基因治疗将极大地受益于暂时调节治疗性基因表达的方法。核糖开关是小RNA元件,已经研究了它们在通过配体结合打开或关闭转基因表达中的潜在用途。我们比较了几种四环素和托伊霉素诱导的ON核糖开关对药物反应性转基因表达的影响。四环素依赖性K19核糖开关显示出最佳的控制，我们成功地将其应用于不同的转基因。基因表达的诱导是6到10倍，剂量依赖性，可逆,并在添加临床相关四环素剂量后数小时内发生，使用质粒或腺相关病毒(AAV)载体。为了提高交换能力，我们进一步优化了基因盒，以控制潜在的心血管疾病治疗基因的表达，VEGF-B使用两个或三个核糖开关同时减少泄漏和提高动态范围，核糖开关之间的接头序列改善了它们的功能。核糖开关功能是不依赖启动子的，但是表达盒中的转录后WPRE元件降低了其功能。优化的构建体是在转基因的3'末端具有100bp接头序列的双核糖开关。我们的研究揭示了核糖开关功能的显着差异，并为优化表达盒设计提供了重要方面。这些发现将有利于核糖开关的进一步研究和开发。
Gene therapy would greatly benefit from a method to regulate therapeutic gene expression temporally. Riboswitches are small RNA elements that have been studied for their potential use in turning transgene expression on or off by ligand binding. We compared several tetracycline and toyocamycin-inducible ON-riboswitches for a drug responsive transgene expression. The tetracycline-dependent K19 riboswitch showed the best control and we successfully applied it to different transgenes. The induction of gene expression was 6- to 10-fold, dose-dependent, reversible, and occurred within hours after the addition of a clinically relevant tetracycline dose, using either plasmid or adeno-associated virus (AAV) vectors. To enhance the switching capacity, we further optimized the gene cassette to control the expression of a potential therapeutic gene for cardiovascular diseases, VEGF-B. Using two or three riboswitches simultaneously reduced leakiness and improved the dynamic range, and a linker sequence between the riboswitches improved their functionality. The riboswitch function was promoter-independent, but a post-transcriptional WPRE element in the expression cassette reduced its functionality. The optimized construct was a dual riboswitch at the 3\' end of the transgene with a 100 bp linker sequence. Our study reveals significant differences in the function of riboswitches and provides important aspects on optimizing expression cassette designs. The findings will benefit further research and development of riboswitches.
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