以模型为指导设计基于 microRNA 的基因回路,支持将转基因货物精确植入不同的原代细胞中

Kasey S Love, Christopher P Johnstone, Emma L Peterman, Stephanie Gaglione, Kate E Galloway
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摘要

要实现工程细胞在治疗应用中的潜力,转基因必须在治疗效果窗口内表达。拷贝数的差异和其他外在噪声源会导致转基因表达的差异,并限制合成基因回路的性能。在治疗方面,转基因的超生理表达会损害工程表型并导致毒性。为了确保转基因的窄范围表达,我们设计并鉴定了基于单转录本、microRNA 的非相干前馈环路--Compact microRNA-Mediated Attenuator of Noise and Dosage (ComMAND)。我们调整了 ComMAND 的输出曲线,并建立了系统模型,以探索其他调整策略。通过将 ComMAND 与双基因实现进行比较,我们强调了单转录本架构所提供的精确控制,尤其是在拷贝数相对较低的情况下。我们的研究表明,ComMAND 能严格调控慢病毒的转基因表达,并能精确控制原代人类 T 细胞、原代大鼠神经元、原代小鼠胚胎成纤维细胞和人类诱导多能干细胞的表达。最后,ComMAND 能有效地将与临床相关的转基因 FMRP1 和 FXN 的水平设定在一个狭窄的窗口内。总之,ComMAND 是一种结构紧凑的工具,非常适合精确指定治疗载体的表达。
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Model-guided design of microRNA-based gene circuits supports precise dosage of transgenic cargoes into diverse primary cells
To realize the potential of engineered cells in therapeutic applications, transgenes must be expressed within the window of therapeutic efficacy. Differences in copy number and other sources of extrinsic noise generate variance in transgene expression and limit the performance of synthetic gene circuits. In a therapeutic context, supraphysiological expression of transgenes can compromise engineered phenotypes and lead to toxicity. To ensure a narrow range of transgene expression, we design and characterize Compact microRNA-Mediated Attenuator of Noise and Dosage (ComMAND), a single-transcript, microRNA-based incoherent feedforward loop. We tune the ComMAND output profile, and we model the system to explore additional tuning strategies. By comparing ComMAND to two-gene implementations, we highlight the precise control afforded by the single-transcript architecture, particularly at relatively low copy numbers. We show that ComMAND tightly regulates transgene expression from lentiviruses and precisely controls expression in primary human T cells, primary rat neurons, primary mouse embryonic fibroblasts, and human induced pluripotent stem cells. Finally, ComMAND effectively sets levels of the clinically relevant transgenes FMRP1 and FXN within a narrow window. Together, ComMAND is a compact tool well-suited to precisely specify expression of therapeutic cargoes.
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