Model-guided design of microRNA-based gene circuits supports precise dosage of transgenic cargoes into diverse primary cells.

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.