Bruter A V, Kalashnikova M V, Prytyko A P, Belyavsky A V
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia.
Blokhin National Cancer Research Center, Ministry of Health of the Russian Federation, Moscow, 115478 Russia.
Mol Biol (Mosk). 2020 May-Jun;54(3):487-496. doi: 10.31857/S0026898420030040.
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.
质粒介导的基因治疗作为一种安全且相对廉价的治疗策略，却因转基因表达的快速沉默而备受困扰。这种沉默严重降低了质粒载体的长期效率。我们之前构建了一种低CpG的pMBR2质粒载体，该载体能在体外支持间充质干细胞中转基因的长期表达。在将载体电穿孔导入小鼠后肢肌肉并通过流体动力学方法导入肝脏后，研究了pMBR2载体对野生型小鼠分泌性碱性磷酸酶基因（mSEAPTwt）及其不含CpG的版本（mSEAP0）的长期表达情况。在一年的时间里测量了血液中的mSEAP水平。使用pMBR2 - mSEAP0构建体时，腿部肌肉中的mSEAP水平在前两个月增加了超过2.5倍，并在实验结束前一直高于初始水平。使用对照pCDNA3.1 - mSEAP0构建体时观察到的表达水平要低得多。pMBR2 - mSEAPwt的表达在6个月后降至约40%，此后保持在相似水平。在小鼠肝脏中，pMBR2 - mSEAP0的表达在最初18周内大约减半，然后缓慢下降至最终的17%水平。pMBR2 - mSEAPwt的表达最初降至18%，此后保持在约10%。相比之下，pCDNA3.1 - mSEAP0的表达在2周后急剧降至5%，并在实验的其余时间内保持在几乎为零的水平。因此，载体和转基因都应显著降低CpG含量，以确保在肝脏中质粒介导的表达得以延长，而将载体的CpG含量降至最低就足以在骨骼肌中表达。结果还表明，与位于转录单元外部相比，S/MAR元件位于转录单元内部会导致分泌蛋白而非细胞质蛋白的水平显著降低。
