Love Kasey S, Johnstone Christopher P, Peterman Emma L, Gaglione Stephanie, Birnbaum Michael E, Galloway Kate E
Department of Biological Engineering, MIT, 25 Ames St., Cambridge, MA 02139, USA.
Department of Chemical Engineering, MIT, 25 Ames St., Cambridge, MA 02139, USA.
Cell Syst. 2025 Apr 22:101269. doi: 10.1016/j.cels.2025.101269.
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 developed a single-transcript, microRNA-based incoherent feedforward loop called compact microRNA-mediated attenuator of noise and dosage (ComMAND). We experimentally tuned the ComMAND output profile, and we modeled the system to explore additional tuning strategies. By comparing ComMAND to two-gene implementations, we demonstrate the precise control afforded by the single-transcript architecture, particularly at 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 frataxin (FXN) and fragile X messenger ribonucleoprotein 1 (Fmr1) within a narrow window. Overall, ComMAND is a compact tool well suited to precisely specify the expression of therapeutic cargoes. A record of this paper's transparent peer review process is included in the supplemental information.
在治疗背景下，转基因的超生理表达可能会损害工程化表型并导致毒性。为确保转基因表达范围狭窄，我们开发了一种基于单转录本、微小RNA的非相干前馈环，称为紧凑微小RNA介导的噪声和剂量衰减器（ComMAND）。我们通过实验调整了ComMAND的输出谱，并对该系统进行建模以探索其他调整策略。通过将ComMAND与双基因实施方案进行比较，我们证明了单转录本结构所提供的精确控制，尤其是在低拷贝数情况下。我们表明，ComMAND能严格调节慢病毒中的转基因表达，并精确控制原代人T细胞、原代大鼠神经元、原代小鼠胚胎成纤维细胞和人诱导多能干细胞中的表达。最后，ComMAND有效地将临床相关转基因frataxin（FXN）和脆性X信使核糖核蛋白1（Fmr1）的水平设定在一个狭窄的范围内。总体而言，ComMAND是一种紧凑的工具，非常适合精确指定治疗性货物的表达。本文透明同行评审过程的记录包含在补充信息中。
