转染效率，表型，分化潜能和肿瘤嗜性均未改变。在所有规模的转染中均观察到了高重复性。经过治疗修饰的MSC仅在5FC存在下，对TMZ敏感和TMZ耐药的U251-MG和U87-MG细胞系均显示出强大的细胞毒性。此方法的有效性已通过其他特征明​​确且临床上注明患者来源的GBM细胞得到了进一步验证。另外，证实了对皮下TMZ抵抗性U-251MG肿瘤的生长的长期抑制（> 30天）。总而言之，这种高效的非病毒工作流程可以潜在地实现治疗性MSC的可扩展翻译，以治疗TMZ耐药性GBM和本研究范围之外的其他应用。