Molecular Microbiology and Gene Therapy Unit, Institute of Medical Microbiology, University of Regensburg, Franz-Josef-Strauss-Allee 11, Regensburg, Germany.
Gene Ther. 2011 Feb;18(2):189-98. doi: 10.1038/gt.2010.134. Epub 2010 Oct 14.
Advanced gene delivery techniques can be combined with rational gene design to further improve the efficiency of plasmid DNA (pDNA)-mediated transgene expression in vivo. Herein, we analyzed the influence of intragenic sequence modifications on transgene expression in vitro and in vivo using murine erythropoietin (mEPO) as a transgene model. A single electro-gene transfer of an RNA- and codon-optimized mEPOopt gene into skeletal muscle resulted in a 3- to 4-fold increase of mEPO production sustained for >1 year and triggered a significant increase in hematocrit and hemoglobin without causing adverse effects. mEPO expression and hematologic levels were significantly lower when using comparable amounts of the wild type (mEPOwt) gene and only marginal effects were induced by mEPOΔCpG lacking intragenic CpG dinucleotides, even at high pDNA amounts. Corresponding with these observations, in vitro analysis of transfected cells revealed a 2- to 3-fold increased (mEPOopt) and 50% decreased (mEPOΔCpG) erythropoietin expression compared with mEPOwt, respectively. RNA analyses demonstrated that the specific design of the transgene sequence influenced expression levels by modulating transcriptional activity and nuclear plus cytoplasmic RNA amounts rather than translation. In sum, whereas CpG depletion negatively interferes with efficient expression in postmitotic tissues, mEPOopt doses <0.5 μg were sufficient to trigger optimal long-term hematologic effects encouraging the use of sequence-optimized transgenes to further reduce effective pDNA amounts.
先进的基因传递技术可以与合理的基因设计相结合，进一步提高质粒 DNA（pDNA）介导的转基因在体内的表达效率。本文以鼠红细胞生成素（mEPO）为转基因模型，分析了基因内序列修饰对转基因在体外和体内表达的影响。单次电基因转移 RNA 和密码子优化的 mEPOopt 基因到骨骼肌中，导致 mEPO 产生持续增加 3-4 倍，持续>1 年，同时使红细胞压积和血红蛋白显著增加，而无不良影响。使用相同量的野生型（mEPOwt）基因时，mEPO 表达和血液学水平明显较低，而缺乏基因内 CpG 二核苷酸的 mEPOΔCpG 仅引起轻微影响，即使在高 pDNA 量时也是如此。与这些观察结果一致，转染细胞的体外分析显示，与 mEPOwt 相比，（mEPOopt）分别增加了 2-3 倍，（mEPOΔCpG）减少了 50%的促红细胞生成素表达。RNA 分析表明，转基因序列的特异性设计通过调节转录活性和核加细胞质 RNA 量而不是翻译来影响表达水平。总之，CpG 耗竭会对有丝分裂后组织的有效表达产生负面影响，而 mEPOopt 剂量<0.5μg 足以引发最佳的长期血液学效应，鼓励使用序列优化的转基因进一步减少有效 pDNA 量。
