Department of Chemical and Biological Engineering, University of Sheffield, Sheffield, UK.
Research and Early Development, REGENXBIO Inc., Rockville, Maryland, USA.
Biotechnol Bioeng. 2021 May;118(5):2001-2015. doi: 10.1002/bit.27713. Epub 2021 Mar 3.
Age-related macular degeneration (AMD) associated with dysfunction of retinal pigment epithelial (RPE) cells is the most common cause of untreatable blindness. To advance gene therapy as a viable treatment for AMD there is a need for technologies that enable controlled, RPE-specific expression of therapeutic genes. Here we describe design, construction and testing of compact synthetic promoters with a pre-defined transcriptional activity and RPE cell specificity. Initial comparative informatic analyses of RPE and photoreceptor (PR) cell transcriptomic data identified conserved and overrepresented transcription factor regulatory elements (TFREs, 8-19 bp) specifically associated with transcriptionally active RPE genes. Both RPE-specific TFREs and those derived from the generically active cytomegalovirus-immediate early (CMV-IE) promoter were then screened in vitro to identify sequence elements able to control recombinant gene transcription in model induced pluripotent stem (iPS)-derived and primary human RPE cells. Two libraries of heterotypic synthetic promoters varying in predicted RPE specificity and transcriptional activity were designed de novo using combinations of up to 20 discrete TFREs in series (323-602 bp) and their transcriptional activity in model RPE cells was compared to that of the endogenous BEST1 promoter (661 bp, plus an engineered derivative) and the highly active generic CMV-IE promoter (650 bp). Synthetic promoters with a highpredicted specificity, comprised predominantly of endogenous TFREs exhibited a range of activities up to 8-fold that of the RPE-specific BEST1 gene promoter. Moreover, albeit at a lower predicted specificity, synthetic promoter transcriptional activity in model RPE cells was enhanced beyond that of the CMV-IE promoter when viral elements were utilized in combination with endogenous RPE-specific TFREs, with a reduction in promoter size of 15%. Taken together, while our data reveal an inverse relationship between synthetic promoter activity and cell-type specificity, cell context-specific control of recombinant gene transcriptional activity may be achievable.
年龄相关性黄斑变性（AMD）与视网膜色素上皮（RPE）细胞功能障碍有关，是无法治愈的失明的最常见原因。为了将基因治疗推进为 AMD 的可行治疗方法，需要能够实现治疗基因的受控、RPE 特异性表达的技术。在这里，我们描述了具有预定义转录活性和 RPE 细胞特异性的紧凑型合成启动子的设计、构建和测试。对 RPE 和光感受器（PR）细胞转录组数据的初始比较分析确定了与转录活性 RPE 基因特异性相关的保守和过表达转录因子调节元件（TFRE，8-19bp）。然后，在体外筛选 RPE 特异性 TFRE 和源自通用巨细胞病毒立即早期（CMV-IE）启动子的 TFRE，以鉴定能够在模型诱导多能干细胞（iPS）衍生和原代人 RPE 细胞中控制重组基因转录的序列元件。使用多达 20 个离散 TFRE 串联（323-602bp）从头设计了两种异质合成启动子文库，以改变预测的 RPE 特异性和转录活性，并将它们在模型 RPE 细胞中的转录活性与内源性 BEST1 启动子（661bp，加上工程衍生）和高度活跃的通用 CMV-IE 启动子（650bp）进行了比较。具有高预测特异性的合成启动子主要由内源性 TFRE 组成，其活性范围高达内源性 BEST1 基因启动子的 8 倍。此外，尽管预测特异性较低，但当在病毒元件与内源性 RPE 特异性 TFRE 结合使用时，合成启动子在模型 RPE 细胞中的转录活性增强超过 CMV-IE 启动子，而启动子大小减小了 15%。总之，虽然我们的数据显示合成启动子活性与细胞类型特异性之间存在反比关系，但可以实现重组基因转录活性的细胞上下文特异性控制。
