Abstract

The development of ‘smart’ cell-based therapeutics requires cells that first recognize conditions consistent with disease (e.g. inflammation) and then subsequently release therapeutic proteins, thereby reducing potential toxicity from otherwise continuous expression. Promoters containing NF-κB response elements are often used as reporters of inflammation; however, endogenous promoters have crosstalk with other pathways, and current synthetic promoters have many exact sequence repeats of NF-κB response elements which make them both difficult to synthesize and inherently genetically unstable. Herein, a synthesis-friendly inflammation-inducible promoter (named SFNp) was created by the packing of 14 NF-κB response elements, which have no repeats >9 bp, followed by a minimal cytomegalovirus promoter. In stably expressing human embryonic kidney 293 cells, we assessed the ability of SFNp to inducibly transcribe genes for reporting expression, changing cell morphology, and performing cell fusion. These experiments represent simple milestones for potentially using SFNp in the development of cell-based therapeutics. As strongly repeated DNA can compromise the long-term stability of genetic circuits, new designs used in ‘smart’ cell therapy will become more reliant on synthesis-friendly components like SFNp.

中文翻译：

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创造适合细胞治疗的合成友好型炎症诱导启动子

摘要

“智能”细胞疗法的发展需要细胞首先识别与疾病一致的条件（例如炎症），然后释放治疗性蛋白质，从而降低其他连续表达的潜在毒性。含有 NF-κB 反应元件的启动子通常被用作炎症的报告分子；然而，内源性启动子与其他途径有串扰，并且目前合成的启动子具有许多精确的 NF-κB 反应元件序列重复，这使得它们既难以合成，又在遗传上不稳定。在这里，合成友好的炎症诱导启动子（命名为 SFNp）是通过包装 14 个 NF-κB 反应元件产生的，这些元件没有重复 >9 bp，然后是最小的巨细胞病毒启动子。在稳定表达人胚肾 293 细胞时，我们评估了 SFNp 诱导转录基因以报告表达、改变细胞形态和进行细胞融合的能力。这些实验代表了在开发基于细胞的疗法中可能使用 SFNp 的简单里程碑。由于强烈重复的 DNA 会损害基因回路的长期稳定性，“智能”细胞疗法中使用的新设计将更加依赖于合成友好的成分，如 SFNp。