Abstract

Plasmid-mediated gene therapy, being a safe and relatively inexpensive therapeutic strategy, is plagued by a fast silencing of transgene expression. The silencing severely reduces the long-term efficiency of plasmid vectors. We have earlier constructed a low-CpG pMBR2 plasmid vector supporting prolonged expression of transgenes in mesenchymal stem cells in vitro. Long-term expression from the pMBR2 vector was studied for the wild-type mouse secreted alkaline phosphatase gene (mSEAPTwt) and its version devoid of CpGs (mSEAP0) after vector electroporation into mouse hindlimb muscles and hydrodynamic delivery to the liver. The mSEAP levels in the blood were measured over one year. With the pMBR2-mSEAP0 construct, the mSEAP levels in leg muscles increased more than 2.5-fold in the first two months and remained higher than the initial level until the end of the experiment. Far lower expression levels were observed with the control pCDNA3.1-mSEAP0 construct. Expression from pMBR2-mSEAPwt decreased to about 40% after 6 months and remained at similar levels thereafter. In the mouse liver, expression from pMBR2-mSEAP0 was approximately halved within the first 18 weeks and then decrease slowly to the final 17% level. Expression from pMBR2-mSEAPwt initially dropped to 18% and remained at approximately 10% thereafter. In contrast, expression from pCDNA3.1-mSEAP0 sharply dropped to 5% after 2 weeks and remained at nearly zero levels throughout the rest of the experiment. Thus, both vector and transgene should have significantly reduced CpG contents to ensure prolonged plasmid-mediated expression in the liver, while minimizing the vector CpG content is sufficient for expression in skeletal muscles. The results suggested additionally that the localization of S/MAR elements within the transcription unit, in contrast to their outside location, results in significant reduction of the level of secreted, but not cytoplasmic, proteins.

中文翻译：

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体内质粒表达的维持主要取决于载体和转基因的CpG含量

摘要

质粒介导的基因治疗是一种安全且相对便宜的治疗策略，但转基因表达的快速沉默困扰着他们。沉默严重降低了质粒载体的长期效率。我们早先已经构建了一个低CpG pMBR2质粒载体，该载体在体外支持间充质干细胞中转基因的延长表达。研究了pMBR2载体对野生型小鼠分泌的碱性磷酸酶基因（mSEAPTwt）及其不含CpGs（mSEAP0）的版本的pMBR2载体的长期表达，将载体电穿孔到小鼠后肢肌肉中并将其流体动力传递至肝脏。一年中测量了血液中的mSEAP水平。使用pMBR2-mSEAP0构建体，腿部肌肉中的mSEAP水平增加了2倍以上。在最初的两个月中提高了5倍，并且一直高于初始水平，直到实验结束。用对照pCDNA3.1-mSEAP0构建体观察到低得多的表达水平。6个月后，pMBR2-mSEAPwt的表达降低至约40％，此后保持相似水平。在小鼠肝脏中，来自pMBR2-mSEAP0的表达在前18周内大约减半，然后缓慢降低至最终17％的水平。pMBR2-mSEAPwt的表达最初下降至18％，此后保持在约10％。相比之下，pCDNA3.1-mSEAP0的表达在2周后急剧下降至5％，在整个实验的其余部分中几乎保持在零水平。因此，载体和转基因的CpG含量均应显着降低，以确保质粒介导的肝脏中延长的表达，同时将载体的CpG含量降至最低，足以在骨骼肌中表达。结果还表明，S / MAR元件在转录单位内的定位与其外部位置相反，导致分泌的蛋白水平显着降低，但胞质蛋白水平却没有显着降低。