A longstanding challenge in gene therapy is expressing a dosage-sensitive gene within a tight therapeutic window. For example, loss of MECP2 function causes Rett syndrome, while its duplication causes MECP2 duplication syndrome. Viral gene delivery methods generate variable numbers of gene copies in individual cells, creating a need for gene dosage-invariant expression systems. Here, we introduce a compact miRNA-based, incoherent feed-forward loop circuit that achieves precise control of Mecp2 expression in cells and brains, and improves outcomes in an AAV-based mouse model of Rett syndrome gene therapy. Single molecule analysis of endogenous and ectopic Mecp2 mRNA revealed precise, sustained expression across a broad range of gene dosages. Delivered systemically in a brain-targeting AAV capsid, the circuit strongly suppressed Rett behavioral symptoms for over 24 weeks, outperforming an unregulated gene therapy. These results demonstrate that synthetic miRNA-based regulatory circuits can enable precise in vivo expression to improve the safety and efficacy of gene therapy.

中文翻译：

---

用于定量基因治疗的合成剂量补偿 miRNA 电路

基因治疗的一个长期挑战是在严格的治疗窗口内表达剂量敏感的基因。例如，MECP2 功能丧失会导致 Rett 综合征，而 MECP2 功能重复则会导致 MECP2 重复综合征。病毒基因递送方法在单个细胞中产生不同数量的基因拷贝，这就产生了对基因剂量不变表达系统的需求。在这里，我们引入了一种紧凑的基于 miRNA 的非相干前馈环路，可实现细胞和大脑中 Mecp2 表达的精确控制，并改善基于 AAV 的 Rett 综合征基因治疗小鼠模型的结果。对内源性和异位 Mecp2 mRNA 的单分子分析揭示了在广泛的基因剂量范围内精确、持续的表达。该电路通过大脑靶向 AAV 衣壳进行全身递送，在超过 24 周的时间内强烈抑制 Rett 行为症状，优于不受监管的基因疗法。这些结果表明，基于合成 miRNA 的调控电路可以实现体内精确表达，从而提高基因治疗的安全性和有效性。